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a recent systematic analysis showed that in 2011 , 314 ( 296 - 331 ) million children younger than 5 years were mildly , moderately or severely stunted and 258 ( 240 - 274 ) million were mildly , moderately or severely underweight in the developing countries . in iran a study among 752 high school girls in sistan and baluchestan showed prevalence of 16.2% , 8.6% and 1.5% , for underweight , overweight and obesity , respectively . the prevalence of malnutrition among elementary school aged children in tehran varied from 6% to 16% . anthropometric study of elementary school students in shiraz revealed that 16% of them suffer from malnutrition and low body weight . snack should have 300 - 400 kcal energy and could provide 5 - 10 g of protein / day . nowadays , school nutrition programs are running as the national programs , world - wide . national school lunch program in the united states there are also some reports regarding school feeding programs in developing countries . in vietnam , school base program showed an improvement in nutrient intakes . in iran a national free food program ( nffp ) is implemented in elementary schools of deprived areas to cover all poor students . however , this program is not conducted in slums and poor areas of the big cities so many malnourished children with low socio - economic situation are not covered by nffp . although the rate of poverty in areas known as deprived is higher than other areas , many students in deprived areas are not actually poor and can afford food . hence , nutritional value of the nffp is lower than the scientific recommended snacks for this age group . furthermore , lack of variety of food packages has decreased the tendency of children toward nffp . on the other hand , the most important one is ministry of education ( moe ) of iran , which is responsible for selecting and providing the packages for targeted schools . the ministry of health ( moh ) is supervising the health situation of students and their health needs . welfare organizations , along with charities , have the indirect effect on nutritional status of students by financial support of their family . provincial governors have also the role of coordinating and supervising all activities of these organizations . parent - teacher association is a community - based institution that participates in school 's policy such as nffp . in addition to these organizations , nutritional literacy of students , their parents and teachers , is a very important issue , which could affect nutritional status of school age children . therefore , the present study was conducted with the aim of improving the nffp , so that by its resources all poor children will be covered even in big cities . moreover , all food packages were replaced by nutritious and diverse packages that were accessible for non - poor children . according to the aim of this study and multiple factors that could affect the problem , public health advocacy has been chosen as the best strategy to deal with this issue . therefore , the present study determines the effects of nutrition intervention in an advocacy process model on the prevalence of underweight in school aged children in the poor area of shiraz , iran . this interventional study has been carried out between 2009 and 2010 in shiraz , iran . this survey was approved by the research committee of shiraz university of medical sciences . in coordination with education organization of fars province two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . in those schools all students ( 2897 , 7 - 13 years old ) were screened based on their body mass index ( bmi ) by nutritionists . according to convenience method all students divided to two groups based on their economic situation ; family revenue and head of household 's job and nutrition situation ; the first group were poor and malnourished students and the other group were well nourished or well - off students . for this report , the children 's height and weight were entered into center for disease control and prevention ( cdc ) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards . the significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . for implementing the interventions , the advocacy process model weight was to the nearest 0.1 kg on a balance scale ( model # seca scale ) . standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer . advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders . the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts . the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies : three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge . education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) . accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children . after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . for educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support . healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on . nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron . low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions . the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean . each student that was malnourished and poor has been taken into account for free food and nutritious snacks . demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire . this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province . statistical analyses were performed using the statistical package for the social sciences ( spss ) software , version 17.0 ( spss inc . , the results are expressed as mean sd and proportions as appropriated . in order to determine the effective variables on the malnutrition status paired t test was used to compare the end values with baseline ones in each group . in this project , the who z - score cut - offs used were as follow : using bmi - for - age z - scores ; overweight : > + 1 sd , i.e. , z - score > 1 ( equivalent to bmi 25 kg / m ) , obesity : > + 2 sd ( equivalent to bmi 30 kg / m ) , thinness : < 2 sd and severe thinness : < 3 sd . this interventional study has been carried out between 2009 and 2010 in shiraz , iran . this survey was approved by the research committee of shiraz university of medical sciences . in coordination with education organization of fars province two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . in those schools all students ( 2897 , 7 - 13 years old ) were screened based on their body mass index ( bmi ) by nutritionists . according to convenience method all students divided to two groups based on their economic situation ; family revenue and head of household 's job and nutrition situation ; the first group were poor and malnourished students and the other group were well nourished or well - off students . for this report , the children 's height and weight were entered into center for disease control and prevention ( cdc ) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards . the significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . for implementing the interventions , weight was to the nearest 0.1 kg on a balance scale ( model # seca scale ) . standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer . advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders . the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts . the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies : three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge . duration of intervention was 6 months . education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) . accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . obviously , student 's families had remarkable effect on children 's food habit . it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children . after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support . healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on . nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron . low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions . the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean . each student that was malnourished and poor has been taken into account for free food and nutritious snacks . demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire . this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province . advocacy group formation : this step was started with stakeholder analysis and identifying the stakeholders . the team was formed with representatives of all stakeholders include ; education organization , welfare organization , deputy for health of shiraz university , food and cosmetic product supervisory office and several non - governmental organizations and charities . situation analysis : this was carried out by use of existing data such as formal report of organizations , literature review and focus group with experts . the prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . accordingly , three sub - groups were established : research and evaluation , education and justification and executive group . designing the strategies : three strategies were identified ; education and justification campaign , nutritional intervention ( providing nutritious , safe and diverse snacks ) and networking . performing the interventions : interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group ( poor and malnourished students ) was utilized the package free of charge . education and justification intervention : regarding the literature review and expert opinion , an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level ( degree ) . accordingly , education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . obviously , student 's families had remarkable effect on children 's food habit . it leads the educational group to hold several meeting with the student 's parents to justify them about the project and its benefit for their children . after these meetings , parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . educate fifteen talk show programs in tv and radio , 12 published papers in the local newspaper , have implemented to mobilize the community and gain their support . healthy diet , the importance of breakfast and snack in adolescence , wrong food habits among school age children , role of the family to improve food habit of children were the main topics , in which media campaign has focused on . nutritional intervention : the snack basket of the students was replaced with traditional , nutritious and diverse foods . in general , the new snack package in average has provided 380 kcal energy , 15 g protein along with sufficient calcium and iron . low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . research and evaluation : in this step , the literacy and anthropometric indices ( bmi ) of students were assessed before and after the interventions . the reference for anthropometric measures was the world health organization / national center for health statistics ( who / nchs ) standards and the cut - offs were - two standard deviations ( sd ) from the mean . each student that was malnourished and poor has been taken into account for free food and nutritious snacks . demographic information , height , weight and knowledge of the students were measured by use of a validated and reliable ( cronbach 's alpha was 0.61 ) questionnaire . this project is granted by shiraz university of medical sciences , charities and welfare organization and education organization of fars province . statistical analyses were performed using the statistical package for the social sciences ( spss ) software , version 17.0 ( spss inc . , chicago , il , usa ) . the results are expressed as mean sd and proportions as appropriated . in order to determine the effective variables on the malnutrition status paired t test was used to compare the end values with baseline ones in each group . two - sided p < 0.05 was considered to be statistically significant . in this project , the who z - score cut - offs used were as follow : using bmi - for - age z - scores ; overweight : > + 1 sd , i.e. , z - score > 1 ( equivalent to bmi 25 kg / m ) , obesity : > + 2 sd ( equivalent to bmi 30 kg / m ) , thinness : < 2 sd and severe thinness : < 3 sd . study population contains 2897 children ; 70.8% were primary school students and 29.2% were secondary school students . 2336 ( 80.5% ) out of total students were well - off and 561 children ( 19.5% ) were indigent . 19.5% of subjects were in case group ( n = 561 ) and 80.5% were in the control group ( n = 2336 ) . the mean of age in welfare group was 10.0 2.3 and 10.5 2.5 in non - welfare group . demographic characteristics of school aged children in shiraz , iran table 2 shows the frequency of subjects in different categories of bmi for age in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model in shiraz , iran . the frequency of subjects with bmi lower than < 2 sd decreased significantly after intervention among non - welfare girls ( p < 0.01 ) . however , there were no significant decreases in the frequency of subjects with bmi lower than < 2 sd boys . when we assess the effect of intervention in total population without separating by sex groups , we found no significant change in this population [ table 3 ] . bmi for age for iranian students aged 7 - 14 years based on gender according to who growth standards 2007 bmi for age for iranian students aged 7 - 14 years according to who growth standards 2007 in non - welfare and welfare groups of total population table 4 has shown the prevalence of normal bmi , mild , moderate and severe malnutrition in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model . according to this table there were no significant differences in the prevalence of mild , moderate and severe malnutrition among girls and boys . table 4 also shows the mean of all anthropometric indices changed significantly after intervention both among girls and boys . the pre- and post - test education assessment in both groups showed that the student 's average knowledge score has been significantly increased from 12.5 3.2 to 16.8 4.3 ( p < 0.0001 ) . bmi , height and weight in non - welfare and welfare groups of school aged children separately in males and females before and after a nutrition intervention based on advocacy process model in shiraz , iran according to study 's finding the odds ratio ( or ) of sever thinness and thinness in non - welfare compared with welfare is 3.5 ( or = 3.5 , confidence interval [ ci ] = 2.5 - 3.9 , p < 0.001 ) . furthermore , the finding showed or of overweight and obesity in welfare compared to non - welfare is 19.3 ( or = 19.3 , ci = 2.5 - 3.9 , p = 0.04 ) . the result of this community intervention study revealed that nutrition intervention based on advocacy program had been successful to reduce the prevalence of underweight among poor girls . this study shows determinant factor of nutritional status of school age children was their socio - economic level . according to our knowledge , this is the first study , which determines the effect of a community intervention based on advocacy process on the malnutrition indices in a big city ( shiraz ) in iran . the other program in iran ( nffp ) is specified to deprived area and is not conducted in big cities . allocating millions of dollars to nffp by government , selecting the malnourished students through an active screening system at primary and middle schools , paying attention of policy makers to student 's nutrition have provided the opportunity to combat the problem . however , negligence of under - poverty line , providing poor snacks in terms of nutritional value and lack of variety are the main defects of this program . advocacy by definition is a blending of science , ethics and politics for comprehensive approaching health issues . by using advocacy program in california among the high school students for improving their nutrition and physical activity angeles unified school district participants emphasized on nutrition classes for families as well as students in addition to other interventions . in the present study another study revealed that evaluability assessment gave stakeholders the opportunity to reflect on the project and its implementation issues . it seems that in iran , free food program among the students not only is needed in deprived areas , but also it should be performed in big cities such as shiraz . at baseline , no significant difference was founded among wealthy students between the pre- and post - nutritional status intervention . in contrast , the numbers of students who have malnutrition decreased from 44% to 39.4% , which was identified as a significant among impecunious girls students . there was also a significant increase in the proportion of children with bmi that was normal for age ( 2 to + 1 sd ) most of the published community interventions showed better results among females compared with males . this difference in the impact of nutritional interventions between male and female might be related to the different age of puberty in the female population compared to the male population . in the age range of the present study female although , there is no nffp in big cities of iran , there are some programs for improving the nutritional status such as providing free milk in schools . a recent publication has shown that school feeding programs focus on milk supplementation had beneficial effects on the physical function and school performances specifically among girls in iran . the results of the mentioned study showed an improvement in the weight of children , psychological test 's scores and the grade - point average following this school feeding program . the intervention in the present study had focused on the snack intake in the school time . there are some reports regarding the nutrition transition in iran , which shows the importance of nutrition intervention to provide more healthy eating dietary habits among welfare groups of adolescents . hence , nutrition intervention especially in the form of nutrition education is needed in big cities and among welfare children and adolescents . although a study among iranian adolescents showed that dietary behavior of adolescents does not accord to their knowledge , which emphasize on the necessity of community intervention programs . a recent study regarding the major dietary pattern among iranian children showed the presence of four major dietary patterns , in which fast food pattern and sweet pattern as two major dietary patterns can be mentioned among iranian children . in advocacy program audience 's analysis accordingly , one of the prominent strategies in this study was working with media and was meeting with parent - teacher association that both of them were secondary target audiences . we also took into account policy makers in different levels , from national to local as primary audiences . advocacy team had several meetings with management and planning organization at national level and education organization of the fars province as well as principal of the targeted schools . providing nutritious snacks need contribution of private sector such as food industries or factories , but their benefits should be warranted . another choice was community involvement ; which can be achieved by female health volunteers who are working with the health system . advocacy team by using the support of charities and female health volunteers could establish a local factory that produced student 's snacks based on the new definition . however , there are some challenges on the way of expanding this program . mass production of the proposed snacks according to different desires and cultures and getting involvement of food industries with respect to marketing issues is one of those challenges . moreover , providing a supportive environment in order to change the food habits of the students and their parents among the wide range of the population require a sustainable and continuous inter - sector collaboration . although in a limited number of schools , in our study , interventions and advocacy program was successful , expanding this model to another areas around the country depends on convincing the policy makers at national level . in this regard , advocacy team should prepare evidenced based profile and transitional planning to convince the policy makers for improving the rule and regulation of nffp . the same as this study in other studies have also emphasized that there must be efforts to strengthen the capacity within the schools to deal with the nutritional problems either overweight , obesity or malnutrition by using of educational and nutritional intervention . assessing the dietary adherence is very important in nutrition intervention among population . as this population was children and adolescents we had a limitation in the blood sample collection to assess the subject 's dietary adherence . furthermore , this intervention was only focused on the intake of snack in school time and we did not have comprehensive information on the dietary intake of children and adolescents after school all over the day . the investigators propose further investigation in different areas of the country based on socio - cultural differences in order to make necessary modification and adapt this model to other areas . regarding the nutritional needs of the school age children , provision of a good platform for implementing and expanding this efficient model to the whole country based upon the socio - economic situation of each region is advisable to the moh and the moe . community nutrition intervention based on the advocacy process model is effective on reducing the prevalence of underweight specifically among female school aged children . it occurs in more than 50% of patients and may reach 90% in certain types of cancers , especially in patients undergoing chemotherapy and/or radiation therapy.1 anemia is defined as an inadequate circulating level of hemoglobin ( hb ) ( hb < 12 g / dl ) and may arise as a result of the underlying disease , bleeding , poor nutrition , chemotherapy , or radiation therapy . preliminary studies suggest that survival and loco - regional control after radiation therapy , especially in head and neck cancers , may be compromised by anemia.24 anemia often worsens symptoms such as fatigue , weakness , and dyspnea , and thus may have a negative effect on quality of life ( qol ) and performance status in patients with cancer . thus , to improve physical functioning , qol , and prognosis in patients with cancer , it would be reasonable to take a proactive approach in identifying populations who need treatment for cancer - associated anemia ( caa ) and provide timely management . blood transfusion is an effective way to replace depleted hb within a short period , but the effect is , unfortunately , temporary and can cause serious adverse risks and increased mortality . in randomized clinical trials in patients with caa , erythropoiesis - stimulating agents ( esas ) produced significant increases in hb level , decreased transfusion requirements , and improved qol.57 however , 30%50% of patients do not respond to such agents . in addition , the use of esas often causes concern about severe adverse reactions.6,8 in several studies , esas were found to shorten overall survival time , or time to tumor progression in patients whose hb level reached more than 12 g / dl . these studies included patients with different primary cancers , such as breast , lung , head and neck , cervix , and lymphomas.911 the lack of response to erythropoietin stimulation in patients with cancer is partly attributed to the functional iron deficiency state , in which the high rate of erythropoiesis exceeds the delivery of usable iron , despite adequate iron stores.12 absolute iron deficiency , in contrast , occurs when iron delivery is impaired because iron stores are depleted ( serum ferritin , < 100 ng / ml ; transferring saturation , < 20%).13 hepcidin , a peptide hormone produced by the liver , is up - regulated in chronic inflammatory states including cancer . hepcidin inhibits iron transport across cell membranes , thus decreasing the accessibility of stored iron and gastrointestinal absorption of dietary iron , leading to an increased frequency of iron - restricted erythropoiesis.1416 many randomized trials examined the role of intravenous ( iv ) iron in addition to esas in the treatment of anemia in patients with cancer . many of these studies showed improvement in esa response , time to maximal response , reduction in esa dose , and improvement in qol parameters ( when measured ) in favor of the combination over esas alone . the observed benefit was independent of baseline iron parameters.1721 one study found a 36% reduction in the number of patients transfused.21 this pilot study assessed the efficacy and feasibility of iv iron monotherapy in patients with cancer who have anemia and who are undergoing treatment with chemotherapy and/or radiation therapy without the use of esas . patients received the study treatment for 12 weeks followed by a 4-week follow - up period . eligible patients were at least 18 years old , about to start a cycle of chemotherapy and/or radiation therapy within 1 week of inclusion , and had a nonmyeloid malignancy , hb levels of 11.0 g / dl or less , a life expectancy of more than 24 weeks , and an eastern cooperative oncology group performance status of 02 . patients were also required to have a serum ferritin level of 100 ng / ml or higher or transferrin saturation ( tsat ) levels of 15% or higher and to have received no esas or iv iron therapy within 30 days and no oral iron therapy ( 27 mg / day or more ) within 7 days before enrollment . patients were excluded for leukoerythroblastic features on blood film , hemolysis , gastrointestinal bleeding , folate or vitamin b12 deficiency , elevated serum ferritin ( 900 ng / ml ) or transferrin saturation ( tsat ) ( 35% ) levels , pregnancy or lactation , liver dysfunction ( grade 2 or higher based on national cancer institute common toxicity criteria ) , renal dysfunction ( serum creatinine levels 2.0 mg / dl ) , active infection requiring systemic antibiotics , personal or family history of hemochromatosis , comorbidities precluding study participation , hypersensitivity to iv iron , red blood cell transfusion within the last 2 weeks , or any investigational agent within 30 days before enrollment . patients were not allowed to take any vitamin , mineral , or herbal supplements containing 27 mg or more of iron per day or 100 mg vitamin c per day during the study or follow - up period . blood transfusions were permitted at the primary physician s discretion if hb levels decreased to 8 g / dl or less , and such patients were considered treatment failures . written informed consent was provided by all patients before study participation , and the protocol and supporting documents were approved by the institutional review board of king hussein cancer center . the study was conducted in accordance with the declaration of helsinki and good clinical practice as contained in the us code of federal regulations that governs the protection of human subjects and the obligations of clinical investigators . patients received 200 mg ferric hydroxide sucrose diluted in 100 ml normal saline and infused over the course of 1 hour weekly for a total of 12 weeks . the first dose was given during the first clinic visit ( 4 days from the initiation of chemotherapy or radiation therapy ) . tsat was monitored , as protocol mandated withholding iron therapy when tsat levels were higher than 50% . at the first clinic visit ( week 1 ; baseline ) , a blood sample was obtained for laboratory assessments before the study treatment was started . patients attended weekly clinic visits for treatment and assessment ; and returned for follow - up visits at week 14 which included a complete physical examination . complete blood count and tsat were done every 3 weeks , and again 2 weeks after last treatment ( week 14 ) . complete laboratory assessment ( hb , serum ferritin , reticulocyte count , transferrin , tsat , serum iron , total iron binding capacity , red cell indices , white blood cell count with differential , platelet count , and serum chemistries ) were done at week 1 and at week 14 ( end of study ) . adverse events were assessed at each clinic visit until study completion or withdrawal , and during the 30 days after the last study treatment . hb test results were presented as mean , median , and range through all 12 weeks . comparison between means of hb level were made between the baseline hb and hb levels in the following weeks , using t - test . a significance criterion of p < 0.05 was used in the analysis . all analyses were performed using sas version 9.1 ( sas institute inc , cary , nc , usa ) . patients received the study treatment for 12 weeks followed by a 4-week follow - up period . eligible patients were at least 18 years old , about to start a cycle of chemotherapy and/or radiation therapy within 1 week of inclusion , and had a nonmyeloid malignancy , hb levels of 11.0 g / dl or less , a life expectancy of more than 24 weeks , and an eastern cooperative oncology group performance status of 02 . patients were also required to have a serum ferritin level of 100 ng / ml or higher or transferrin saturation ( tsat ) levels of 15% or higher and to have received no esas or iv iron therapy within 30 days and no oral iron therapy ( 27 mg / day or more ) within 7 days before enrollment . patients were excluded for leukoerythroblastic features on blood film , hemolysis , gastrointestinal bleeding , folate or vitamin b12 deficiency , elevated serum ferritin ( 900 ng / ml ) or transferrin saturation ( tsat ) ( 35% ) levels , pregnancy or lactation , liver dysfunction ( grade 2 or higher based on national cancer institute common toxicity criteria ) , renal dysfunction ( serum creatinine levels 2.0 mg / dl ) , active infection requiring systemic antibiotics , personal or family history of hemochromatosis , comorbidities precluding study participation , hypersensitivity to iv iron , red blood cell transfusion within the last 2 weeks , or any investigational agent within 30 days before enrollment . patients were not allowed to take any vitamin , mineral , or herbal supplements containing 27 mg or more of iron per day or 100 mg vitamin c per day during the study or follow - up period . blood transfusions were permitted at the primary physician s discretion if hb levels decreased to 8 g / dl or less , and such patients were considered treatment failures . written informed consent was provided by all patients before study participation , and the protocol and supporting documents were approved by the institutional review board of king hussein cancer center . the study was conducted in accordance with the declaration of helsinki and good clinical practice as contained in the us code of federal regulations that governs the protection of human subjects and the obligations of clinical investigators . patients received 200 mg ferric hydroxide sucrose diluted in 100 ml normal saline and infused over the course of 1 hour weekly for a total of 12 weeks . the first dose was given during the first clinic visit ( 4 days from the initiation of chemotherapy or radiation therapy ) . tsat was monitored , as protocol mandated withholding iron therapy when tsat levels were higher than 50% . at the first clinic visit ( week 1 ; baseline ) , a blood sample was obtained for laboratory assessments before the study treatment was started . patients attended weekly clinic visits for treatment and assessment ; and returned for follow - up visits at week 14 which included a complete physical examination . complete blood count and tsat were done every 3 weeks , and again 2 weeks after last treatment ( week 14 ) . complete laboratory assessment ( hb , serum ferritin , reticulocyte count , transferrin , tsat , serum iron , total iron binding capacity , red cell indices , white blood cell count with differential , platelet count , and serum chemistries ) were done at week 1 and at week 14 ( end of study ) . adverse events were assessed at each clinic visit until study completion or withdrawal , and during the 30 days after the last study treatment . hb test results were presented as mean , median , and range through all 12 weeks . comparison between means of hb level were made between the baseline hb and hb levels in the following weeks , using t - test . a significance criterion of p < 0.05 was used in the analysis . all analyses were performed using sas version 9.1 ( sas institute inc , cary , nc , usa ) . twenty - five patients ( 17 women and 8 men ) were eligible , consented , and included in the study ; their mean age ( standard deviation , [ sd ] ) was 56 years ( 13.0 years ) . chemotherapy varied according to the primary cancer and included anthracycline , platinum , taxanes , cyclophosphamide , high - dose ifosfamide , vincristine , vinblastine , bleomycin , and others . many of the included patients had their chemotherapy treatment as second- or third - line therapy . patients characteristics , including age , primary tumor , and active anticancer treatment are summarized in table 1 . one patient died during the study from his tumor ( after week 2 ) , and five patients withdrew from the study because of inconvenience ( three after week 3 , and two after week 4 ) . nineteen ( 76.0% ) patients completed a minimum of three treatments , 15 ( 60.0% ) completed nine treatments , and 14 ( 56.0% ) completed all twelve planned weekly treatments . as seen in table 2 , the mean hb level of the 25 patients at baseline was 9.6 g / dl ( median , 9.9 g / dl ; range , 6.9 g / dl10.9 g / dl ) . for the 15 patients who completed at least nine treatments , the mean change in their hb level was 1.7 g / dl ( median , 1.1 g / dl ; range , 1.9 g / dl to 3.2 g / dl ) . for the 14 patients who completed the whole treatment period ( 12 weeks ) , the mean hb level change was 2.1 g / dl ( median , 1.3 g / dl ; range , 0.2 g / dl to 4.6 g / dl ; p = 0.0007 ) . eight ( 42.1% ) of the 19 patients who completed at least three iron infusions had a more than 1 g / dl increase in their hb level . hemoglobin level changes for the 14 patients who completed twelve iron infusions are shown in figure 1 . no iv iron - related adverse events were reported among patients during the study or the follow - up period . tsat was monitored during the study period , and no patients had tsat levels increase to more than 50% . the highest ferritin level among patients who completed at least nine iv iron treatments was 1,170 ng / ml ; the mean level at the end of study period for the whole group was 379 ng / ml . five ( 20.0% ) patients received blood transfusions and were considered treatment failures ( three after week 3 , transfused at hb levels of 6.9 g / dl , 7.8 g / dl , and 5.4 g / dl ; one after week 4 , transfused at an hb level of 8.2 g / dl ; and one after week 9 , transfused at an hb level of 7.2 g / dl ) . low hb levels are associated with diminished qol and possibly decreased overall survival.2 successful treatment of anemia has undeniable benefits for patients , often yielding dramatic symptomatic improvement . although the role of esas is well - established in treating caa , big concerns were recently raised about the negative effect of esas on survival in some patients with cancer.911 concerns about the risk for thromboembolism in patients with cancer with higher hb levels who are receiving esa were also addressed in many trials.22,23 in addition , the possible immunosuppressive effects of blood product transfusions that may have relevance to neoplasia progression were addressed before.24 , 25 in our pilot study , we tested the feasibility of using iron supplementation alone to treat anemia in patients with cancer who are undergoing chemotherapy without the use of esas or blood transfusion , which could be a valid alternative , especially for patients with curable cancers . oral iron is easier to administer and relatively inexpensive , but low patient adherence , poor enteral absorption , and poor tolerance because of a wide range of troublesome gastrointestinal adverse effects limit its overall effectiveness.26 anemia of chronic disease may occur in patients with cancer and is associated with an increase in hepcidin levels , which decreases oral iron absorption and bone marrow iron use , negating any possible effect of regular doses of oral iron.15 iv iron therapy significantly improves response to epoetin alfa when compared with oral iron or no iron in anemic patients with cancer who are receiving chemotherapy.1721 oral iron supplements with esas showed no significant benefit over esas alone in treating caa.21 sodium ferric gluconate and iron sucrose appear to have more favorable safety profiles over iron dextran . a large prospective safety comparison trial failed to show serious anaphylactoid reactions,27 which is confirmed in our study , in which no patients developed reactions and no patients withdrew from the study because of adverse effects . given that the mean hb increase using esas with iv iron in one large controlled trial was 2.4 g / dl,21 the results obtained in our study are clinically significant . these findings should be further confirmed and better assessed in larger studies , in which questions such as the optimal timing of iv iron therapy with respect to chemotherapy and the optimal total dose of iv iron should be determined . the use of iv iron monotherapy was recently reviewed by a group in germany that studied the use of ferric carboxymaltose to replace esa and blood transfusions as a treatment for caa . iron - deficient patients treated with ferric carboxymaltose alone ( n = 233 ) had a median of 1.4 g / dl increase in hemoglobin levels compared with those receiving additional treatment with esas ( n = 46 ; median , 1.6 g / dl ) . our study , however , is peculiar in using iron therapy in a non - iron - deficiency state.28 iron overload after iv iron therapy , with potential concerns about the risk of developing secondary cancers and infection , might be raised . the highest serum ferritin level in the present study in patients who completed at least 9 weeks of iv iron therapy was 1,170 ng / ml . most of the literature addressing cancer and infections in iron - overloaded patients comes from patients with hemochromatosis or patients who are undergoing hemodialysis . published reviews report an increase in hepatocellular carcinoma only in patients with hemochromatosis after they develop cirrhosis.29 similarly data supporting the association between iv iron therapy and higher infection rate are weak and not well - supported.30 in fact , anemia itself is a risk factor for infections in patients receiving hemodialysis.31 a multivariate analysis of associations between iron and mortality in more than 58,000 patients receiving hemodialysis reported no increased death rate from serum ferritin levels as high as 1,200 ng / ml.30 the increasing cost of therapy in patients with cancer is of grave concern , which could be an additional benefit of iv iron over the use of esas in such patients . to further address many of the questions raised , our team is planning a bigger trial for iv iron in patients with cancer who have anemia to confirm the results discussed in this pilot trial . in addition , we will be looking into predictors of response to iv iron , such as serum hepcidin level . iv iron therapy alone is safe and may be effective in improving hb levels in patients with cancer who are undergoing active anticancer therapy . further randomized trials are needed to address many of the questions raised in our pilot study .

background : the present study was carried out to assess the effects of community nutrition intervention based on advocacy approach on malnutrition status among school - aged children in shiraz , iran.materials and methods : this case - control nutritional intervention has been done between 2008 and 2009 on 2897 primary and secondary school boys and girls ( 7 - 13 years old ) based on advocacy approach in shiraz , iran . the project provided nutritious snacks in public schools over a 2-year period along with advocacy oriented actions in order to implement and promote nutritional intervention . for evaluation of effectiveness of the intervention growth monitoring indices of pre- and post - intervention were statistically compared.results:the frequency of subjects with body mass index lower than 5% decreased significantly after intervention among girls ( p = 0.02 ) . however , there were no significant changes among boys or total population . the mean of all anthropometric indices changed significantly after intervention both among girls and boys as well as in total population . the pre- and post - test education assessment in both groups showed that the student 's average knowledge score has been significantly increased from 12.5 3.2 to 16.8 4.3 ( p < 0.0001).conclusion : this study demonstrates the potential success and scalability of school feeding programs in iran . community nutrition intervention based on the advocacy process model is effective on reducing the prevalence of underweight specifically among female school aged children . backgroundanemia in patients with cancer who are undergoing active therapy is commonly encountered and may worsen quality of life in these patients . the effect of blood transfusion is often temporary and may be associated with serious adverse events . erythropoiesis - stimulating agents are not effective in 30%50% of patients and may have a negative effect on overall survival.aimsto assess the efficacy and feasibility of intravenous iron therapy in patients with cancer who have non - iron - deficiency anemia and who are undergoing treatment with chemotherapy without the use of erythropoiesis - stimulating agents.methodsadult patients with solid cancers and non - iron - deficiency anemia were included . ferric sucrose at a dose of 200 mg was given in short intravenous infusions weekly for a total of 12 weeks . hemoglobin level was measured at baseline , every 3 weeks , and 2 weeks after the last iron infusion ( week 14 ) . adverse events related to intravenous iron were prospectively reported.resultsof 25 patients included , 19 ( 76.0% ) completed at least three iron infusions and 14 ( 56.0% ) finished the planned 12 weeks of therapy . the mean hemoglobin level of the 25 patients at baseline was 9.6 g / dl ( median , 9.9 g / dl ; range , 6.9 g / dl 10.9 g / dl ) . the mean change in hemoglobin level for the 15 patients who completed at least 9 treatments was 1.7 g / dl ( median , 1.1 g / dl ; range , 1.9 g / dl to 3.2 g / dl ) ; it reached 2.1 g / dl ( median , 1.3 g / dl ; range , 0.2 g / dl to 4.6 g / dl ; p = 0.0007 ) for the 14 patients who completed all 12 weekly treatments . five ( 20.0% ) patients were transfused and considered as treatment failures . no treatment - related adverse events were reported.conclusionintravenous iron treatment alone is safe and may reduce blood transfusion requirements and improve hemoglobin level in patients with cancer who are undergoing anticancer therapy . further randomized studies are needed to confirm these findings .

tardive dystonia ( td ) , a rarer side effect after longer exposure to antipsychotics , is characterized by local or general , sustained , involuntary contraction of a muscle or muscle group , with twisting movements , generally slow , which may affect the limbs , trunk , neck , or face . td has been shown to develop in about 3% of patients who have had long - term exposure to antipsychotics . . the low risk of td for atypical antipsychotics is thought to result from their weak affinity for dopamine receptors . compared with typical , atypical antipsychotic agents have a greater affinity for serotonin 5-ht2a than dopamine d2 receptors , with a low propensity to induce td . among this olanzapine is thought to have preferential action at mesolimbic over nigrostriatal dopaminergic pathways and is , therefore , associated with a very low incidence of extrapyramidal symptom ( eps ) . furthermore , a retrospective analysis of controlled multicentric trials suggested that olanzapine also improves preexisting symptoms of tardive movements . we report a case of 20-year - old male , belonging to lower socioeconomic class , educated up to 2 standard , presented with severe unilateral dystonic left sided neck movements [ figure 1 ] . careful history exploration revealed he was taking risperidone 2 mg irregularly for 2 months and then olanzapine 5 mg for another 4 months . picture of neck dystonia of patient at 19 years , the patient presented with occasional anger outbursts , getting provoked on small matters and beating family members , running away from home , screaming episodes occasionally , fearfulness , sleep disturbance for 2 days ; which was precipitated after fever . according to the mother , one friend might have threatened / made fun of him actually and after that patient stopped going out of house , and displayed above mentioned symptoms . this was interpreted as psychosis with persecutory ideas , and he was treated with risperidone 2 mg / day for 2 months and then with olanzapine 5 mg / day for 4 months . in last two follow - ups patient did not present himself , and mother reported unusual neck movements , which were taken as a part of his overall psychopathology and not taken seriously , slight intermittent neck movements reported were missed as part of adolescent behavior problems mimicking some hero in movies . as neck dystonia increased , the patient had a severe disability as patient had to keep his hands behind his head for the support . the movement would decrease when the patient was lying down and was absent during sleep . he even stopped taking food due to severe neck movements making chewing and swallowing difficult . his birth and early developmental milestones were normal . during 210 years of age patient was inattentive and mildly hyperactive . other siblings were educated with master 's degree , and patient was also sent to school , but due to inattention and restlessness , he did not pass 2 standard after three attempts . he left the schooling . with average executive functioning and life skills , he worked as an unskilled laborer in the neighborhood shops as a helping hand . , he was found to be getting over familiar , cheerful , moody , and short tempered . sometimes , the patient had inappropriate social judgment ; for which his friends made fun of him , and teased him . on mental status assessment , routine investigations , thyroid function tests , electroencephalogram , fundus examination , cervical x - ray , magnetic resonance imaging brain were normal . after consulting neurophysician , wilson 's disease and other secondary causes of dystonia were ruled out . the patient was treated with clonazepam 1 mg total dissolved solid ( tds ) , tetrabenazine 25 mg tds , trihexiphenidyl 2 mg bipolar disorder ( bd ) . after 2 months , there was some improvement of around 30% . baclofen 10 mg was added ; increased up to 20 mg , trihexyphenidyl reduced to 2 mg . with little improvement after 4 months of treatment for dystonia , levodopa + carbidopa ( 100 + 25 ) was added by neurophysician and increased up to tablet tds and baclofen omitted . after 12 months of treatment , patient has improved around 90% with tetrabenazine 75 mg , levodopa + carbidopa ( 100 + 25 ) - tablet bd , and clonazepam 1 mg bd . earlier case reports reported td developing with high - dose atypical antipsychotics such as olanzapine 20 mg or aripiprazole 15 mg with longer duration of exposure of around 1215 months in established psychiatric illness like schizophrenia or any other psychotic illness . eps in general and tardive dyskinesia , in particular , have been extensively studied in schizophrenia . even though a number of studies suggest that bipolar patients experience higher rates of eps ( parkinsonism , dystonia , akathisia ) and td compared to patients with a diagnosis of schizophrenia , research within the bd population has been limited . the risk is found to be 3 to 5 times higher in elderly patients compared to young patients . in addition to age , the risk is directly proportional to : female gender , daily and total dose of the antipsychotic drug , presence of mood disorder , the use of anticholinergics with neuroleptics , previous physical therapies ( electroconvulsive therapy ) , the presence of other physical illness such as diabetes or an organic disorder , younger age of exposure , and the presence of extrapyramidal symptoms early in treatment . this patient 's severe dystonic neck movements developed within short period of 6 months of exposure to atypical antipsychotics risperidone 2 mg and then olanzapine 5 mg only , which can cause minimal extrapyramidal side effects . in this case , risk factors for developing serious disabling td were neuroleptic exposure , borderline intellectual functioning , externalizing behavior , probable misdiagnosis , and overlooking early indicators of side effects . this case highlights dangers of casually prescribing low dose second generation antipsychotics in patient with hyperthymic temperament and borderline intellectual functioning with vague short lasting presenting complaints ; probably misdiagnosed as psychosis ; leading to such severe adverse effects because patients with organic brain damage are more prone to develop adverse effects like td . thus , judicious use of antipsychotics , with detailed and frequent assessments is important , and emergent stereotyped behavior or unexplained movements must be examined carefully and taken seriously . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . lepidoptera include agricultural pests that , through feeding and other activities , negatively affect stored grains , food and fiber crops [ 2 , 3 ] . since a single lepidoptera adult can produce hundreds of eggs , and their primary food source is typically plant material , they can cause significant damage to agricultural crops . although biological agents can help manage these insect pests , insecticides currently are essential for large - scale effective and economical pest control . these insecticides can also affect non - target organisms , including pollinators , and their application not only disrupts natural ecosystems but also reduces yields of crops that rely on pollination [ 5 , 6 ] . the non - target effect of some pesticides is in part due to their effects on insect immunity , which is necessary for insect survival in natural environments . for example , currently used pesticides have been shown to affect cellular [ 710 ] and humoral [ 11 , 12 ] immune responses and interfere with grooming behavior [ 13 , 14 ] . these effects on immunity are likely non - specific and negatively impact the health of both the target pest and beneficial arthropods . therefore , there is a need for novel target - specific approaches to control insect pests without affecting beneficial arthropods . although immune pathways can be generally and non - specifically inhibited by pesticides , they also are a likely source of candidate molecules that could be inhibited for target - specific insect control since multiple classes of insect immunity genes , including signaling pathways , can be under strong selection for diversification . fundamental mechanisms of innate immunity comprising cellular and humoral pathways are conserved throughout the animal kingdom and are controlled by signaling pathways activated by various stimuli [ 17 , 18 ] , including pathogen recognition by immune surveillance systems . despite this overall conservation , aspects of immune systems are subject to strong selection to evolve in response to varying pathogen exposure and to pathogen evolution of virulence determinants that modulate immunity [ 15 , 1921 ] . such co - evolutionary dynamics can promote diversification of conserved elements of immunity as well as the recruitment of novel effectors . as such , the investigation of insect immune pathways and mechanisms of pathogen modulation can yield insights into components that may be susceptible to inhibition . for example , the insect pathogen xenorhabdus nematophila suppresses cellular and humoral immunity in the lepidopteran moths manduca sexta and spodoptera exigua [ 23 , 24 ] but not in the dipteran fly drosophila melanogaster , suggesting the stage of immunity suppressed by x. nematophila may be absent from d. melanogaster , but present in lepidoptera . since dipteran flies serve as pollinators [ 26 , 27 ] , decomposers , food sources for other animals , and pest control agents , capitalizing on the possible differences between dipteran and lepidopteran immune signaling cascades will help in the identification of targets for pest - specific inhibition . with this knowledge in hand , pest management can be achieved by developing small molecule inhibitors of these targets that will suppress pest insect immunity and lead to increased susceptibility to environmental pathogens . indeed , many insecticides may contribute to insect ( target and non - target ) death by modulating aspects of immunity . the feasibility of targeted pest control via insect immune inhibition has been established for termites ; a small molecule inhibitor of an immune surveillance protein led to faster termite death upon exposure to various pathogens . much of our current knowledge of insect immune signaling pathways and receptor and effector function is based on the premiere model organism d. melanogaster , for which there are extensive genetic tools and several fully sequenced genomes . well - established lepidopteran insect models such as the silkworm bombyx mori and the tobacco hornworm m. sexta also have been widely used to study insect immunity . these organisms have been particularly useful for investigating hemolymph proteins and hemocyte function because of their relatively large larval size and hemolymph volume . many insects in the order lepidoptera are easy to rear in laboratory conditions , and new tools such as rna interference have been implemented successfully to study genetics of their immune systems [ 30 , 31 ] . also , their immune signaling pathways are gradually being revealed by genomic and transcriptomic data [ 3238 ] . based on these model insect systems a fairly detailed picture of immunity , from pathogen detection to effector function , is emerging , though many gaps remain , particularly with regard to components that are unique to different insect orders . here we review aspects of insect immunity with an emphasis on the similarities and distinctions between d. melanogaster and representative lepidoptera . in insects , the cellular immune response includes phagocytosis , nodulation and encapsulation [ 3942 ] and the humoral response involves the expression of antimicrobial peptides ( amps ) [ 43 , 44 ] as well as the pro - phenol oxidase ( propo ) proteolytic cascade that results in formation of melanized nodules and toxic reactive compounds [ 45 , 46 ] . amps are small cationic peptides that insert into and disrupt microbial membranes , thereby killing and clearing pathogens . they are synthesized by hemocytes and to a greater extent in fat body from which they are released into the insect hemolymph rapidly after microbial infection [ 43 , 47 ] . amps are also expressed in extra - embryonic tissues of eggs , which may help protect the developing embryo from infection . amps are a conserved component of immunity in plants and animals and while they have diverse structures most can be assigned to larger families such as cecropins , attacins , defensins and diptericins . their diversity and immune effector function as well as their variant representation among insects ( table 1 ) have made them a central focus in the study of invertebrate pathology [ 30 , 52 ] . in d. melanogaster amp synthesis each of these pathways is activated by detection of microbial components via different pattern recognition receptors ( prrs ) that trigger , through complex regulatory cascades , nuclear factor kappa b ( nf-b ) dependent transcription of the genes encoding amps . after amps are translated in the cytoplasm they are released into the hemolymph where their high concentrations and broad activity are thought to enhance clearance of invading microorganisms from the insect . bioinformatic and experimental data support the existence of the amp - inducing toll and imd pathways in lepidopterans , though not all components have been identified in model organisms such as m. sexta [ 32 , 35 ] . the conserved presence of amps in immunity coupled with the possibility that certain elements of their induction pathways may vary among insects enhances the probability that microbially - induced amp expression could be inhibited in a pest - specific manner . as such , for the remainder of this review we focus on pathways leading to amp gene expression . in d. melanogaster , transcription of amp - encoding genes is activated by the nf-b family transcription factors in response to infection [ 6569 ] with distinct nf-b family transcription factors responding to the toll and immune deficiency ( imd ) signal transduction pathways . in response to toll pathway activation , the nf-b inhibitor cactus is phosphorylated and degraded allowing its targets , the nf-b factors dif and dorsal , to be translocated to the nucleus . imd pathway activity culminates in the nf-b factor relish being activated by a stimulus - induced proteolytic cleavage . in the case of dif and dorsal , gram - positive bacterial and fungal infections primarily serve as the stimuli that induce degradation of cactus through the toll signaling pathway . in general , gram - negative bacterial infections of d. melanogaster stimulate the proteolytic cleavage of relish through the imd pathway . once in the nucleus , these transcription factors drive the transcription of immune effectors , including amp genes whose promoters contain nf-b binding sites [ 70 , 72 ] . overall , nf-b proteins and their dna - binding specificities are conserved among organisms , including those lepidoptera studied to date [ 73 , 74 ] . however , the nfb - binding regions for inhibitor of b ( ib ) proteins ( e.g. , cactus ) are not conserved , suggesting diversification and co - adaptation between ib and nfb pairs . also , recent work indicates that nf-b nuclear co - regulators may contribute to species - specific regulation of amp gene expression . therefore , modulation of inhibitors and nuclear - co - regulators of nf-b - dependent transcription may be one avenue by which target - specific immune suppression could be achieved . in d. melanogaster , nf-b - dependent amp induction through the toll and imd pathways is activated by detection of microbial components via different pattern recognition receptors ( prrs ) . prrs are soluble or membrane - bound proteins that bind to specific microbe associated molecular patterns ( mamps ) such as lipopolysaccharide ( lps ) , lipoteichoic acid ( lta ) , peptidoglycan ( pgn ) or -1,3-glucan that are released from or are found on the cell surfaces of bacteria or fungi . upon interaction with mamps , prrs can directly agglutinate pathogens or trigger proteolytic signaling cascades and cytokine release , which in turn lead to the activation of downstream cellular and humoral pathways , including pro - po activation and amp gene expression [ 16 , 65 , 76 ] . pgn recognition proteins ( pgrps ) and -1,3-glucanase - related proteins ( grps ) were discovered in the lepidopteran silkworm ( b. mori ) by assaying for plasma components that activate the propo cascade . pgrps were subsequently shown to be conserved across mammals and insects , and in d. melanogaster their role in the induction of amp gene expression through toll and imd pathways in response to pgn has been well documented [ 7983 ] . similarly , grps have been shown to induce amp expression through toll pathway in response to fungal infections [ 79 , 84 ] . in contrast , there is a dearth of literature linking specific pgrps or grps to amp induction in lepidoptera . such a link is possible , since pgn and -1,3-glucan can activate amp gene expression in m. sexta and b. mori [ 8590 ] and multiple infection - induced pgrp- and grp - encoding genes have been identified in lepidoptera [ 32 , 38 , 54 , 55 , 9194 ] . however , there are numerous hints that lepidoptera and diptera may have evolved divergent mechanisms of linking pathogen detection to conserved toll and imd signal transduction cascades . first , a genome comparison between b. mori and d. melanogaster failed to identify 1:1 pgrp orthologs . similarly , b. mori gram - negative binding protein ( gnbp ) and m. sexta microbe binding protein ( mbp ) , members of the -1,3-glucanase - related protein superfamily [ 76 , 95 ] , appear to be distantly related to d. melanogaster gnbps , suggesting divergence of this group of proteins . m. sexta mbp expression is strongly up - regulated in fat body after immune challenge and shows specific binding to lta , lps , dap - pgn . also , in contrast to the situation in d. melanogaster , highly purified lps and lta are inducers of amp gene expression in lepidoptera , though not as potently as crude lps ( with contaminating pgn ) or purified pgn [ 85 , 90 , 96 , 97 ] . this raises the possibility that different mamps or combinations of mamps are most efficacious in eliciting amp gene expression in lepidoptera relative to diptera . also , since purified lps can elicit amp expression in lepidoptera but not d. melanogaster , lepidoptera have either a distinct repertoire of prrs responsible for lps - dependent triggering of imd or toll pathways , or an as - yet undiscovered pathway that links lps to amp induction . testing these ideas awaits the identification of the suite of prrs and signal transduction pathways responsible for transducing lps , lta , pgn , or combinatorial microbial signals to amp gene expression . one class of lepidopteran prr that may mediate infection - dependent induction of amps is the c - type lectins ( ctls ) , ca - dependent , secreted proteins that have carbohydrate - binding capabilities . similar to some ctls of d. melanogaster , several ctls of m. sexta and b. mori [ 54 , 99 ] are reported to mediate induction of cellular responses and the propo cascade . although the nomenclature quickly becomes confusing , ctls include lipopolysaccharide - binding protein ( lbp ) . m. sexta iml-1 binds to gram - positive and gram - negative bacteria as well as yeast , iml-2 shows specific binding to lps , iml-3 and iml-4 show specific binding to lps and lta , and iml-3 can also bind laminarin , a -1,3-glucan [ 103 , 104 ] . diversity in ctl carbohydrate - binding specificities may result in lineage - specific pathogen recognition - signal transduction connections . of particular relevance to the theme of this review are prrs present in lepidoptera but not diptera ( table 1 ) . in general , both orders of insects encode grps and pgrps . however , specific representatives of each class are restricted to lepidoptera ( table 1 ) . for example , the lepidopteran grp-2 , which binds fungal cell wall -1,3 glucans and lta , is absent from diptera . such derived grp and pgrps may contribute to lepidopteran - specific transduction of signals to downstream pathways . other lepidoptera - specific prrs are hemolin and hemolymph proteinase-14 precursor ( prohp14 ) ( table 1 ) . like iml c - type lectins , hemolin is an lps- and lta - binding prr with roles in mediating cellular responses and as an opsonin to enhance phagocytosis . hp14 has been shown to detect and bind a broad range of mamps , and may coordinate with grp1 or grp2 to activate propo [ 60 , 106 ] . the potential role of the prrs discussed above in mediating the expression of amp genes remains to be determined , and further study of the lepidoptera - specific immune surveillance proteins and divergent activities of conserved prrs likely will yield novel avenues for pest - control . d. melanogaster has both mamp - dependent and mamp - independent routes to activate the toll pathway . in mamp - dependent toll induction , bacterial lys - pgn ( typical of gram - positive bacteria ) is detected by pgrp - sa or pgrp - sd ( in the presence of gnbp-1 ) , while yeast or fungal -1,3-glucan is detected by gnbp-3 [ 85 , 108 , 109 ] ( figure 1 ) . mamp - independent stimuli are virulence determinants , such as proteases and chitinases , secreted by microbes and dubbed mamps and mamp - independent stimuli each trigger a distinct proteolytic cascade that both culminate in cleavage of the cytokine sptzle by the serine protease sptzle processing enzyme ( spe ) . interaction of active sptzle c - terminal domain ( c-106 ) with the surface - localized toll receptor triggers an intracellular signal transduction terminating in induced expression of amps and cellular responses . some of the basic components of the toll pathway are present in lepidoptera ( figure 2 ) . m. sexta hemocytes express an infection - induced toll - like receptor and the genome of b. mori encodes 14 genes predicted to encode toll - like receptors as well as homologs of each of the intracellular components of toll - dependent signaling [ 85 , 114 , 115 ] . both m. sexta and b. mori encode homologs of the d. melanogaster toll - activating cytokine sptzle ( figure 2 ) . also , for both m. sexta and b. mori there is experimental evidence linking the toll pathway with amp induction [ 116118 ] . in m. sexta , toll pathway results in expression of several antimicrobial peptides , including attacin-1 , cecropin-6 , moricin and lysozyme . in addition , the transcript level of hemolin , a pattern recognition protein exclusive to lepidopterans ( table 1 ) , is induced by injection of activated sptzle - c108 into larvae . despite the conservation of certain aspects of the toll pathway , the extracellular cascades that lead to sptzle activation may have diverged between d. melanogaster and the two lepidoptera ( figure 1 ) . for example , in contrast to what is known in d. melanogaster , the m. sexta toll pathway is activated by gram - negative - associated mamps [ 115 , 118 ] . also , the genome of b. mori lacks 1:1 orthologs of grass , spirit and persephone , the d. melanogaster serine proteases responsible for mamp / prr - dependent and mamp - independent cleavage of spe ( figure 1 ) . progress has been made in identifying a m. sexta proteolytic cascade that results in processing pro - sptzle into its active c - terminal domain ( c-108 ) . the direct cleavage is mediated by hemolymph proteinase ( hp ) 8 [ 11 , 13 ] , a homolog of d. melanogaster spe , in turn , hp8 is processed into its active form by hp6 . hp6 is most similar to d. melanogaster persephone protease , which activates spe in response to mamp - independent stimuli [ 110 , 113 ] . this hemolymph proteinase is activated in response to gram - positive or gram - negative bacteria and in response to -1,3-glucan . however , the prrs and proteolytic cascade that transduce mamp signals to amp induction are unknown ( figure 1 ) . the findings reviewed above demonstrate that while the overall architecture of the toll pathway is conserved among insects , the specific identities of proteolytic cascade members are distinct and many gaps remain in our understanding of toll activation in lepidoptera . filling these gaps should reveal potential lineage - specific molecules that can serve as targets to hinder the activation of the toll pathway in agricultural pests . in d. melanogaster the imd pathway also contributes to amp gene induction and is triggered by direct interaction of dap - pgn , a mamp typical of gram - negative bacteria , with the transmembrane receptor pgrp - lc [ 82 , 83 , 125 , 126 ] . for example , pgrp - le can act as an intracellular receptor for monomeric pgn and its truncated form can enhance pgrp - lc - mediated recognition . dap - pgn / pgrp - lc interaction activates intracellular imd , which then recruits fas - associated death domain ( fadd ) and death - related ced-3/nedd2-like protein ( dredd ) to form a complex [ 129 , 130 ] . current evidence supports the idea that dredd , a caspase - like molecule , cleaves the nf-b transcription factor relish . imd also appears to activate a phosphorelay : the transforming growth factor- ( tgf)-activated kinase 1 ( tak1 ) phosphorylates the ikb kinase ( ikk ) relish cleavage into its activated amino - terminal transcriptional regulator domain allows its translocation into the nucleus , where it activates amp gene expression . the translocation of relish into the nucleus is regulated by two recently discovered components of this pathway : inhibitor of apoptosis 2 ( iap2 ) and transforming growth factor - activated kinase 1 ( tak1)-binding protein 2 ( tab2 ) [ 132 , 133 ] . both , iap2 and tab2 act upstream of relish and downstream of imd , while iap2 functions downstream of tak1 [ 132 , 134 ] . of particular importance to amp gene expression is iap2 , the knockdown of which hampers the sustained expression of amp genes . while iap2 and tab2 are necessary for imd signal transduction , the gene product of pirk , a recently characterized gene , interacts directly with imd and pgrp - lc . pirk overexpression analyses revealed that it acts as a negative regulator by reducing the expression of the amp genes attacin b , cecropin b , and diptericin b , which are all under the control of the imd pathway . most of the information available about the imd pathway in lepidoptera comes from bioinformatics ; orthologs of all intracellular components of the imd pathway have been found in b. mori [ 54 , 137 ] and m. sexta . however , few experiments have been done to characterize the molecular mechanisms leading to activation of the imd pathway in these insects . in m. sexta several genes of the imd pathway , including those encoding imd , fadd , tak1 , dredd and relish are up regulated in fat body of immune challenged 5th instar larvae and in the midgut of b. mori during the wandering stage . genes encoding lysozyme , moricin and defensin amps also were up regulated in the midgut of b. mori in the wandering stage , consistent with the possibility that amp induction is imd - mediated . finally , in the lepidopteran beet armyworm spodoptera exigua , rnai - mediated knockdown of relish expression resulted in loss of cecropin induction upon fungal infection , further strengthening the idea that the imd pathway may contribute to amp gene expression in lepidoptera , though perhaps it is triggered by distinct signals . further study is needed to elucidate imd - mediated amp induction in lepidoptera and to reveal any differences there are in this pathway between diptera and lepidoptera . in d. melanogaster , transcription of amp - encoding genes is activated by the nf-b family transcription factors in response to infection [ 6569 ] with distinct nf-b family transcription factors responding to the toll and immune deficiency ( imd ) signal transduction pathways . in response to toll pathway activation , the nf-b inhibitor cactus is phosphorylated and degraded allowing its targets , the nf-b factors dif and dorsal , to be translocated to the nucleus . imd pathway activity culminates in the nf-b factor relish being activated by a stimulus - induced proteolytic cleavage . in the case of dif and dorsal , gram - positive bacterial and fungal infections primarily serve as the stimuli that induce degradation of cactus through the toll signaling pathway . in general , gram - negative bacterial infections of d. melanogaster stimulate the proteolytic cleavage of relish through the imd pathway . once in the nucleus , these transcription factors drive the transcription of immune effectors , including amp genes whose promoters contain nf-b binding sites [ 70 , 72 ] . overall , nf-b proteins and their dna - binding specificities are conserved among organisms , including those lepidoptera studied to date [ 73 , 74 ] . however , the nfb - binding regions for inhibitor of b ( ib ) proteins ( e.g. , cactus ) are not conserved , suggesting diversification and co - adaptation between ib and nfb pairs . also , recent work indicates that nf-b nuclear co - regulators may contribute to species - specific regulation of amp gene expression . therefore , modulation of inhibitors and nuclear - co - regulators of nf-b - dependent transcription may be one avenue by which target - specific immune suppression could be achieved . in d. melanogaster , nf-b - dependent amp induction through the toll and imd pathways is activated by detection of microbial components via different pattern recognition receptors ( prrs ) . prrs are soluble or membrane - bound proteins that bind to specific microbe associated molecular patterns ( mamps ) such as lipopolysaccharide ( lps ) , lipoteichoic acid ( lta ) , peptidoglycan ( pgn ) or -1,3-glucan that are released from or are found on the cell surfaces of bacteria or fungi . upon interaction with mamps , prrs can directly agglutinate pathogens or trigger proteolytic signaling cascades and cytokine release , which in turn lead to the activation of downstream cellular and humoral pathways , including pro - po activation and amp gene expression [ 16 , 65 , 76 ] . pgn recognition proteins ( pgrps ) and -1,3-glucanase - related proteins ( grps ) were discovered in the lepidopteran silkworm ( b. mori ) by assaying for plasma components that activate the propo cascade . pgrps were subsequently shown to be conserved across mammals and insects , and in d. melanogaster their role in the induction of amp gene expression through toll and imd pathways in response to pgn has been well documented [ 7983 ] . similarly , grps have been shown to induce amp expression through toll pathway in response to fungal infections [ 79 , 84 ] . in contrast , there is a dearth of literature linking specific pgrps or grps to amp induction in lepidoptera . such a link is possible , since pgn and -1,3-glucan can activate amp gene expression in m. sexta and b. mori [ 8590 ] and multiple infection - induced pgrp- and grp - encoding genes have been identified in lepidoptera [ 32 , 38 , 54 , 55 , 9194 ] . however , there are numerous hints that lepidoptera and diptera may have evolved divergent mechanisms of linking pathogen detection to conserved toll and imd signal transduction cascades . first , a genome comparison between b. mori and d. melanogaster failed to identify 1:1 pgrp orthologs . similarly , b. mori gram - negative binding protein ( gnbp ) and m. sexta microbe binding protein ( mbp ) , members of the -1,3-glucanase - related protein superfamily [ 76 , 95 ] , appear to be distantly related to d. melanogaster gnbps , suggesting divergence of this group of proteins . m. sexta mbp expression is strongly up - regulated in fat body after immune challenge and shows specific binding to lta , lps , dap - pgn . also , in contrast to the situation in d. melanogaster , highly purified lps and lta are inducers of amp gene expression in lepidoptera , though not as potently as crude lps ( with contaminating pgn ) or purified pgn [ 85 , 90 , 96 , 97 ] . this raises the possibility that different mamps or combinations of mamps are most efficacious in eliciting amp gene expression in lepidoptera relative to diptera . also , since purified lps can elicit amp expression in lepidoptera but not d. melanogaster , lepidoptera have either a distinct repertoire of prrs responsible for lps - dependent triggering of imd or toll pathways , or an as - yet undiscovered pathway that links lps to amp induction . testing these ideas awaits the identification of the suite of prrs and signal transduction pathways responsible for transducing lps , lta , pgn , or combinatorial microbial signals to amp gene expression . one class of lepidopteran prr that may mediate infection - dependent induction of amps is the c - type lectins ( ctls ) , ca - dependent , secreted proteins that have carbohydrate - binding capabilities . similar to some ctls of d. melanogaster , several ctls of m. sexta and b. mori [ 54 , 99 ] are reported to mediate induction of cellular responses and the propo cascade . although the nomenclature quickly becomes confusing , ctls include lipopolysaccharide - binding protein ( lbp ) . m. sexta iml-1 binds to gram - positive and gram - negative bacteria as well as yeast , iml-2 shows specific binding to lps , iml-3 and iml-4 show specific binding to lps and lta , and iml-3 can also bind laminarin , a -1,3-glucan [ 103 , 104 ] . diversity in ctl carbohydrate - binding specificities may result in lineage - specific pathogen recognition - signal transduction connections . of particular relevance to the theme of this review are prrs present in lepidoptera but not diptera ( table 1 ) . in general , both orders of insects encode grps and pgrps . however , specific representatives of each class are restricted to lepidoptera ( table 1 ) . for example , the lepidopteran grp-2 , which binds fungal cell wall -1,3 glucans and lta , is absent from diptera . such derived grp and pgrps may contribute to lepidopteran - specific transduction of signals to downstream pathways . other lepidoptera - specific prrs are hemolin and hemolymph proteinase-14 precursor ( prohp14 ) ( table 1 ) . like iml c - type lectins , hemolin is an lps- and lta - binding prr with roles in mediating cellular responses and as an opsonin to enhance phagocytosis . hp14 has been shown to detect and bind a broad range of mamps , and may coordinate with grp1 or grp2 to activate propo [ 60 , 106 ] . the potential role of the prrs discussed above in mediating the expression of amp genes remains to be determined , and further study of the lepidoptera - specific immune surveillance proteins and divergent activities of conserved prrs likely will yield novel avenues for pest - control . d. melanogaster has both mamp - dependent and mamp - independent routes to activate the toll pathway . in mamp - dependent toll induction , bacterial lys - pgn ( typical of gram - positive bacteria ) is detected by pgrp - sa or pgrp - sd ( in the presence of gnbp-1 ) , while yeast or fungal -1,3-glucan is detected by gnbp-3 [ 85 , 108 , 109 ] ( figure 1 ) . mamp - independent stimuli are virulence determinants , such as proteases and chitinases , secreted by microbes and dubbed mamps and mamp - independent stimuli each trigger a distinct proteolytic cascade that both culminate in cleavage of the cytokine sptzle by the serine protease sptzle processing enzyme ( spe ) . interaction of active sptzle c - terminal domain ( c-106 ) with the surface - localized toll receptor triggers an intracellular signal transduction terminating in induced expression of amps and cellular responses . some of the basic components of the toll pathway are present in lepidoptera ( figure 2 ) . m. sexta hemocytes express an infection - induced toll - like receptor and the genome of b. mori encodes 14 genes predicted to encode toll - like receptors as well as homologs of each of the intracellular components of toll - dependent signaling [ 85 , 114 , 115 ] . both m. sexta and b. mori encode homologs of the d. melanogaster toll - activating cytokine sptzle ( figure 2 ) . also , for both m. sexta and b. mori there is experimental evidence linking the toll pathway with amp induction [ 116118 ] . in m. sexta , toll pathway results in expression of several antimicrobial peptides , including attacin-1 , cecropin-6 , moricin and lysozyme . in addition , the transcript level of hemolin , a pattern recognition protein exclusive to lepidopterans ( table 1 ) , is induced by injection of activated sptzle - c108 into larvae . despite the conservation of certain aspects of the toll pathway , the extracellular cascades that lead to sptzle activation may have diverged between d. melanogaster and the two lepidoptera ( figure 1 ) . for example , in contrast to what is known in d. melanogaster , the m. sexta toll pathway is activated by gram - negative - associated mamps [ 115 , 118 ] . also , the genome of b. mori lacks 1:1 orthologs of grass , spirit and persephone , the d. melanogaster serine proteases responsible for mamp / prr - dependent and mamp - independent cleavage of spe ( figure 1 ) . progress has been made in identifying a m. sexta proteolytic cascade that results in processing pro - sptzle into its active c - terminal domain ( c-108 ) . the direct cleavage is mediated by hemolymph proteinase ( hp ) 8 [ 11 , 13 ] , a homolog of d. melanogaster spe , in turn , hp8 is processed into its active form by hp6 . hp6 is most similar to d. melanogaster persephone protease , which activates spe in response to mamp - independent stimuli [ 110 , 113 ] . this hemolymph proteinase is activated in response to gram - positive or gram - negative bacteria and in response to -1,3-glucan . however , the prrs and proteolytic cascade that transduce mamp signals to amp induction are unknown ( figure 1 ) . the findings reviewed above demonstrate that while the overall architecture of the toll pathway is conserved among insects , the specific identities of proteolytic cascade members are distinct and many gaps remain in our understanding of toll activation in lepidoptera . filling these gaps should reveal potential lineage - specific molecules that can serve as targets to hinder the activation of the toll pathway in agricultural pests . in d. melanogaster the imd pathway also contributes to amp gene induction and is triggered by direct interaction of dap - pgn , a mamp typical of gram - negative bacteria , with the transmembrane receptor pgrp - lc [ 82 , 83 , 125 , 126 ] . for example , pgrp - le can act as an intracellular receptor for monomeric pgn and its truncated form can enhance pgrp - lc - mediated recognition . dap - pgn / pgrp - lc interaction activates intracellular imd , which then recruits fas - associated death domain ( fadd ) and death - related ced-3/nedd2-like protein ( dredd ) to form a complex [ 129 , 130 ] . current evidence supports the idea that dredd , a caspase - like molecule , cleaves the nf-b transcription factor relish . imd also appears to activate a phosphorelay : the transforming growth factor- ( tgf)-activated kinase 1 ( tak1 ) phosphorylates the ikb kinase ( ikk ) relish cleavage into its activated amino - terminal transcriptional regulator domain allows its translocation into the nucleus , where it activates amp gene expression . the translocation of relish into the nucleus is regulated by two recently discovered components of this pathway : inhibitor of apoptosis 2 ( iap2 ) and transforming growth factor - activated kinase 1 ( tak1)-binding protein 2 ( tab2 ) [ 132 , 133 ] . both , iap2 and tab2 act upstream of relish and downstream of imd , while iap2 functions downstream of tak1 [ 132 , 134 ] . of particular importance to amp gene expression is iap2 , the knockdown of which hampers the sustained expression of amp genes . while iap2 and tab2 are necessary for imd signal transduction , the gene product of pirk , a recently characterized gene , interacts directly with imd and pgrp - lc . pirk overexpression analyses revealed that it acts as a negative regulator by reducing the expression of the amp genes attacin b , cecropin b , and diptericin b , which are all under the control of the imd pathway . most of the information available about the imd pathway in lepidoptera comes from bioinformatics ; orthologs of all intracellular components of the imd pathway have been found in b. mori [ 54 , 137 ] and m. sexta . however , few experiments have been done to characterize the molecular mechanisms leading to activation of the imd pathway in these insects . in m. sexta several genes of the imd pathway , including those encoding imd , fadd , tak1 , dredd and relish are up regulated in fat body of immune challenged 5th instar larvae and in the midgut of b. mori during the wandering stage . genes encoding lysozyme , moricin and defensin amps also were up regulated in the midgut of b. mori in the wandering stage , consistent with the possibility that amp induction is imd - mediated . finally , in the lepidopteran beet armyworm spodoptera exigua , rnai - mediated knockdown of relish expression resulted in loss of cecropin induction upon fungal infection , further strengthening the idea that the imd pathway may contribute to amp gene expression in lepidoptera , though perhaps it is triggered by distinct signals . further study is needed to elucidate imd - mediated amp induction in lepidoptera and to reveal any differences there are in this pathway between diptera and lepidoptera . insecticides are necessary to guarantee effective insect pest management in agricultural settings . however , the cost and off - target effects of these insecticides directly and indirectly increase economic burden ; the latter by affecting beneficial arthropods such as pollinators . the study of insect immunity can provide tools for the development of target - specific cost - effective approaches to control agricultural pests . directed suppression of pest immune defenses is predicted to render them susceptible to environmental and applied biocontrol pathogens , as recently demonstrated in termites by bulmer and colleagues . the studies summarized above suggest that many aspects of insect immunity , including recognition factors and serine proteases , have diverged between d. melanogaster and lepidoptera . continued comparative immunity of a broad array of species from diptera , lepidoptera , and other insect orders will reveal possible candidate immunity factors for target - specific approaches that will enable the effective control of insect pests . however , before such approaches can be realized , the details of lepidopteran immune signaling pathways must be elucidated . the relatively large sizes of last instar larvae of many lepidopteran species will facilitate biochemical approaches to such studies , while the establishment of immune - inducible lepidopteran cell lines such as the uga - cie1 cell line can enable the characterization of molecular mechanisms leading to imd pathway activation and its contribution to amp gene expression . finally , ongoing investigations into the immune - modulatory mechanisms of entomopathogens will help identify key steps in immunity that are susceptible to manipulation , contributing to the development of natural , cost - effective , non - toxic alternatives to chemical insecticides currently used for pest management .

tardive dystonia ( td ) is a serious side effect of antipsychotic medications , more with typical antipsychotics , that is potentially irreversible in affected patients . studies show that newer atypical antipsychotics have a lower risk of td . as a result , many clinicians may have developed a false sense of security when prescribing these medications . we report a case of 20-year - old male with hyperthymic temperament and borderline intellectual functioning , who developed severe td after low dose short duration exposure to atypical antipsychotic risperidone and then olanzapine . the goal of this paper is to alert the reader to be judicious and cautious before using casual low dose second generation antipsychotics in patient with no core psychotic features , hyperthymic temperament , or borderline intellectual functioning suggestive of organic brain damage , who are more prone to develop adverse effects such as td and monitor the onset of td in patients taking atypical antipsychotics . many lepidopteran insects are agricultural pests that affect stored grains , food and fiber crops . these insects have negative ecological and economic impacts since they lower crop yield , and pesticides are expensive and can have off - target effects on beneficial arthropods . a better understanding of lepidopteran immunity will aid in identifying new targets for the development of specific insect pest management compounds . a fundamental aspect of immunity , and therefore a logical target for control , is the induction of antimicrobial peptide ( amp ) expression . these peptides insert into and disrupt microbial membranes , thereby promoting pathogen clearance and insect survival . pathways leading to amp expression have been extensively studied in the dipteran drosophila melanogaster . however , diptera are an important group of pollinators and pest management strategies that target their immune systems is not recommended . recent advances have facilitated investigation of lepidopteran immunity , revealing both conserved and derived characteristics . although the general pathways leading to amp expression are conserved , specific components of these pathways , such as recognition proteins have diverged . in this review we highlight how such comparative immunology could aid in developing pest management strategies that are specific to agricultural insect pests .

syncope is caused by transient diffuse cerebral hypoperfusion and is characterized by transient loss of consciousness with a rapid onset followed by spontaneous and complete recovery . clinical features of syncope may include myoclonic jerks which are often multifocal and asynchronous , convulsions , and urinary incontinence , making it difficult to differentiate from epileptic seizure by clinical features alone . significant fluctuations in cerebral perfusion pressure are prevented by autoregulation of cerebral circulation , but there may be conditions where such mechanism may not compensate adequately . cough syncope , a rare form of syncope , may be a result of transient failure of the cerebral autoregulatory mechanism to cope with sudden decrease in cerebral blood flow . we present an unusual case of recurrent cough syncope , which was initially diagnosed and treated as seizures , in the context of a left - sided glomus jugulare tumor , a benign paraganglioma . a 43-year - old right - handed woman with history of glomus jugulare tumor in the left jugular fossa with intracranial extension into the posterior cranial fossa was transferred from another hospital for recurrent seizure - like spells . she had a 90% surgical resection of the tumor done in 2011 followed by radiation therapy in september 2012 . her episodes occurred multiple times a day ( 7 per day on average ) during wakeful state . they were triggered by coughing ( usually a bout of cough ) and were characterized by staring and unresponsiveness as well as stiffening of the body with mild shaking of both upper extremities . she was diagnosed with epileptic seizures but continued to have episodes during treatment with the antiepileptic drugs ( aeds ) phenytoin , levetiracetam , and lamotrigine . escalation of aed therapy made her increasingly drowsy , and she was on all three aforementioned aeds at the time of presentation . her physical examination was remarkable for excessive drowsiness , mild dysarthria , right sixth cranial nerve palsy , mild hypertonia with hyperreflexia in the lower extremities ( left more than right ) , and bilateral ( left more than right ) ankle clonus . she had a lumbar puncture done at the outside hospital , and the opening cerebrospinal fluid ( csf ) pressure was reported to be 25 cm . blood work was also unremarkable except for mild anemia ( hemoglobin : 9.4 g / dl ) , mild hyponatremia ( 132 meq / l ) , and mild hypokalemia ( 3.1 meq / l ) . antiepileptic drug levels were within therapeutic range ( free phenytoin : 1.3 g / ml , levetiracetam : 5.9 g / ml , and lamotrigine : 2.3 g / ml ) . all started with a bout of cough when the patient was lying in bed ( in supine or in lateral position ) which was followed by brief ( less than a minute ) distal upper extremity tremor and subtle proximal upper extremity myoclonic jerks and prolonged unresponsiveness for up to 10 min . all of these episodes were associated with hypotension ( 7278/3147 mm of hg as revealed by continuous arterial pressure monitoring ) and bradycardia ( 5459 bpm ) . the eeg during the spells was characterized by generalized synchronous and asynchronous high amplitude 1- to 2-hz delta activity which progressed to generalized attenuation and then transitioned to generalized delta activity again with recovery ( fig . 1 ) . a head ct showed recurrence of the glomus jugulare tumor and communicating hydrocephalus . an external ventricular drain ( evd ) after placement of the evd , her drowsiness gradually started to improve , and episodes decreased in frequency to one per day . 3 showed an enhancing t2 hyperintense left skull base mass in the region of the left jugular foramen with extension into the posterior cranial fossa and below the base of the skull . brain imaging showed evidence of hydrocephalus that had increased compared with her previous brain imaging done 2 months back . her mental status continued to improve , and she had only one mild episode triggered by cough during the next two days before her discharge . repeat surgical resection of the tumor was recommended by the otolaryngology team , which the patient declined . based on the clinical features and eeg findings , the episodes observed in our patient are most consistent with cough syncope . the mechanism underlying cough syncope is not definitively established , but it is postulated that coughing increases intrathoracic and intraabdominal pressures leading to a transient increase in icp . increased icp , in turn , causes a decrease in cerebral perfusion pressure which , if it drops below a critical level , may result in global cerebral hypoperfusion leading to syncope . transient cerebral circulatory arrest has been demonstrated by transcranial doppler measurements during cough syncope . our patient also had a drop in blood pressure and heart rate but probably not sufficient to cause syncope by itself . cough syncope has been associated with posterior fossa mass lesions or tonsillar herniation and with hydrocephalus . it may be speculated that bouts of cough caused transient herniation of cerebellar tonsils obstructing csf flow that further contributed to the increase in icp during coughing . decrease in frequency of events following placement of evd to relieve icp lends support to this notion . paragangliomas are rare tumors of extraadrenal chromaffin cell origin that most commonly occur in the head and neck region . catecholamine - hypersecreting paraganglionomas are uncommon in the head and neck region , and most patients ( 95% ) with hypersecreting paraganglionomas have hypertension . hypotension accompanying syncope observed in our patient was not orthostasis - related ( the patient was always supine during spells ) and was most likely related to cough . identified a subset of patients with cough syncope who lacked a blood pressure overshoot ( expected response ) after the relief of straining during valsalva maneuver . the authors postulate that cough syncope in these patients might be the result of delayed recovery from hypotension that follows a paroxysm of cough , and this was likely contributing to global cerebral hypoperfusion in our patient . this case highlights the fact that cough syncope , a rare form of syncope , may be associated with intracranial mass lesions that indirectly exaggerate the increase in icp in response to cough . glomus caroticum tumor presenting as recurrent unexplained syncope and posterior fossa meningioma presenting as recurrent cough syncope have been described . recurrent cough syncope should trigger search for factors , including brain tumors , with the potential to cause transient elevation in icp . this case also illustrates an important role for ceeg monitoring with video in distinguishing syncope from seizures in cough syncope cases . world - wide , infertility affects 1015% of couples who are trying to conceive , and about 15% of these cases are caused by male factors , which affect 1 out of 20 men in the general population . most cases of male infertility are idiopathic , apart from several etiologies , such as obstruction of deferent duct , varicocele , sexual dysfunction , and cryptorchidism . although assisted reproductive technology ( art ) has helped many sterile couples to conceive , non - obstructive azoospermia ( noa ) , which accounts for a considerable proportion of male infertility , has a dramatically lower rate of sperm retrieval and clinical pregnancy . the etiological mechanism of noa is unknown , but factors such as oxidative stress were considered to have effects on spermatogenesis , and some antioxidants have been effective in protecting spermatogenesis . therefore , it is helpful to explore the underlying pathogenesis of noa in these patients . micrornas are a class of small rnas that do not code amino acid sequences , but they play fundamental roles in regulating gene expression after transcription . lian et al . found 154 down - regulated mirnas and 19 up - regulated mirnas in testes of noa patients compared to fertile males , by using microarray technologies . furthermore , some of the mirnas have been shown to affect the proliferation , apoptosis , and dna damage in germ cells [ 911 ] . mir-210 is one of the 19 up - regulated mirnas in testes of noa patients , located within the genomic loci of transcript ak123483 . it can be induced by hypoxia , and plays an essential role in cell adaptation to hypoxia . mir-210 also affects regulation of diverse physiological processes , such as angiogenesis , cell survival , proliferation , cell cycle arrest , protein modification , and dna damage repair . although mir-210 has been shown to be involved in regulation of physiological processes in various diseases and to be an up - regulated mirna in testes of noa patients , it remains unknown how mir-210 affects spermatogenesis . hence , the aim of this study was to investigate the underlying mechanisms by which mir-210 is involved in the pathogenesis of spermatogenesis . we enrolled 25 patients ( aged 1841 years ) with azoospermia ( proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university ) . pathological examinations were performed on each testicular specimen . combined with clinical features , 4 patients were diagnosed as having sertoli - cell - only syndrome ( scos ) , 7 patients were diagnosed as having maturation arrest ( ma ) , 8 patients were diagnosed as having hypospermatogenesis , and the other 6 patients were diagnosed as having obstructive azoospermia ( oa ) . all patients provided informed consent before their participation in this study . our local medical ethics committee approved this study before it began . to examine the location of insulin - like growth factor ii ( igf2 ) in human testicular tissues , we performed immunohistochemistry staining to detect the igf2 expression . tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax . after the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al . , sections were incubated with igf2 antibody ( abcam ) overnight at 4c and biotinylated secondary antibody ( abcam ) for 2 h at room temperature . to detect expression of mir-210 , rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . . briefly , rna extraction was performed following a standard trizol protocol , real - time pcr was carried out with the abi step one system ( applied biosystems ) , and the sybr premix ex taq ii kit ( takara bio , inc . ) was used . primers for q - rt pcr were as follows : forward primer : 5-caataactgtgcgtgtgacagc-3 reverse primer : 5-tatggttttgacgactgtgtgat-3 forward primer : 5-cagcacatatactaaaattggaacg-3 reverse primer : 5-acgaatttgcgtgtcatcc-3 western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells . anti - igf2 ( abcam ) was used for western blot analysis , and we used -actin as a loading control to detect expression of igf2 . we supplemented the medium with 10% fetal bovine serum ( life technology inc . ) , 1% antibiotics ( 100 units / ml penicillin , and 100 ug / ml streptomycin , life technology inc . ) . cells were incubated at 37c in a humidified incubator with 5% co2 . to transfect oligonucleotides and plasmids into nt-2 cells , lipofectamine rnaimax ( invitrogen ) and fugene hd ( roche ) all processes were performed in accordance with the protocols supplied by manufacturers . in this study , all the experiments were performed independently at least 3 times . we enrolled 25 patients ( aged 1841 years ) with azoospermia ( proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university ) . pathological examinations were performed on each testicular specimen . combined with clinical features , 4 patients were diagnosed as having sertoli - cell - only syndrome ( scos ) , 7 patients were diagnosed as having maturation arrest ( ma ) , 8 patients were diagnosed as having hypospermatogenesis , and the other 6 patients were diagnosed as having obstructive azoospermia ( oa ) . all patients provided informed consent before their participation in this study . to examine the location of insulin - like growth factor ii ( igf2 ) in human testicular tissues , we performed immunohistochemistry staining to detect the igf2 expression . tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax . after the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al . , sections were incubated with igf2 antibody ( abcam ) overnight at 4c and biotinylated secondary antibody ( abcam ) for 2 h at room temperature . rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . . briefly , rna extraction was performed following a standard trizol protocol , real - time pcr was carried out with the abi step one system ( applied biosystems ) , and the sybr premix ex taq ii kit ( takara bio , inc . ) was used . primers for q - rt pcr were as follows : forward primer : 5-caataactgtgcgtgtgacagc-3 reverse primer : 5-tatggttttgacgactgtgtgat-3 forward primer : 5-cagcacatatactaaaattggaacg-3 reverse primer : 5-acgaatttgcgtgtcatcc-3 western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells . anti - igf2 ( abcam ) was used for western blot analysis , and we used -actin as a loading control to detect expression of igf2 . we supplemented the medium with 10% fetal bovine serum ( life technology inc . ) , 1% antibiotics ( 100 units / ml penicillin , and 100 ug / ml streptomycin , life technology inc . ) . cells were incubated at 37c in a humidified incubator with 5% co2 . to transfect oligonucleotides and plasmids into nt-2 cells , lipofectamine rnaimax ( invitrogen ) and fugene hd ( roche ) the igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2 , which is only produced from the paternal allele . the maternal allele is transcriptionally silent . to clarify the location of igf2 in human testicular tissues , we found igf2 located in spermatocytes in the testes of patients with oa ( figure 1 ) . because igf2 is located in spermatocytes of the testis , we detected the expression of igf2 in cases with ma , hypospermatogenesis , and oa , but not in the scos patients . we found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients , which was considered as the control group with normal spermatogenesis , although without a significant difference ( figures 2 , 3 ) , possibly because there were fewer samples and longer preservation times of some samples . quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma , hypospermatogenesis , and oa . we found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients ( figure 4 ) . however , due to errors in the rna extraction in the preliminary experiment , 3 testis samples ( 1 each ) from ma , hypospermatogenesis , and oa patients were damaged and were not tested . in the targetscan database , because the 3utr of the igf2-mrna has a putative mir-210-binding site , igf2 was predicted to be a potential target of mir-210 . to identify whether the igf2 gene was targeted by mir-210 directly , renilla luciferase reporters , which include the wild - type full - length 3utr forms of mir-210 seeding sites , figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells , and inhibiting mir-210 expression increased activity of the reporter renilla luciferase . expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells , and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 ( figures 6 ) . the igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2 , which is only produced from the paternal allele . the maternal allele is transcriptionally silent . to clarify the location of igf2 in human testicular tissues , we found igf2 located in spermatocytes in the testes of patients with oa ( figure 1 ) . because igf2 is located in spermatocytes of the testis , we detected the expression of igf2 in cases with ma , hypospermatogenesis , and oa , but not in the scos patients . we found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients , which was considered as the control group with normal spermatogenesis , although without a significant difference ( figures 2 , 3 ) , possibly because there were fewer samples and longer preservation times of some samples . quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma , hypospermatogenesis , and oa . we found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients ( figure 4 ) . however , due to errors in the rna extraction in the preliminary experiment , 3 testis samples ( 1 each ) from ma , hypospermatogenesis , and oa patients were damaged and were not tested . in the targetscan database , because the 3utr of the igf2-mrna has a putative mir-210-binding site , igf2 was predicted to be a potential target of mir-210 . to identify whether the igf2 gene was targeted by mir-210 directly , renilla luciferase reporters , which include the wild - type full - length 3utr forms of mir-210 seeding sites , were used . figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells , and inhibiting mir-210 expression increased activity of the reporter renilla luciferase . expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells , and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 ( figures 6 ) . during recent decades several studies have focused on the effects of mirnas on spermatogenesis in male infertility [ 911,17 ] . however , it was not understood how mir-210 , which is one of the up - regulated mirnas in testes of patients with noa , was involved in spermatogenesis in male infertility . the transformation of diploid spermatogonia into mature haploid cells in spermatogenesis is a complex biological process in the testes of males . the insulin / igf system takes part in the processes of cell proliferation , cell growth , differentiation , and survival , which affects nearly every organ in the body . also , insulin / igf plays an important role in the proper function of the testis in males . igf2 binds to igf1r and insr - a with a high affinity and binds to insr - a / igf1r , insr - b / igf1r , but with lower affinity . found that in inactivated insr and igf1r , there was a 79% reduction in daily sperm production in adult mouse testes by a conditional ko approach . taken together , the aforementioned data suggest that igf2 might be involved in the process of spermatogenesis . to examine the specific mechanism by which mir-210 is associated with the process of spermatogenesis , quantitative real - time pcr was performed to detect mir-210 expression . we found that mir-210 was significantly up - regulated in the testes of subjects with ma and hypospermatogenesis patients compared to oa . these results agree with findings of lian et al . using microarray technologies performed in noa and normal controls . several studies have suggested that this mirna could be mediated by hypoxia and participate in various types of regulation of angiogenesis , cell survival , proliferation , cell cycle arrest , and protein modification [ 1214 ] . furthermore , some researchers even found that mir-210 might be considered as one of the indicated markers in some diseases , such as clear cell renal cell carcinoma and acute myeloid leukemia . in the present study we found that igf2 was targeted by mir-210 directly in the in vitro experiment in nt2 cells , and mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility . firstly , as some errors occurred in the rna extraction in the preliminary experiment , mir-210 of 3 testes samples were damaged and not detected in the subsequent quantitative real - time pcr experiment , which might have affected our results . secondly , we did not investigate the functions of mir-210 and igf2 in vitro or in vivo , and we plan to do this in future research . we demonstrated that mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility . future mechanistic studies on the role of mir-210/igf2 in the process of spermatogenesis in male infertility will provide new insights into the diagnosis and management of male infertility .

we present an unusual case of recurrent cough syncope in a 43-year - old woman , which was initially thought to be seizures . syncopal episodes were triggered by paroxysms of cough and were characterized by unresponsiveness and myoclonic jerks in her extremities . she had a left - sided glomus jugulare tumor that extended into the posterior cranial fossa with evidence of worsening communicating hydrocephalus on brain imaging . we postulate that bouts of cough produced increased intracranial pressure both by raising intrathoracic and intraabdominal pressures as well as by transient obstruction to cerebrospinal fluid flow secondary to intermittent tonsillar herniation during cough . this resulted in diffuse decrease in cerebral blood flow causing syncope . the patient 's syncopal episodes decreased in frequency once an external ventricular drain was placed followed by a ventriculoperitoneal shunt . search for factors that can increase intracranial pressure seems warranted in patients with recurrent cough syncope . backgroundmicrornas ( mirnas ) play pivotal roles in spermatogenesis . microrna-210 ( mir-210 ) expression was up - regulated in the testes of sterile men with non - obstructive azoospermia ( noa ) . however , the underlying mechanisms of mir-210 involved in the spermatogenesis in patients with noa are unknown.material/methodsexpression of mir-210 and insulin - like growth factor ii ( igf2 ) in the testes of noa cases ( only including maturation arrest and hypospermatogenesis ) were detected in this study . we carried out in vitro experiments to determine if igf2 was directly targeted by mir-210 in nt2 cells.resultscompared with obstructive azoospermia ( oa ) as normal control , our results suggest that mir-210 was significantly up - regulated in testis of patients with noa ( p<0.05 ) , and igf2 was down - regulated , but without a significant difference . the results also indicated that igf2 was directly targeted by mir-210 in nt2 cells.conclusionsthe results showed that mir-210 was involved in spermatogenesis by targeting igf2 in male infertility .

midwife - led primary delivery care for low - risk pregnant women during labor has been reported to have various advantages , such as increased odds of high maternal satisfaction and a decrease of unnecessary medical interventions [ 18 ] . although the maternity care system for low - risk pregnant women peculiar to one country can not easily be compared with those in other countries , consumer demands for the humanization of obstetric care have arisen in various countries [ 18 ] . to date , we have found no evidence that midwife - led primary obstetric care is unsafe for low - risk pregnant women in comparison with obstetric care with the favorable cooperation of obstetricians and midwives in japan [ 912 ] . in addition , about 85% of low - risk pregnant women request that they give birth while receiving midwife - led primary delivery care . therefore , safe midwife - led delivery care with the backup of obstetricians may also be required for low - risk pregnant women in japan . if complications occur or threaten to occur during the primary midwife - led delivery care , the midwives have to refer the woman to obstetricians at the same or a neighboring hospital or private obstetric clinic as soon as possible . this is because , in deliveries managed by independent midwives in japan , many intervention measures , such as oxytocin infusion , epidural anesthesia , episiotomy , suture , and instrumental delivery , are not available based on japanese legal restrictions . in our institute , one of the main tokyo city perinatal centers , there are 3 japanese systems of midwife - led delivery care , as follows : ( 1 ) those intending to give birth at home managed by midwives who do not belong to our hospital , ( 2 ) those planning to give birth on futons ( i.e. , japanese - style bedding ) in japanese tatami mat delivery rooms in our hospital managed by the same midwives who do not belong to our hospital , and ( 3 ) those planning to give birth in japanese tatami mat delivery rooms managed by midwives who belong to our hospital . the objective of this study was to describe trends in transfers and perinatal outcomes among labors using these 3 japanese systems of midwife - led primary delivery care . the protocol for this analysis was approved by the ethics committee of the japanese red cross katsushika maternity hospital . in addition , informed consent for analysis from a retrospective database was obtained from each subject during their hospital visit . in our hospital , pregnant women who are initially considered low - risk at 3436 weeks of gestation can choose freely between the 3 systems of midwife - led care and obstetric shared care . in the midwife - led care units , midwives can practice autonomously and are fully accountable for their own practice , unsupervised by obstetricians . factors used to exclude women from the low - risk group comprise the following [ 912 ] : ( 1 ) medical history : pregnancy - induced hypertension , chronic hypertension , diabetes mellitus , renal disease , idiopathic thrombocytopenia , and other systemic illnesses ; ( 2 ) gynecological history : history of infertility therapies of in vitro fertilization , congenital uterine anomalies , uterine myomatosis , and adnexal anomaly ; ( 3 ) obstetric history : narrowing of the pelvic outlet , cephalopelvic disproportion , previous cesarean section , previous anal sphincter injury , previous postpartum hemorrhage 1,000 ml with blood transfusion , previous manual removal of placenta , previous gestational diabetes , and history of severe preeclampsia ; ( 4 ) complications during the present pregnancy : multiple pregnancy , nonvertex presentation , obesity ( maternal body mass index before pregnancy 25 and/or during the third trimester 28 ) , anemia ( hemoglobin < 9.0 g / dl ) , epilepsy with treatment , polyhydramnios , oligohydramnios , low - set placenta , placenta previa , fetal growth restriction , heavy for date fetus , gestational diabetes , and pregnancy - induced hypertension ; when risk factors are present , those women are managed by obstetricians and midwives ; ( 5 ) complications during labor : intrauterine infection , thick meconium staining , prolongation of labor such as active - phase dilation < 1 cm / hour and duration of second stage of labor 2 hours , prolonged rupture of membranes ( 24 hours ) , uterine inertia , arrest of labor , and fetal heart rate abnormality such as a nonreassuring fetal status . when these factors are present , the women are transferred to be managed mainly by obstetricians ( obstetric shared care ) in a standard western - style delivery room or surgery room in our hospital . a retrospective study was performed to examine trends in transfers and perinatal outcomes among labors that started using the 3 systems of midwife - led primary delivery care . in this study , student 's t - test was used for continuous variables and the test for categorical variables . odds ratios ( ors ) and 95% confidence intervals ( cis ) were also calculated . differences with p between 2009 and 2012 , a total of 678 low - risk women were placed in the 3 forms of midwife - led primary delivery care at the onset of labor pains and/or rupture of membranes at 3741 weeks of gestation . of these , 123 ( 18% ) intended to give birth at home , 88 ( 13% ) planned to give birth in the japanese tatami mat rooms in our hospital managed by midwives who do not belong to our hospital , and 467 ( 59% ) planned to give birth managed by the midwives belonging to our hospital . table 1 shows the clinical descriptions of the 678 pregnant women initially considered as low - risk for receiving our midwife - led primary delivery care systems . there were no significant differences in the maternal age or parity among the 3 groups . table 2 shows the rate of transfers in the 3 groups of the midwife - led primary delivery care systems . the total rate of transfers in the system run by the midwives belonging to our hospital ( 56% ) was higher than in the other 2 systems run by the independent midwives ( 31% in planned home birth : or 1.87 , 95% ci 1.23.0 , p < 0.01 ; 38% in planned hospital birth : or 2.51 , 95% ci 1.73.8 , p < 0.01 ) . in addition , the timing of transfers in the system run by the midwives belonging to our hospital ( before the second stage of labor : 52% ) was earlier than those in the other 2 systems ( 21% in the planned home birth : or 4.12 , 95% ci 2.66.6 , p < 0.01 ; 20% in planned hospital birth : or 4.29 , 95% ci 2.57.4 , p < 0.01 ) . however , if classified into nulliparous and parous women , there were no significant differences in the rate of transfers among the 3 groups , as shown in table 1 . in addition , among the 3 groups there were no significant differences in the rate of the main 2 indications for transfer : fetal heart rate abnormality and failure to progress . the main indications for transfer after delivery were maternal postpartum hemorrhage and neonatal respiratory distress associated with asphyxia . table 3 shows the obstetric and neonatal outcomes in the pregnant women initially considered as low - risk for receiving our midwife - led primary delivery care systems . there were no significant differences in these outcomes among the 3 groups our obstetric care system involves the division of women in labor into low- and high - risk groups [ 912 ] . the women who are initially considered low - risk can choose freely between midwife - led care and obstetric shared care . if complications occur or risk factors arise during labor in the primary midwife - led care , they are transferred to obstetric shared care . this may be the first report concerning the differences in the timing of transfers from midwife - led care to obstetric shared care among the 3 systems of midwife - led primary delivery care in japan . in this study , there was no evidence that the primary midwife - led care is unsafe for low - risk pregnant women in any of these 3 midwife - led delivery care systems . however , there were no significant differences in the timing of referrals from midwife - led care to obstetric shared care between the system led by midwives who belong to our hospital ( hospital midwifery system ) and the systems led by the midwives who do not belong to our hospital . in the hospital midwifery system , the timing of transfers seemed to be the earliest due to the ease of transfer within the same hospital and administrator setting . on the other hand , the rate of transfers after delivery with the other 2 systems was higher than that in the hospital midwifery care . during the period , the main indications for transfers were maternal postpartum hemorrhage and/or neonatal respiratory distress associated with asphyxia . fortunately , the difference was not associated with adverse obstetric or neonatal outcomes ; however , unfortunately , they led to early mother - to - child separation , especially in cases of planned home birth because healthy puerperal women or newborns can not be transferred from home to hospital according to japanese law . although home birth might be very comfortable , those involved must be prepared for mother - to - child separation in cases of referrals after delivery . the major limitations of this study were the small sample size and lack of long - term follow - up of mothers and children to consider the potential of the findings based on our own context . there were no cases of fetal / neonatal death under the midwife - led delivery care . the most evaluated outcome under midwife - led delivery was the satisfaction of pregnant women with the development of mother - child relationships after delivery . in addition , there might be some bias related to the backgrounds in the selection of the systems because this was not a randomized trial study . therefore , a further large prospective study with long - term follow - up may be needed . there were no significant differences in perinatal outcomes among the 3 systems ; however , there were some differences in the status of the transfers to the obstetric shared care . diabetes decreases the overall life expectancy and cause a heavy burden on public health ( 1 ) . moreover , the asia - pacific region is considered to be on the verge of an emerging diabetes epidemic ( 2 ) . the development of type 2 diabetes is affected by genetic and environmental determinants ( 3 ) . recently , one study investigated whether common variants of functional and positional candidate genes , including adrb3 , pparg , enpp1 and capn10 , were determinants of type 2 diabetes ( 4 ) . enpp1 , also called k121q , has a glutamine substitution for lysine at codon 121 ( 5 ) . type 2 diabetes is characterized by insulin resistance ( 6 ) , and enpp1 plays an important role in insulin resistance ( 7 , 8) . enpp1 interacts with -subunit of the insulin receptor to interrupt signaling ( 9 ) . in previous studies , the k121q polymorphism of the human pc-1 gene was strongly associated with insulin resistance ( 1 , 3 , 7 , 10 - 12 ) . however , there was no association between insulin resistance and the k121q variant ( 13 , 14 ) . in addition , there were discrepancies for the impact of enpp1 polymorphism on obesity between ethnic groups ( 5 , 10 , 13 , 15 - 19 ) . obesity increases the concentration of insulin in plasma and is the major contributor of insulin resistance ( 20 ) . obesity appears to be an effect modifier of type 2 diabetes in d1057 carriers ( 21 ) . the association of obesity with the genetic variant of the insulin receptor substrate was identified in other studies ( 21 , 22 ) . in the chinese han population , the pc-1 q121 allele was associated with insulin resistance . in women , carriers of the q allele had an increased risk for obesity development ( 3 ) . in caucasians and african - americans , 121q carriers had an association with increased body mass index ( bmi ) ( 23 ) , and the three - allele risk type haploid qdeltg with the q allele increased the risk for obesity ( 24 ) however , in the danish population , there were no differences in the distribution of frequencies of dominant types ( kk wild type and kq / qq variant type ) and alleles ( 19 ) . the complexity of type 2 diabetes is related to factors such as genetic heterogeneity , interactions between genes , and the modulating role played by the environment ( 4 ) . in spite of these limitations , studies of type 2 diabetes and genetic factors of obesity can predict the risks for development of both type 2 diabetes and obesity in order to assist primary prevention , and korea is an appropriate country for such studies because of the homogeneity of racial composition and lifestyle ( 25 ) . therefore , the aim of this study was to analyze the presence of the enpp1 polymorphism , not studied yet in korean population , to identify the association between genotypes and allele with type 2 diabetes and obesity . this company is an electric power company located at kori , yonggwang , ulchin , wolsung , and seoul in korea . there were 195 male workers ( age 48.26.7 yr , bmi 24.672.64 kg / m ) who were diagnosed as diabetics during medical examinations conducted from march 2004 to october . the 1,750 male workers ( age 45.27.7 yr , bmi 24.772.64 kg / m ) in the control group were selected randomly . subjects were included if they met one of the criteria below and their onset age was older than 20 yr old to exclude type 1 diabetes : 1 ) blood - sugar level before a meal exceeded 126 mg / dl twice or more ; 2 ) blood - sugar level before a meal exceeded 126 mg / dl once or more and blood - sugar level two hours after a meal exceeded 200 mg / dl ; 3 ) those who reported that he had a history of diabetes in the questionnaire and being taken oral hypoglycemic agents . the workers included in the obesity group were those whose bmi was 25 kg / m or more . after collecting a blood sample from vein in fasting ( 8 hr ) status , we measured the blood - sugar level , insulin , and lipid profile . we also measured height and weight to calculate bmi . if fasting glucose levels were greater than 126 mg / dl , we checked the 2 hr post - prandial plasma glucose level at each site within 1 month . height and weight were measured by autoanalyzer ( health guard , fanics , seoul , korea ) . the fasting blood level was analyzed by glucose - oxidase assay using an autochemistry analyzer . to determine the lipid profile , total cholesterol was analyzed by enzyme assay using cholesterol oxidase ( cod ) , high density lipoprotein ( hdl ) cholesterol by glycerol phosphate oxidase assay , low density lipoprotein ( ldl ) cholesterol by direct surfactant assay . we carried out this study under the approval by the ethnics committee of the asan medical center , and obtained written consent from all subjects , providing subjects with sufficient explanation to obtain informed consent . we extracted genomic dna from buffy coats using the generall blood sv kit ( general biosystem , seoul , korea ) and following the instructions suggested by the manufacturer . the method of genotyping used to identify the k121q polymorphism in enpp1 exon 4 was polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) , using dna treated with a restriction enzyme after pcr , based on the paper reported by abate et al . we carried out student 's t - test to analyze the effects of genotypes on biochemical parameters using genotypes as factors . the hardy - weinberg equilibrium was computed based on the goodness - of - fit test . we also investigated the differences in frequencies of genotypes between type 2 diabetic and normal groups , and between obesity and normal groups by fisher 's exact test . the spss 12.0 ( for window ) statistical software package was used for statistical analysis . this company is an electric power company located at kori , yonggwang , ulchin , wolsung , and seoul in korea . there were 195 male workers ( age 48.26.7 yr , bmi 24.672.64 kg / m ) who were diagnosed as diabetics during medical examinations conducted from march 2004 to october . the 1,750 male workers ( age 45.27.7 yr , bmi 24.772.64 kg / m ) in the control group were selected randomly . subjects were included if they met one of the criteria below and their onset age was older than 20 yr old to exclude type 1 diabetes : 1 ) blood - sugar level before a meal exceeded 126 mg / dl twice or more ; 2 ) blood - sugar level before a meal exceeded 126 mg / dl once or more and blood - sugar level two hours after a meal exceeded 200 mg / dl ; 3 ) those who reported that he had a history of diabetes in the questionnaire and being taken oral hypoglycemic agents . the workers included in the obesity group were those whose bmi was 25 kg / m or more . after collecting a blood sample from vein in fasting ( 8 hr ) status , we measured the blood - sugar level , insulin , and lipid profile . we also measured height and weight to calculate bmi . if fasting glucose levels were greater than 126 mg / dl , we checked the 2 hr post - prandial plasma glucose level at each site within 1 month . height and weight were measured by autoanalyzer ( health guard , fanics , seoul , korea ) . the fasting blood level was analyzed by glucose - oxidase assay using an autochemistry analyzer . to determine the lipid profile , total cholesterol was analyzed by enzyme assay using cholesterol oxidase ( cod ) , high density lipoprotein ( hdl ) cholesterol by glycerol phosphate oxidase assay , low density lipoprotein ( ldl ) cholesterol by direct surfactant assay . we carried out this study under the approval by the ethnics committee of the asan medical center , and obtained written consent from all subjects , providing subjects with sufficient explanation to obtain informed consent . we extracted genomic dna from buffy coats using the generall blood sv kit ( general biosystem , seoul , korea ) and following the instructions suggested by the manufacturer . the method of genotyping used to identify the k121q polymorphism in enpp1 exon 4 was polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) , using dna treated with a restriction enzyme after pcr , based on the paper reported by abate et al . we carried out student 's t - test to analyze the effects of genotypes on biochemical parameters using genotypes as factors . the hardy - weinberg equilibrium was computed based on the goodness - of - fit test . we also investigated the differences in frequencies of genotypes between type 2 diabetic and normal groups , and between obesity and normal groups by fisher 's exact test . the spss 12.0 ( for window ) statistical software package was used for statistical analysis . the frequencies of the kk type , kq type , and qq type in enpp1 k121q were 82.1% , 17% and 0.9% , respectively . the homozygous type of q carrier ( qq type ) was added to the heterozygous type ( kq type ) because the frequency of qq type was very low ( 0.9% ) . we investigated the differences in age , blood pressure , bmi , and results of clinical examinations according to genotypes of enpp1 k121q between the type 2 diabetic and non - diabetic groups ( table 1 ) . when type 2 diabetics and non - diabetics were pooled ( n=1,945 ) , there were no significant differences in bmi , systolic pressure , diastolic pressure , glucose value in fasting status , total cholesterol , ldl cholesterol , hdl cholesterol , triglyceride , c - reactive protein , and homa - ir between the kk type and kq+qq type . in addition , there were no significant differences in the same characteristics above between type 2 diabetic group and non - diabetic groups ( table1 ) , and between the obese group and non - obese groups ( table2 ) . the frequencies of genotypes were in accordance with the hardy - weinberg equilibrium ( p=0.85 ) . the odds ratio of having a kq+qq genotype was 0.85 for diabetics versus non - diabetics . the odds ratio of having a q allele was 0.91 for diabetics versus non - diabetics . however , there was no significant difference in the genotypic and allelic distribution between type 2 diabetics and non - diabetics ( table3 ) . the odds of a kq+qq genotype were 0.93 for obese versus non - obese subjects . furthermore , the odds of having a q allele were 0.96 for obese versus non - obese subjects . however , there was no significant difference in the genotypic and allelic distribution between obese and non - obese ( table4 ) . the frequency of the kk type in enpp1 k121q genotypes was 82.1% , that of kq+qq type was 17.9% and that of q allele was 9.4% . there was no statistically significant difference in the distribution among these genotypes and alleles ( p=0.81 , p=0.89 , respectively ) , although the 121q carrier and q allele in obese and/or diabetics seemed to differ slightly from those in the non - obese non - diabetics ( reference group ) . in determining the prevalence of the q allele carriers ( kq and qq subjects ) and q allele , there were no significant differences in the genotypic and allelic distribution with the respect to any phenotypes ( data not shown ) . after adjusting for the effects of obesity , the probability of type 2 diabetes in kq+qq type was 0.858 ( data not shown ) and that in q allele was 1.095 , showing no significant difference ( table 5 ) . moreover , after adjusting for the effects of type 2 diabetes , the probability of developing obesity in the kq+qq type was 0.936 ( data not shown ) and that in the q allele was 1.038 , showing no significant difference ( table 6 ) . the results of studies of the association between enpp1 k121q variants and both type 2 diabetes and obesity in several races are disparate . we carried out this study to investigate the association of k121q variants with type 2 diabetes and obesity in korean male workers . insulin resistance is a major component of the pathogenesis of type 2 diabetes ( 27 ) , and insulin receptor kinase activity is impaired in muscle and other insulin - sensitive tissue of many type 2 diabetic patients ( 28 ) , and then a potential inhibitor of the insulin receptor tyrosine kinase is identified as the plasma - cell membrane differentiation antigen-1 ( pc-1 ) ( 29 ) . therefore , it is significant to analyze enpp1 ( pc-1 ) polymorphism . in the previous studies of the association between type 2 diabetes and polymorphism in enpp1 , the k121q missense mutation increased the odds ratio ( or ) for type 2 diabetes in dominican ( 10 ) , south asian , caucasian ( 16 ) , finnish ( 18 ) , and french populations ( 24 ) . moreover , according to a meta - analysis of the association between enpp1 k121q variant and type 2 diabetes , the odds ratios were 1.30 ( 95% confidence interval [ ci ] , 1.13 - 1.50 ) ( 16 ) and 1.17 ( 95% ci , 1.10 - 1.25 ) ( 19 ) , showing significant association . in this study , 121q was not associated with type 2 diabetes , showing consistent results with those in the japanese population ( 5 ) , danish caucasians ( 13 ) , oji - cree population ( 17 ) , finnish population ( 18 ) , and danish white subjects ( 19 ) . most type 2 diabetes in koreans is characterized by non - obesity , thus the enpp1 k121q mutant relevant to insulin resistance possibly could be a candidate gene that is not appropriate to explain susceptibility to type 2 diabetes . this can be a possible explanation for the lack of association between enpp1 121q carrier and type 2 diabetes in this study . obesity is a main risk factor for the development of type 2 diabetes ( 20 ) and there is linear association between obesity and type 2 diabetes ( 3 ) . in previous studies of the association between obesity and polymorphism in enpp1 , 121q carriers and/or q allele were associated with obesity in the chinese han population ( bmi of obesity group 27 kg / m ) ( 3 ) , caucasians ( bmi of obesity group > 90th percentile ) , african - american adults ( bmi of obesity group > 80th percentile ) ( 23 ) , french population ( bmi of obesity group 95th percentile ) ( 24 ) , and dominican population ( bmi of obesity group 30 kg / m ) ( 10 ) . however , in this study ( bmi of obesity group 25 kg / m ) , there was no difference in distribution between obesity and 121q carriers and presence of the q allele . this result was consistent with those from a study of 7,333 danes ( 19 ) and a spanish population ( 14 ) in which the bmi of the obesity group was 25 kg / m or higher . on the other hand , in matsuoka 's study , the percentage of subjects whose bmi was 30 kg / m or higher was too low ( 2.5% ) to investigate the effect of k121q genotype on obesity . the results of the present study showed that the frequencies of the q allele was 8.7% in the type 2 diabetic group and 9.2% in the obesity group , which was lower than those in finnish and swedish populations ( 12.9 - 15.1% ) ( 11 ) , danish caucasians ( 14 - 16% ) ( 13 ) , south asians in chennai ( 14% ) , caucasians in dallas ( 16% ) , south asians in dallas ( 19% ) ( 16 ) , the dominican population ( 54.2% ) ( 10 ) and , black children ( 77% ) ( 30 ) . the frequencies of the q allele investigated in this study might have a smaller statistical power to explain any association with either type 2 diabetes or obesity with 121q carriers ( kq+qq ) and/or the q allele . in conclusion , the present study suggests that the enpp1 k121q polymorphism was not associated with type 2 diabetes and obesity . the results of negative associations in this study might be attributable to the low prevalence of obesity , relatively younger age , and low frequencies of the 121q carriers . large and prospective studies are needed to confirm this preliminary observation in the korean population .

objective . the objective of this study was to describe the recent clinical characteristics of labor using 3 systems of japanese midwife - led primary delivery care , as follows : ( 1 ) those intending to give birth at home managed by midwives who do not belong to our hospital , ( 2 ) those planning to give birth in our hospital managed by the same midwives , and ( 3 ) those planning to give birth managed by midwives who belong to our hospital . methods . a retrospective cohort study was performed . results . there were no significant differences in the obstetric or neonatal outcomes among the 3 groups . the rate of transfers during labor with the system involving midwives belonging to our hospital was higher than those with the other 2 systems . in addition , the timing of transfers in the system with the midwives belonging to our hospital was earlier than with the other 2 systems . among the 3 groups , there were no significant differences in the rate of the main 2 indications for transfers : fetal heart rate abnormality and failure to progress . conclusion . there were no significant differences in perinatal outcomes among the 3 systems ; however , there were some differences in the status of transfers to obstetric shared care . type 2 diabetes is characterized by insulin resistance , and enpp1 plays an important role in insulin resistance . we investigated the association of the enpp1 k121q polymorphism with both diabetes and obesity ( body mass index [ bmi ] ) in korean male workers . the study design was case - control . subjects were 1,945 male workers ( type 2 diabetes , 195 ; non - diabetes , 1,750 ) of nuclear power plants who received examinations from march to october in 2004 . we collected venous blood samples under fasting ( 8 hr ) conditions , calculated bmi by height and weight , and assessed relevant biochemical factors . the results of this study demonstrated that the enpp1 121q genotype ( kq+qq types ) was not associated with type 2 diabetes ( odds ratios [ or ] , 0.854 ; 95% confidence interval [ ci ] , 0.571 - 1.278 ) or obesity ( or , 0.933 ; 95% ci , 0.731 - 1.190 ) . in addition , the frequency of the q allele was not related to type 2 diabetes ( or , 0.911 ; 95% ci , 0.630 - 1.319 ) or obesity ( or , 0.962 ; 95% ci , 0.767 - 1.205 ) . we concluded that the enpp1 121q allele is not a critical determinant for either diabetes or obesity in korean males . the discordance between the results of this study and those derived from studies of dominican , south asian , caucasian , finnish , and french populations might be due to differences in genetic backgrounds between these populations .

lipid apheresis provides a safe and effective means of treating patients with severe hyperlipidemia . it functions by first separating plasma from blood cells with a cell separator and then using either the adsorption of apolipoprotein b by affinity columns containing anti - apolipoprotein b antibodies or dextran sulphate , or their precipitation at low ph by heparin . lipid apheresis allows patients to attain lower levels of low - density lipoprotein ( ldl ) , which are usually not attainable with traditional drug therapy alone , while leaving high - density lipoprotein ( hdl ) levels generally unaffected . when used in conjunction with statins and other lipid - lowering drugs , lipid apheresis may also induce the regression of coronary atherosclerotic plaque in familial hypercholesterolemia ( fh ) patients . fh is a group of autosomal dominant genetic defects resulting in elevated serum ( ldl ) cholesterol levels . in the heterozygous state , fh is a relatively common but serious genetic disorder , with an incidence of about 1 in 500 persons in the general population . fh has been associated with an increased risk for atherosclerosis , premature coronary heart disease , and heart failure [ 3 , 4 ] . fh is caused by a mutation affecting apolipoprotein b , proprotein convertase subtilisin kexin type 9 ( pcsk9 ; an enzyme involved in ldl receptor degradation ) , or , most commonly , the ldl receptor gene , resulting in defective ldl receptors and/or a diminished number of ldl receptors [ 7 , 8 ] . these mutations cause ldl to be catabolized at a slower rate and thus accumulate in the circulation . currently , fh is treated using a variety of cholesterol - lowering drugs , most notably statins or hmg - coa reductase inhibitors . for many patients , however , statins are not a viable treatment option , because of either intolerance or ineffectiveness . lipid apheresis is an alternative form of treatment for these fh patients as well as those who have persistently elevated ldl levels despite treatment . because apheresis is performed at only a few highly specialized centers in relatively low volume , there is very little literature discussing the effectiveness of lipid apheresis on the reduction of lipid profiles and the prevention of future cardiac events . this study , therefore , reports the experience in a single metropolitan center of treating patients with hyperlipidemia with lipid apheresis . retrospective chart reviews were performed and questionnaire surveys were given to active lipid apheresis patients at the minneapolis heart institute ( mhi ) at abbott northwestern hospital ( anw ) , minneapolis , minnesota . mhi and anw are divisions of allina health , a large healthcare provider in minnesota and western wisconsin . patients were identified through an electronic health record ( ehr ) screen of ambulatory patients representing all patients seen at all allina health metro area and regional locations between 2009 and 2012 ( epic systems , verona wi ) . of these patients , criteria to qualify for apheresis were based on the united states food and drug administration ( fda ) approval recommendations . currently , the fda supports ldl apheresis for patients who , after six months , do not have an adequate response to diet therapy and maximum drug therapy , due to either ineffectiveness or intolerance , and meet the following criteria : functional homozygotes with an ldl cholesterol > 500 mg / dl without cad , functional heterozygotes with ldl cholesterol > 300 mg / dl without cad , functional heterozygotes with ldl cholesterol > 200 mg / dl with documented coronary heart disease . functional homozygotes with an ldl cholesterol > 500 mg / dl without cad , functional heterozygotes with ldl cholesterol > 300 mg / dl without cad , functional heterozygotes with ldl cholesterol > 200 mg / dl with documented coronary heart disease . the date of birth , gender , date of apheresis initiation , lipid disorder diagnosis , apheresis frequency , and family history of cardiac events were recorded . patients were noted as having fh if the active problem list contained a diagnosis of fh . to determine which patients had fh , we used the national lipid association ( nla ) criteria for an 80% probable fh diagnosis , using the highest ldl recorded in the patient chart as follows : age < 20 and ldl > 190 mg / dl , age 2029 and ldl > 220 mg / dl , and age 30 and ldl > 250 mg / dl . potential homozygous fh ( hofh ) patients were defined as having an untreated ldl > 500 mg / dl or a treated ( on statin ) ldl over > 300 mg / dl , in addition to clinical evidence of xanthomas before age of 10 years or having two parents with heart disease or high lipids . identifiable secondary causes for marked hyperlipidemia were excluded from the analysis by examining the ehr chart of each potential homozygote . current cholesterol lowering medications were also recorded , focusing on the use of statins , colesevelam ( welchol ) , ezetimibe ( zetia ) , niacin , and aspirin . a significant cardiovascular event was defined as a myocardial infarction ( mi ) , a percutaneous transluminal coronary angioplasty ( ptca ) or stenting procedure , or a coronary artery bypass graft ( cabg ) using ehr documented icd-9 criteria . cardiac events were separated by their occurrence before or after the patient began apheresis , and the total number of events was recorded for each group . multiple cardiac events occurring at the same hospitalization , such as mi followed by ptca , were counted as a single event for cardiac event rate calculation . pre- and postapheresis cardiac event rates were calculated by adding the total number of cardiac events and dividing by the total person years during each time period . the preapheresis time period describes the time from the first documented ehr visit to the date of apheresis initiation . the postapheresis time period describes the time from the date of apheresis initiation to the study date . unverifiable events noted in the ehr but occurring prior to the first documented ehr visit were noted but excluded from the cardiac event rate calculation . mean acute ldl reductions were calculated by averaging all recorded ldl values prior to and immediately after the treatment sessions . mean acute total cholesterol , hdl cholesterol , and triglyceride reductions were calculated by using lipid profile from the most recent treatment session . ldl apheresis was performed at abbott northwestern hospital using the kaneka liposorber la-15 system ( kaneka medical products ) . the system consists of the kaneka ma-03 machine , the integrated sulflux kp-05 plasma separator , which consists of porous hollow fibers , to separate the plasma from the whole blood , and two disposable liposorber la-15 adsorption columns to adsorb apolipoprotein b - containing lipoproteins from patient plasma . patients confirmed information in their ehr such as risk factors , answered questions relating to their awareness of fh and if their family had been previously tested for it , provided their level of satisfaction with their apheresis program , and indicated their interest in learning more about alternative treatments . the data from the questionnaires was cross - referenced with the data from the patient charts to ensure accuracy . descriptive statistics are displayed as means and sds for continuous variables ; number and percentage with characteristic are given for categorical variables . categorical variables were analyzed using pearson 's chi - square or fisher 's exact tests . continuous variables were analyzed using student 's t - test . a value of p < 0.05 was considered significant , and p values are two - sided where possible . all statistical calculations and plots were done with stata 11.2 ( college station , tx ) . institutional review board approval was obtained for data collection , follow - up , and data analysis . of these , 8 ( 72.7% ) were male , 10 ( 90.9% ) were caucasian , 1 ( 9.1% ) was african american , 10 ( 90.9% ) carried the diagnosis of fh , with 2 ( 18.2% ) patients identified as probable homozygotes , and 1 ( 9.1% ) was diagnosed as having familial combined hyperlipidemia . the average age of patients was 65.6 9.3 years , and patients had been on apheresis for an average of 6.2 7.0 years . four ( 36.4% ) patients were currently on statins while the other 7 ( 63.6% ) had a history of statin intolerance . five of 11 ( 45.5% ) patients were on a nonstatin cholesterol lowering medications , including 1 ( 9.1% ) on colesevelam ( welchol ) , 3 ( 27.3% ) on ezetimibe ( zetia ) , and 1 ( 9.1% ) on niacin . maximum ldl levels ranged from 211 to 448 mg / dl with a mean ( sd ) value of 298 80.7 mg / dl in the study group . since our ehr was implemented in 2005 , it is possible we may be underestimating the highest lifetime ldl for each patient . of the 11 participants , 9 completed the questionnaire in its entirety ; 1 patient provided answers to all questions but did not disclose risk factors and 1 patient did not complete the questionnaire . all of the patients indicated that they were aware that they likely had fh and 7 patients indicated that their immediate family had been tested for fh . the patients self - reported a total of 44 cardiac events before apheresis and 8 cardiac events after apheresis . of the 10 patients that completed the questionnaire , 4 patients were currently on statins while the other 6 were statin intolerant . eight patients ( 72.7% ) had a cardiac event documented by ehr , with 43 cardiac events occurring overall ( table 4 ) . self - reported events which were unable to be verified via the ehr were excluded from the cardiac event rate analysis . thirty - four cardiac events were documented before apheresis in 8 patients compared with 9 events in 5 patients after apheresis . after excluding cardiac events that were unverifiable , 14 cardiac events were documented in the preapheresis time period and 7 were documented in the postapheresis time period . the cardiac event rates were calculated to be 0.23 ( 0.13 , 0.39 ) events per person year in the preapheresis group and 0.10 ( 0.041 , 0.21 ) events per person year in the postapheresis group ( p = 0.064 ) . patients were observed for an average of 7.6 5.9 years before apheresis and 6.2 4.7 years after apheresis , with 60.6 total patient years before apheresis and 67.8 patient years after apheresis . this study was conducted to gain more information on lipid apheresis and evaluate the effectiveness in lowering lipid values . in addition , through chart review and patient survey , we attempted to gain a greater understanding of this patient population in terms of traditional risk factors , family awareness and screening , statin and other cholesterol medication uses , desire for additional treatment options , and ultimately cardiac events . our study shows that apheresis markedly lowers total cholesterol , ldl cholesterol , triglycerides and , to a much lesser degree , hdl cholesterol . there was a small , but statistically significant , reduction in hdl values after apheresis . many of these patients were statin intolerant and some had been using nonstatin cholesterol medications . importantly , 10/11 ( 90.9% ) participants indicated a desire to learn more about other potential treatment options , indicating that this population may indeed experience fatigue of this procedure . although taken from a small study population , our data suggests a reduction in cardiac event rate after apheresis . while not statistically significant , our data shows a strong trend towards event rate reduction . this statistical insignificance is likely explained by the study 's small sample size . with a larger population it is also important to note that the risk for cardiac events increases with age . ldl apheresis has been shown to improve endothelium dependent vasodilation [ 11 , 12 ] , microvascular flow , and myocardial perfusion . some studies [ 2 , 15 , 16 ] have also shown a significant reduction in angiographic cad , but others have not . these studies have been small , primarily nonrandomized trials . the ldl - apheresis atherosclerosis regression study ( laars ) looked at the change in plaque characteristics of patients undergoing apheresis compared to drug therapy over a period of two years . in that period , 7 out of 21 patients on apheresis had a cardiac event compared with 3 out of 21 on medication only . while this study found that apheresis arrested the progression of atherosclerosis the fh regression study found that ldl apheresis combined with simvastatin was more effective than colestipol plus simvastatin in reducing ldl cholesterol and lipoprotein ( a ) but was less effective at influencing coronary atherosclerosis . another study found that , out of 18 patients , 3 had myocardial infarctions , 1 underwent a cabg , and 12 needed coronary angioplasties within two years of beginning a combination therapy of apheresis , statins , and probucol . before beginning the combination therapy , 11 had experienced a mi , 5 had undergone a cabg , and 13 had undergone an angioplasty . the heparin - induced extracorporeal ldl precipitation ( help ) study found that help is suitable for reducing ldl concentrations and may work to reduce the burden of atherosclerosis , as there were no myocardial infarctions and a low coronary intervention rate in patients who began apheresis . due to the expensive nature of apheresis , a randomized , controlled clinical trial is needed to truly gauge the effectiveness of apheresis in reducing the occurrence of cardiac events . if apheresis is not deemed effective or is minimally effective , as some of these studies suggest , other types of treatment , such as lomitapide , mipomersen , or pcsk9 inhibitors , should be pursued . while satisfaction was generally high in our survey , patients specifically cited that this satisfaction was based on the results of apheresis and not on the process itself . many patients complained about the invasive nature of apheresis , citing bruises from the procedure and the inconvenience of reporting for treatment every two weeks . additionally , almost all patients were interested in learning more about alternative treatments , suggesting that they would prefer an alternative treatment which could match the results provided by apheresis . this study had several limitations . since lipid apheresis is an advanced treatment for an uncommon genetic disease , the limited number of patients available to participate in the study led to a small sample size . the event rate reduction was not statistically significant but showed a strong trend toward cardiac event rate reduction before and after apheresis . by defining the observational period initial time point as the first documented ehr visit , we excluded 20 events before apheresis and 2 events after apheresis from the cardiac event rate calculation . the lipid - lowering effects of apheresis are best expressed as reductions in interval means . although lipid apheresis was performed every two weeks , ldl values were not measured ever two weeks due to clinical practices . this inconsistency in measurement intervals prevents the use of more advanced measures to accurately track the effect apheresis has on ldl measurements . finally , this study focused on active apheresis patients and therefore did not include patients who had stopped apheresis or were deceased . lipid apheresis can reliably reduce ldl , non - hdl cholesterol , triglyceride , and total cholesterol levels in fh patients . our data suggest that lipid apheresis shows a strong , but not statistically significant , trend towards the reduction of cardiac events . apheresis is a viable treatment for fh patients , especially those that are statin - intolerant , due to its lipid lowering nature and its apparent reduction of cardiac events . however , there is a need for alternative treatments which are less invasive and provide easier patient access . agenesis of the inferior vena cava ( ivc ) as a cause of recurrent deep vein thrombosis ( dvt ) is uncommon . a 33-year - old male with no family history of thrombophilia , who had experienced multiple recurrent episodes of dvt over a 15-year period of unknown cause , was admitted into our hospital because of cellulitis in the right leg . congenital absence of the ivc could be a rare risk factor for idiopathic dvt , especially in young individuals . venous thromboembolism ( vte ) , which includes deep vein thrombosis ( dvt ) and pulmonary embolism , has an incidence of 1 to 3 per 1000 individuals per year in western populations.1 congenital anomalies of the inferior vena cava ( ivc ) are uncommon , and have been associated with the development of venous thrombosis of the lower limbs.2 congenital anomalies of the ivc has been reported as a risk factor for dvt , especially in individuals < 30 years old , and a concomitant thrombophilic disorder has been found in such individuals.3 we report a case of recurrent dvt in a 33-year - old man with agenesis of the ivc . the patient had experienced recurring episodes of idiopathic dvt in the right leg for 15 years . a 33-year - old man was admitted to the internal medicine department , holy family hospital , nazareth , israel , because of cellulitis in the right leg . one week prior to his admission , he complained about pain and increased local heat in the left ankle and thumb of the right leg . the patient had no history of previous trauma , surgery , insect bites , dysuria , or joint symptoms , and no family history of thrombophilia . he reported that he had ( a ) rheumatic fever without any complications when he was 19 years old , which was treated with penicillin , ( b ) been hospitalized when he was 23 years old because of infected skin ulcers on the right calf , for which he was treated by parenteral antibiotics , and ( c ) recurrent episodes of idiopathic dvt for the last 15 years . he also reported that he had not been treated with warfarin , but he had been on prophylactic enoxaparin therapy for dvt some years ago which has since been stopped and that he had been recently treated with allopurinol and colchicine for a presumed diagnosis of gout . he had been investigated several times for a primary hypercoagulability state , and the results were negative . on examination , the most outstanding clinical findings were swelling of ankles , mild edema , redness , and increased temperature of the right ankle and calf with trophic skin changes ( skin discoloration with ulcers ) , and superficial varicose veins in the lower abdomen ( figure 1 ) . the clinical laboratory findings ( erythrocyte sedimentation rate , leukocyte and platelet counts , and plasma hemoglobulin , plasma protein c , plasma protein s , fibrinogen , and antithrombin iii levels ) , the results of the kidney and liver function tests , and resistance to activated protein c were all normal . polymorphisms of the genes that encode for methylenetetrahydrofolate reductase were not detected , and the factor v leiden and prothrombin mutations g20210a were absent . the results of the clinical immunological studies for complement c3 and c4 and rheumatoid factor were negative , and no circulating titers for antinuclear antibody , antineutrophil cytoplasmic antibody , and cardiolipin antibody were found . cultures from the infected skin ulcers of the right leg were positive for methicillin - resistant staphylococcus aureus ( mrsa ) . ultrasound imaging of the leg veins showed a previous dvt in the right common femoral vein , and dilated superficial inguinal veins . computer tomography with contrast of the abdomen showed agenesis of the infrarenal segment of the ivc ( figure 2 ) with dilated azygos and hemiazygos veins ( figure 3 ) . there were also varicose veins in the abdominal wall and right groin , which were associated with dilated superficial and collateral veins ( figure 4 ) . transthoracic echocardiography of the patient s heart revealed mild atrial enlargement and good systolic function of the left ventricle , and no pathological valvular flows . the patient was diagnosed as having agenesis of the infrarenal segment of the ivc and dvt of the right leg without concomitant risk factors for vte . since we attributed the agenesis of the ivc to be the underlying cause of the recurrent episodes of the dvts , the patient was started on anticoagulant therapy ( subcutaneous enoxaparin 160 mg / day ) for dvt , antibiotic therapy ( intravenous vancomycin 1.5 g / day for mrsa skin infection ) , and referred to a vascular surgeon specialist but the patient refused . at follow - up in the internal medicine clinic , the most outstanding clinical findings were swelling of left ankle , redness , with trophic skin changes , and a mild improvement of the skin ulcers . despite several phone calls for follow - up the normal ivc is composed of 4 segments : hepatic , suprarenal , renal , and infrarenal . since many transformations can occur during the formation of the ivc such anomalies occur in 0.3% of otherwise healthy individuals , and in 0.6% to 2% of patients with other cardiovascular anomalies.4 ruggeri et al reported 10 years ago 4 cases of congenital absence of the ivc in 75 young patients with idiopathic dvt over a 5-year period , and estimated that 5% of young patients with dvt had an anomaly of the ivc.5 venous thrombosis is caused by the presence of isolated or combined risk factors . almost 150 years ago , the nineteenth century pathologist rudolf virchow described 3 critically important causes of venous thrombosis : venous damage , coagulation defect(s ) , and venous stasis.6 individuals with a congenital anomaly of the ivc are typically asymptomatic , and the anomaly is usually detected incidentally during radiological or abdominal procedures . congenital absence of the ivc is infrequently associated with thromboembolic events.5 patients who suffer from congenital anomalies of the ivc usually develop a compensatory circulation through the azygos veins or collateral abdominal veins in order to keep the venous return near normal levels.7 most reported cases of congenital anomalies of ivc cases have been linked to thrombophilia disorders.3,5,7 however , the true prevalence of thrombophilia in congenital anomalies of the ivc is unknown because the screening for thrombophilia in patients with an ivc anomaly was usually incomplete.3 anticoagulants , but not thrombolytic therapy , are usually prescribed for venous thrombosis , but the duration of the anticoagulant therapy is not well established . hence , anticoagulant therapy for an indefinite duration will probably be prescribed , unless vascular reconstructive surgery is done on the anomalous ivc . such surgery has been rarely reported , and its long - term outcome is undetermined.8 congenital anomalies of the ivc may cause recurrent dvt , especially in young individuals .

lipid apheresis is used to treat patients with severe hyperlipidemia by reducing low - density lipoprotein cholesterol ( ldl - c ) . this study examines the effect of apheresis on the lipid panel and cardiac event rates before and after apheresis . an electronic health record screen of ambulatory patients identified 11 active patients undergoing lipid apheresis with 10/11 carrying a diagnosis of fh . baseline demographics , pre- and postapheresis lipid levels , highest recorded ldl - c , cardiac events , current medications , and first apheresis treatment were recorded . patients completed a questionnaire and self - reported risk factors and interest in alternative treatment . there were significant reductions in mean total cholesterol ( 58.4% ) , ldl - c ( 71.9% ) , triglycerides ( 51% ) , high - density lipoprotein ( hdl ) cholesterol ( 9.3% ) , and non - hdl ( 68.2% ) values . thirty - four cardiac events were documented in 8 patients before apheresis , compared with 9 events in 5 patients after apheresis . our survey showed a high prevalence of statin intolerance ( 64% ) , with the majority ( 90% ) of participants indicating an interest in alternative treatment options . our results have shown that lipid apheresis primary effect is a marked reduction in ldl - c cholesterol levels and may reduce the recurrence of cardiac events . apheresis should be compared to the newer alternative treatment modalities in a randomized fashion due to patient interest in alternative options . background : agenesis of the inferior vena cava ( ivc ) as a cause of recurrent deep vein thrombosis ( dvt ) is uncommon.case:a 33-year - old male with no family history of thrombophilia , who had experienced multiple recurrent episodes of dvt over a 15-year period of unknown cause , was admitted into our hospital because of cellulitis in the right leg . computer tomography with contrast of the abdomen showed an absence of ivc.conclusion:congenital absence of the ivc could be a rare risk factor for idiopathic dvt , especially in young individuals .

an exponential rise in alzheimer 's disease ( ad ) prevalence rates is predicted to parallel the aging of baby boomers creating a potentially unsustainable economic burden to the healthcare system . delaying the onset or progression of ad , even modestly , by earlier pharmacological intervention could substantially reduce the economic and psychosocial impact of the illness [ 1 , 2 ] . unfortunately , many ad patients remain undiagnosed or go undetected until the later stages of disease . insights into the underlying pathological mechanisms involving beta - amyloid plaque deposition within the brain have led to the development of a host of antiamyloid agents that are in various stages of clinical investigation . there is now a scientific consensus that the pathological events in ad initiate decades before clinical symptoms become apparent , and if disease modification is realized in the coming decades , the need for improved methods of early detection prior to the overt clinical signs will be accentuated . traditionally , neuropsychological measures , particularly those that tap cognitive abilities subsumed by the hippocampal formation such as episodic memory , have shown usefulness in identifying cognitively normal elders who subsequently develop ad [ 4 , 5 ] . decrements in semantic memory and concept formation have been shown to occur nearly a decade before the development of ad . performance on visual - spatial and verbal memory measures in midlife have also been shown to predict later memory loss . however , individuals with very high premorbid intellectual abilities experiencing incipient cognitive decline may go undetected , and false positives are possible in individuals with a low level of intellectual abilities . also appropriate interpretation of extensive neuropsychological testing requires a high degree of expertise and training , which limits its use in routine clinical settings . the advancement of molecular imaging tracers that bind to amyloid , such as pittsburgh compound b ( pib ) or longer - lived probes ( e.g. , fddnp ) , offers a non - invasive in vivo method to detect and quantify brain amyloid deposition [ 8 , 9 ] . however , this approach for presymptomatic detection is economically impractical for routine use given the current costs and restrictions on medically necessary use . similarly , biomarkers including a142 and phosphorylated tau ( also implicated in ad pathology ) in cerebral spinal fluid ( csf ) can predict subsequent cognitive decline [ 10 , 11 ] , but lumbar puncture carries risks and is inconvenient for wide - scale use in cognitively impaired elderly subjects . blood - based biomarkers have more practical applicability for routine use and are likely to be more cost effective than both csf and imaging procedures . consequently , measurement of a140 and a142 in blood is increasingly being explored and shows potential in identifying individuals at the preclinical stage of ad [ 1214 ] . it has been reported that csf a levels are subject to high diurnal fluctuations with extremely high variability reported over 12 hours . over days and weeks , furthermore , serum contains more a than plasma , possibly due to the release of bound a during the clotting process . hence , serum a appears suitable for use in predicting mci / ad and optimal sensitivity , and specificity is probably achievable if combined with current diagnostic procedures , such as brief neuropsychological testing . in this study , we examined the usefulness of brief neuropsychological tests in combination with blood a140 and a142 as a predictive test for detecting mci / ad in at - risk older adults at a pre - symptomatic stage . such an approach will be more practical for clinical use and be germane in designing large - scale prevention trials . participants included a subset of subjects enrolled in the alzheimer 's disease anti - inflammatory prevention trial ( adapt ) . adapt was a randomized , placebo - controlled , multicenter primary prevention trial sponsored by the national institute on aging . subjects were randomly assigned to one of three groups : celecoxib ( 200 mg b.i.d . ) , naproxen sodium ( 220 mg b.i.d . ) , or placebo . full details of data collection , measurements , and study procedures are available at http://www.jhucct.com/adapt/manall43.pdf and described elsewhere . the inclusion criteria for adapt subjects were age of 70 or older at enrollment , a self - reported family history of ad - like dementia , and normal cognitive performance on a brief battery of neuropsychological tests . recruitment for adapt began in 2002 , and the study was completed in 2007 . in 2005 , the roskamp institute initiated a proteomic ancillary study ( f. crawford , pi ) involving blood draw from these subjects . the inclusion criteria for this ancillary study stipulated that each subject was an active adapt participant and had met all the adapt inclusion and exclusion criteria . a separate consent was also obtained from each subject who participated in the ancillary study . two hundred and fifteen subjects from the roskamp adapt cohort enrolled in the proteomic ancillary study . at the time of blood draw , subjects maintained cognitively normal status as determined by their performance on an annual cognitive assessment battery . blood was collected during the semi - annual followup visits , and the cognitive assessments were performed at the baseline visit and at the annual visits . the time from baseline cognitive testing to the diagnosis of mci / ad was 4.06 years ( 1.3 sd ) . timeframe from baseline cognitive testing to blood draw was 2.25 years ( 0.71 sd ) and from blood draw to diagnosis was 1.79 years ( 1.2 sd ) . the cognitive measures completed at baseline and annual followup included the modified mini - mental state examination ( 3ms ) ; the hopkins verbal learning test - revised ( hvlt - r ) ; digit span ( forward and backward ) from the wechsler adult intelligence scale - revised ( wais - r ) ; a generative verbal fluency test ( supermarket items ) ; the narratives from the rivermead behavioral memory test ( rbmt ) ; the brief visuospatial memory test - revised ( bvmt - r ) . the mini - mental state examination ( mmse ) was extracted from 3ms . alternate forms were utilized annually for the hvlt - r , rbmt , and bvmt - r on each subsequent annual visit . subjects also completed the 30-item geriatric depression scale and a self - rating scale of memory functions . collateral respondents completed the dementia severity rating scale ( dsrs ) . due to significant intercorrelations between these tests , analyses described below are limited to those baseline cognitive tests that were sensitive to early changes ( i.e. , verbal learning and memory ) associated with mci / ad or tests that were similar to those previously shown to be associated with a levels . normative data from the cache county study was used to develop the standardized cut - off scores utilized in adapt . individuals who scored below the cut scores on annual cognitive assessments underwent further dementia workup including physical and neurological examinations , laboratory studies ( i.e. , cbc , chemistry count , sedimentation rate , vitamin b12 and folic acid levels , thyroid test , and syphilis serological test ) , and neuroimaging ( i.e. , mri or ct ) , as applicable . a more comprehensive neuropsychological assessment was also administered by a neuropsychologist as part of the dementia work - up . this battery of tests consisted of the expanded consortium to establish a registry for alzheimer 's disease ( cerad ) battery ; logical memory i and ii of the wechsler memory scale - revised ; benton visual retention test ( benton ) ; a generative fluency test ( animals ) ; control oral word association test ( cowat ; cfl ) ; the trail making test ; symbol digit modalities test ( smdt ) ; shipley vocabulary . following completion of all components of the dementia work - up , a consensus team determined cognitive status using published diagnostic criteria . the diagnosis of ad was made using nincds - adrda and amnestic mild cognitive impairment ( mci ) using petersen criteria . all mci patients were considered to be amnestic mci , as they only had memory impairment , but maintained normal activities of daily living and overall had a well - preserved cognition in other cognitive domains . ample evidence indicates that amnestic mci patients may be in a transitional stage between normal aging and ad with 85% of these subjects converting to ad over a 7-year period . additional evidence comes from an imaging study which demonstrated that the pattern of brain atrophy in amnestic mci patients is typical of that observed in ad patients . it is then reasonable to combine these diagnoses in a single category , thus allowing a large enough numbers to supply statistical power . of the 215 subjects who gave blood for the ancillary study , two developed non - ad dementia , and of the remaining subject pool of 208 used in these analyses , 28 subjects met criteria for either ad ( n = 10 ) or mci ( n = 18 ) in the two years following blood draw . the serum a content was determined , as per manufacturer 's instructions , using the elisa kits for human a140 and a142 and the inter - assay cv , and the intraassay cv was reported to be 10% ( invitrogen , calif ) . dna was extracted from whole blood for apoe genotyping using pure gene kits ( gentra systems , calif ) , and apoe genotyping was performed using previously established methods , as described elsewhere . apoe genotypes were unavailable for 4 individuals , but these were included in the analyses . the data set was range checked , and prior to analyses , the dependent and independent variables were examined for missing data , outliers , and violations of the normalcy assumption . differences among groups on demographic variables , neuropsychological variables , and serum a140 levels were examined using either the student 's t - test or analyses , depending on the type of variable measurement . time - updated cox regression modeling was used to test whether neuropsychological test scores , a , or a combination of both can predict conversion to mci / ad in individuals who were cognitively normal at baseline . potential confounding variables shown to impact risk for cognitive decline included age , education , gender , apoe status , serum creatinine , triglycerides , presence of apoe 4 allele , and history of vascular disease as determined by treatment with statins or antihypertensive medication which were entered as covariates . the latter variables , coded dichotomously , have been previously shown to impact a levels . because previous analyses revealed a nonsignificant increase of ad risk with naproxen in this cohort , we also controlled for this effect . logistic regression modeling was employed to construct receiver operator curves ( roc ) to examine the predictive performance of neuropsychological measures from the baseline visit and serum a levels in diagnoses of mci / ad . roc curve comparisons were based on area under the curve ( auc ) , se , and the associated 95% confidence interval ( ci ) . we subsequently calculated sensitivity of the various models using the predicted probability of each subject by logistic regression modeling with specificity of at least eighty percent . post hoc power calculations using the g - power software for multivariate regression analyses utilized here suggest a power of nearly 100% at the alpha value 0.05 for the current sample size , total number of predictors , and the observed effect size . the mean age and education of the sample was 76.7 ( sd = 3.9 ) and 14.6 ( sd = 2.8 ) years , respectively . the majority of the sample was caucasian ( 98.1% ) , and 51.9% were male . despite the cohort 's self - report of enriched family history , less than one - third of the total sample ( 31.7% ) carried at least one apoe 4 allele , a frequency similar to the general population . comparisons on variables between subjects who remained cognitively normal and those who declined over the short follow - up period are reported in table 1 . although all subjects at enrollment performed within the normal limits based on the established cut - off scores , those that ultimately declined had generally poorer scores on the 3ms , mmse , and all memory measures . the two groups were also significantly different on serum a142 levels and a142/a140 ratios prior to diagnoses of mci / ad . only 23% of the cognitively normal individuals had serum a142 in the lowest quartile compared to the nearly 50% of the diagnostic group ( 44% of mci subjects and 50% of ad subjects ) . time - dependent cox regression analyses were performed to examine the relationship between these cognitive tests and a on the prediction of subsequent conversion to mci / ad . all neuropsychological analyses were adjusted for age , gender , and education , but no adjustment for the study medications was required as these were baseline scores . cox regression analyses show that the model using neuropsychological tests predicted mci / ad ( 2 log - likelihood = 206.51 , = 52.11 , df = 8 , p < .001 ) . significant individual neuropsychological measures were 3ms ( = 0.25 0.06 , wald = 17.78 , p < .001 ) ; generative verbal fluency ( = 0.12 0.04 , wald = 8.09 , p < .004 ) ; hvlt - r scores ( = 0.24 0.11 , wald = 4.58 p < .032 ) . cox regression analysis showed that a142 measured in the lowest two quartiles compared to the highest quartile was a significant individual predictor of conversion to mci / ad in this model ( 2 log - likelihood = 197.47 , = 38.41 , df = 15 , p < .001 ) . the regression analysis utilizing the a142/a140 ratio found similarly significant results ( 2 log - likelihood = 204.69 , = 36.10 , df = 14 , p < .001 ) with the lowest ratios being most predictive of subsequent conversion to mci / ad . the final full model , adjusting for confound and the study medications , included hvlt - r , fluency , 3ms , a142 levels , and a142 quartiles ( 2 log - likelihood = 166.25 , = 74.55 , df = 18 , p < .001 ) with fluency , 3ms , and a142 in the lowest two quartiles as significant individual predictors of mci / ad in the model . similar results were observed when a140 levels and a142 quartiles were substituted in this model with a142/a140 ratios ( 2 log - likelihood = 168.49 , = 72.90 , df = 17 , p < .001 ) . baseline values for the 3ms , hvlt - r , and generative verbal fluency scores were subtracted from those obtained at the 12-month repeat testing to determine if changes in these measures differ by a142 and a142/a140 ratios . in unadjusted analyses , among subjects who converted to mci / ad , the greatest decline for hvlt - r was observed among individuals with the lowest quartile of a142 ( 1.17 , 2.33 sd ) and a142/a140 ratios ( 0.75 , 2.63 sd ) where individuals in the highest quartile of a142 ( 1.33 , 1.86 sd ) and a142/a140 ratios improved by nearly one point ( 0.6 1.82 sd ) . however , these differences were not statistically significant ( p > .05 ) . for the 3ms scores , among subjects who converted to mci / ad , those with a142 in the lowest quartile declined ( 1.83 1.28 sd ) as compared to the highest quartile ( 4.83 1.35 sd ) , and this difference was statistically significant ( f = 3.42 , p = .033 ) . for mci / ad subjects with the lowest quartile of the a142/a140 ratios , the 3ms values remained ultimately unchanged ( 0.16 1.20 sd ) , while the scores improved among those with the highest quartile of the a142/a140 ratios ( 4.33 1.20 sd ) , and these differences were also statistically significant ( f = 3.10 , p = .046 ) . for generative verbal fluency test , a decline was noted in both the lowest quartile ( 4.17 1.40 sd ) and the highest quartile ( 1.17 2.13 sd ) of a142 , and these differences were marginally significant ( f = 2.63 , p = .073 ) . for a142/a140 ratios , a similar pattern was observed , but this difference was not statistically significant . among individuals who remained cognitively normal , while a similar pattern was observed , those with lowest quartile of a142 and a142/a140 ratios had a larger decline than those with the highest quartile for each hvlt - r ( 0.28 0.27 sd versus . 0.14 0.33 sd , respectively . ) and 3ms ( 1.02 0.51 sd versus 0.39 0.44 sd ) . however , due to the small magnitude of the change in these scores , these differences were not statistically significant . no such change was observed for the generative verbal fluency test ( data not shown ) . examination of sensitivity and specificity using roc analysis revealed the auc for neuropsychological testing with age , education , and gender as covariates was 0.83 ( 95% ci [ 0.750.91 ] , p < .001 ) . for a142 ( adjusted for presence of apoe 4 allele , vascular risk factors , and associated medications ) , the auc was 0.79 ( 95% ci [ 0.700.88 ] , p < .001 ) . when neuropsychological testing ( 3ms , hvlt - r , and generative verbal fluency ) and a142 were combined , the auc was increased to 0.91 ( 95% ci [ 0.860.95 ] , p < .001 ) . for the adjusted ( as above ) a142/a140 ratios alone , .001 ) , and when combined with the neuropsychological measures , auc was 0.91 ( 95%ci [ 0.870.96 ] , p < .001 ) . optimal sensitivities with specificity of at least 80% predicted probabilities are shown in table 2 . the highest sensitivity and specificity was achieved using a combination of cognitive scores and a142/a140 ratio , but this finding was driven by a142 . the pathogenesis of ad is initiated before the clinical symptoms of cognitive impairment and functional decline become apparent in its victims . a simple and pragmatic method for identifying older adults at an increased risk for mci / ad who may benefit from targeted prevention is therefore of importance in reducing the burden of ad . the combination of brief neuropsychological tests along with blood - based biomarkers of ad represents a reasonable approach with a potential for wide - scale use . our findings here provide support for this notion and demonstrate that early prediction of risk for developing mci / ad may be feasible via a combination of brief neuropsychological tests and biomarkers in an at - risk cohort . in this subcohort from adapt , measures of global cognitive function ( 3ms ) , episodic memory ( hvlt - r trial 4 ) , language fluency , and serum a142/a140 ratio achieved an excellent accuracy of 91% . furthermore , sensitivity with specificity of at least 80% for the combined measures was superior to neuropsychological measures or to serum a levels alone . we have recently shown that a levels alone can predict mci / ad , but a levels are influenced by vascular disease and associated medications and require adjustment to observe the full impact of a in predictive modeling . we have also shown that in subjects diagnosed with ad , there is an association between measures of language tests of fluency and object naming and a140 and that memory performance is associated with serum a142 . an association between serum a140 and cognitive measures of memory and language has also been reported in cognitively normal older adults . high baseline a142 and a140 with stable a142 over time is shown to be associated with diminishing cognition . more recently , yaffe and colleagues demonstrated that low a142/a140 ratios predict cognitive decline over 9 years . in our study , we demonstrate that low a142 and a142/a140 ratios are associated with cognitive decline even within one year . this is extremely valuable from the clinical perspective , as the ability to identify at - risk individuals within a year prior to the onset can significantly improve the quality of care and the recruitment strategy for prevention trials by redirecting those individuals who may not benefit from preventive therapies towards more suitable clinical intervention . this is demonstrated by recent adapt findings , which suggest that individuals with low baseline cognitive scores converted soon after the trial initiated and that neither naproxen nor celecoxib intervention was beneficial to these individuals . collectively , these findings suggest that combining cognitive tests with blood a may be useful for predicting future mci / ad , which to date has not been explored , particularly as either a or the cognitive tests alone may not have the desired sensitivity or specificity for prediction of future mci / ad . this current work presented here provides evidence that the combination of brief neuropsychological tests and blood a has potential utility in predicting mci / ad at least 2 to 4 years prior to the clinical classification of mci or diagnosis of ad . in addition , our findings also demonstrate the importance of accounting for factors such as apoe , vascular risk factors , and medications when using a in predicting mci / ad . although at present no studies have reported sensitivity and specificity of csf a142 in predicting mci / ad conversion from normal cognition , a large multicenter study has shown that csf a142 predicts transition from mci to ad , while tau alone achieved a high sensitivity ( 83% ) with acceptable specificity ( 72% ) . it is interesting to note that our findings using blood and cognitive tests , a far less invasive method , resulted in higher sensitivities and specificities for predicting cognitive decline in at - risk cognitively normal older adults . despite the limitation that blood sampling was not conducted at the same time point as the cognitive testing , our data provide strong support for further evaluation of this approach , particularly as we have not seen significant fluctuations in a levels over a one - year period ( pers . our study provides support that blood - based a levels may have diagnostic utility when combined with neuropsychological measures . this proposed method warrants further investigation to determine its practical applicability in specialized clinic setting by allied health personal and in routine primary care clinics .

we examined the usefulness of brief neuropsychological tests and serum a as a predictive test for detecting mci / ad in older adults . serum a levels were measured from 208 subjects who were cognitively normal at enrollment and blood draw . twenty - eight of the subjects subsequently developed mci ( n = 18 ) or ad ( n = 10 ) over the follow - up period . baseline measures of global cognition , memory , language fluency , and serum a142 and the ratio of serum a142/a140 were significant predictors for future mci / ad using cox regression with demographic variables , apoe 4 , vascular risk factors , and specific medication as covariates . an optimal sensitivity of 85.2% and specificity of 86.5% for predicting mci / ad was achieved using roc analyses . brief neuropsychological tests and measurements of a142 obtained via blood warrants further study as a practical and cost effective method for wide - scale screening for identifying older adults who may be at - risk for pathological cognitive decline . we compared serum polychlorinated dibenzo - p - dioxins ( pcdds ) and polychlorinated dibenzofurans ( pcdfs ) among residents of two homes to levels among age- and sex - matched comparison subjects . the residents of the two homes consumed contaminated eggs and beef from animals raised at the homes . the animals had greater soil contact than those raised with conventional commercial husbandry practices . the comparison subjects were from a similar rural area , but did not consume home - produced beef and eggs . serum levels of 2,3,7 , 8-substituted tetra- , penta- , and hexacdds and penta- , hexa- , and heptacdfs were increased between 2- and 6-fold in residents from one home ; contaminated eggs and beef were consumed by residents for 2 - 15 years . elevations were less for those in the other index home , where only home - produced eggs were consumed for 2 years ; a 3-fold elevation of 1,2,3,7,8,9-hexacdd as compared to controls was most apparent . very strong bivariate correlations among all of the 2,3,7 , 8 penta- and hexacdds / cdfs were observed . the elevations observed verify that pcdd / pcdf - contaminated food contributed to the body burden of these compounds . the blood levels among the highest exposed participants are generally higher than those observed in other studies of u.s . contaminated - fish consumers and higher than average adipose tissue levels observed in u.s . urban populations . there are sufficient animal toxicologic and human epidemiologic data to recommend that exposures be reduced . in the study area , pentachlorophenol and pentachlorophenol incineration sources have been identified , and the animal contamination and blood elevations probably reflect these sources . soil reference values and site - specific risk assessments should include estimates of exposures to contamination in home - produced animal products . such estimates can be verified with limited pcdd / pcdf sampling of animals and humans.imagesfigure 1figure 2

the wnt/-catenin pathway has been implicated in the development and progression of melanoma and a wide range of cancer types , including colorectal cancer , breast cancer , esophageal carcinoma , and liver cancer [ 13 ] . under normal conditions , increases in expression and binding of certain wnt ligands to frizzled receptor or mutations in specific components of the -catenin degradation assembly deactivate this regulatory mechanism . nuclear -catenin stimulates transcription of a large number of tcf/-catenin responsive genes that include cyclin d1 , c - myc [ 5 , 6 ] , and the melanocyte - specific gene , microphthalmia - associated transcription factor mitf - m . thus , accumulation of nuclear -catenin as observed in several cancer types is considered a marker of canonical wnt/-catenin pathway deregulation and unfavorable prognosis [ 3 , 8 ] . previous studies have reported an association between nuclear -catenin accumulation and melanoma progression and suggested nuclear -catenin to be a marker of poor prognosis [ 1 , 7 ] . however , recent studies have shown that contrary to breast and colon cancer , metastatic progression of melanoma is associated with decreases in nuclear and cytoplasmic -catenin expression [ 9 , 10 ] . moreover , clinical , genetic , and histological studies suggest that nuclear and cytoplasmic -catenin may be used as biomarkers of good prognosis in melanoma [ 1114 ] . recently , hhr6 , a human homologue of the yeast rad6 gene and a principal component of the postreplication dna repair pathway , has been identified as an important regulator of canonical wnt/-catenin signaling [ 15 , 16 ] . hhr6 , referred hereafter as rad6 , stabilizes -catenin by polyubiquitin modifications that render -catenin resistant to 26s proteasomal degradation . furthermore , rad6 is a transcriptional target of -catenin , thus revealing a positive feedback loop between -catenin - mediated activation of rad6 gene expression and rad6-induced -catenin stabilization . rad6 expression is low in normal breast tissues ; however , increases in rad6 protein expression are detected in hyperplastic , ductal carcinoma in situ ( dcis ) and invasive breast carcinomas . we have previously demonstrated a role for rad6 in breast cancer progression through its regulatory effect on the canonical wnt/-catenin pathway . since the decrease / loss of nuclear -catenin [ 9 , 10 ] , rather than increases as in breast cancer , is linked to melanoma progression , it is not known whether rad6 and -catenin work in concert to promote melanoma pathogenesis . furthermore , rad6 expression in the skin has not been investigated , and there are no data on the role of rad6 in the pathogenesis of benign ( nevi ) and malignant ( melanoma ) melanocytic lesions . it is important to address this gap in knowledge because of the unmet medical need for new effective antimelanoma therapies and because rad6 and -catenin have been identified as therapeutic targets [ 19 , 20 ] . in this study , we examined rad6 and -catenin expressions in serial sections of nevi , primary , and metastatic melanomas to determine their potential roles in melanoma development and metastatic progression . our data suggest that membranous relocation of -catenin and upregulation of rad6 are independent markers of melanoma development and progression . we also offer a hypothesis that explains the role of membranous -catenin relocation and decreasing cytoplasmic -catenin in melanoma development , a phenomenon that has been linked to unfavorable prognosis [ 9 , 21 , 22 ] . cases were retrieved from the files of the pinkus dermatopathology laboratory ( pdl ) , a private dermatopathology laboratory located in monroe , mi . preserved paraffin - embedded tissue specimens collected for each case were assigned an accession code that excluded patient identifier information . nevus and primary melanoma cases were selected for study using random numbers generated by a uniform random number generator ( stata / mp 13.1 ) . the study groups consisted of 30 cases of melanocytic nevi , 29 cases of primary cutaneous melanoma , and 29 cases of metastatic cutaneous melanoma . the study includes all metastatic cutaneous melanoma samples that were archived between 2010 and 2012 . the number of cases for each nevus and primary melanoma subtype was determined to reflect the lesion 's relative representation in cases obtained at the pdl during the above period . atypical nevi were diagnosed using criteria originally proposed by clark and lesion architecture as reviewed by roth et al . . primary antibodies used in the study are as follows : ( i ) anti--catenin ( is702 ) was purchased from dako ( glostrup , denmark ) and used in an undiluted form ; and ( ii ) anti - rad6 ( ab31917 ) was purchased from abcam ( cambridge , ma ) and used at a 1 : 500 dilution . in humans , the yeast homologous rad6 gene is duplicated and the proteins encoded by the two genes hhr6a ( or rad6a ) and hhr6b ( rad6b ) from chromosomes xq24-q25 and 5q23-q31 , respectively , share 95% identical amino acid residues . neither ab31917 , our own rad6 antibody , nor any other commercially available anti - rad6 antibody is currently able to distinguish between rad6a and rad6b proteins . therefore , rather than referring as rad6a or rad6b , we refer to the protein detected by the antibody as rad6 . briefly , five - micrometer sections were deparaffinized in xylene and rehydrated in graded ethanol . for antigen retrieval , sections were microwaved in citrate buffer ph 6.0 ( biogenex , san ramon , ca , usa ) for 12 min at 95c and cooled for 30 min prior to immunostaining . sections were incubated with 3% hydrogen peroxide for 15 min , followed by incubation with primary antibody for 60 min . an automated immunostainer ( i6000 ; biogenex ) was utilized for subsequent incubation steps : sections were incubated in multilink biotinylated anti - igg for 20 min , horseradish peroxidase conjugated secondary antibody for 20 min , followed by development with 3-amino-9-ethyl - carbazole for 10 min ( biogenex ) . all incubation steps were performed at room temperature , and sections were washed with tris - buffered saline between incubations . lung and colon cancer tissues were included as positive controls for immunostaining with anti--catenin antibody , and breast cancer tissues were included as positive controls for staining with anti - rad6 antibody . stained sections were independently enumerated by two coauthors ( d. r. mehregan and m. campbell ) , who were blinded to patient medical records for each case . blinded enumeration was performed under light microscopy at 400x magnification , and an ocular grid consisting of a simple square lattice of 100 test points was utilized to count the number of positively and negatively stained melanomas or nevus cells for each section . per section , the total number of positively and negatively stained cells was counted for each of three sequential horizontal fields . the mean value of the three fields was used to estimate the relative density of cells in a specimen . to increase assessment accuracy , all positively and negatively stained melanomas or nevus cells in a visual field when independent readings for positively stained cells differed by 20% for a given section , both evaluators reviewed the section together to establish a consensus reading . a specimen was considered negative if less than 4% of the cells were immunostained for rad6 or -catenin . a tumor was considered to be stained with high intensity if > 50% of the cells in a specimen expressed rad6 or -catenin , similar to the criteria used by mineta et al . . kruskal - wallis tests were used to compare groups on the basis of continuous variables such as age and percent positive cells . chi - square tests for differences in proportions were used to compare groups on the basis of categorical variables such as gender and -catenin localization . spearman 's rank correlation was used to assess the pairwise association between age and percent of rad6 positive and -catenin positive cells . multinomial logistic regression was used to assess the simultaneous association of rad6 and age with diagnostic group . adjustments were made for multiple comparisons using wilcoxon rank sum tests with bonferroni correction for pairwise comparisons . our analysis included 30 individuals diagnosed with nevi , 29 with primary melanoma , and 29 with metastatic melanoma ( table 1 ) . these groups differed marginally with respect to gender ( p = 0.08 ) and significantly in age ( p = 0.0001 ) . significant age differences were observed between individuals with nevi and those with either primary melanomas ( p = 0.02 ) or metastatic melanomas ( p = 0.0001 ) . in contrast , no age difference is observed between individuals with primary and metastatic melanomas ( p = 0.27 ) . significant differences in age were also observed between groups defined by -catenin localization ( p = 0.007 ) . individuals with -catenin localized in the cytoplasm were significantly younger than individuals with -catenin localization at the cell membrane ( p = 0.02 ) and marginally younger than individuals with -catenin localization at both the cytoplasm and the cell membrane ( p = 0.05 ) . when age was categorized as < 50 , 5060 , and > 60 years , there were statistically significant differences in rad6 expression between the groups ( p = 0.0008 ) , although there is substantial variability ( table 2 ) . median rad6 is greater in the group of people older than 60 years compared to the 5060 years old group ( p = 0.04 ) and < 50 years old group ( p = 0.001 ) . a 20% discrepancy of positively stained cells between the two evaluators was observed in fewer than 5% of the cases , and these cases were evaluated together to establish a consensus reading . melanoma development and progression were not associated with significant changes in percentage of specimens expressing -catenin . -catenin staining was observed in 97% of nevi and in all primary and metastatic melanomas . also , the percentages of nevi ( 93% ) , primary melanoma ( 97% ) , and metastatic melanoma ( 93% ) that expressed -catenin in more than 50% of the cells did not differ significantly ( figures 1 , 2 , and 3 ) . however , significant differences were observed between the percentages of nevi ( 59% ) , primary melanoma ( 90% ) , and metastatic melanoma ( 56% ) that expressed -catenin in more than 90% of the cells ( p = 0.02 ; figure 2 ) . these differences were greatly impacted by the percentage of primary melanomas ( 48% ) that expressed -catenin in 100% of the cells , which was approximately twofold higher than the percentages of nevi ( 21% ) or metastatic melanoma ( 26% ) ( data not shown ) . none of the nevi or melanomas expressed -catenin in the nucleus ( figure 1 ) . the percentage of tumors that expressed membranous -catenin increased dramatically from nevi ( 10% ) to primary and metastatic melanomas ( 83% and 93% , resp . ; concurrently with this increase , the percentage of tumors that expressed cytoplasmic -catenin decreased from nevi ( 90% ) to primary and metastatic melanomas ( 45% and 38% , resp . ; in contrast , no significant differences were observed between the percentages of primary and metastatic melanomas that expressed -catenin at either the membrane ( 83% and 93% , resp . ; p = 0.289 ) or the cytoplasm ( 45% and 38% , resp . ; p = 0.633 ; figure 4 ) . while all four nevi types ( junctional , intradermal , compound , and atypical ) expressed -catenin in the cytoplasm , only junctional and atypical nevi expressed -catenin at the plasma membrane . as opposed to nevi types , the three primary melanoma types examined ( superficial spreading , nodular , and lentigo maligna ) did not differ in -catenin localization as -catenin was localized at the plasma membrane , in the cytoplasm , or in both ( figure 4 ) . conversely , all primary melanomas ( 100% ) and the majority of metastatic melanomas ( 96% ) exhibited greater than 50% rad6 expression . the increase in tumor populations expressing rad6 from 37% of nevi to 100% of primary and metastatic melanomas was significant ( p = 0.0001 ; figures 1 , 2 , and 3 ) . melanoma progression from primary to metastatic disease was not associated with changes in the ( i ) percentage of melanomas expressing rad6 ( 100% of primary and metastatic melanomas ) , or ( ii ) percentage of melanomas expressing rad6 in more than 50% of the tumor cells ( 100% and 96% of primary and metastatic melanomas , resp . ) . the increase in proportion of tumor populations , expressing rad6 in more than 50% of the cells in primary melanoma ( 67% ) versus metastatic melanoma ( 79% ) was not significant ( p = 0.37 ; figure 2 ) . this study was not designed to test whether the distribution of the tumor cells positive for rad6 is the same between the subtypes of nevi . however , the percentages of benign tumors that lacked rad6 were similar between atypical nevi ( 62% ) and the group of other three nevi types ( 59% ) . interestingly , only one of the 30 nevi ( atypical nevus , 3% ) expressed rad6 in > 80% of the cells , and none of the primary and metastatic melanomas expressed rad6 in < 40% of the cells ( figure 2 ) . these results prompted us to examine whether rad6 expression can serve as a marker for histological diagnosis of melanoma . using a multiple logistic regression model , we found that the strength of rad6 expression is a strong predictor of melanoma ( p < 0.001 ) even when age group ( p = 0.65 ) and gender ( p = 0.24 ) are included in the model . the model predicts that every 1% increase in rad6 expression results in a 9% increase in the probability that a lesion is melanoma . if we assume that a predicted probability of > 0.5 indicates melanoma , the model with only rad6 has sensitivity of 93% and specificity of 80% . these results are very encouraging ; however , they need to be validated in a larger study . the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin in more than 50% of the cells , whereas only 27% of the same population of nevi expressed rad6 ( figure 2 ) . -catenin and rad6 expressions in nevi were not significantly correlated ( r = 0.06 ; p = 0.77 ) . there is a 2.7-fold difference in the percentage of primary melanomas ( 100% ) expressing rad6 compared to nevi ( 37% ) and virtually no difference in -catenin expression between primary and metastatic melanomas ( 100% ) . accordingly , rad6 and -catenin expressions in primary melanoma were not correlated ( r < 0.001 , p > 0.99 ) . a significant correlation between rad6 and -catenin positive cells however , this association diminished ( r = 0.40 , p = 0.05 ) following the exclusion of two observations which are disproportionally influential ( one with < 50% positive rad6 and one with < 50% positive -catenin ) . this is the first study to characterize rad6 expression in cutaneous benign and malignant melanocytic tumors . in this study , we examined the association between rad6 and -catenin expressions in benign and malignant melanocytic tumors to determine whether rad6 works in concert with -catenin to influence melanoma development and progression . rad6 and -catenin positively regulate each other in breast cancer [ 15 , 18 ] . however , while -catenin has been implicated in the pathogenesis of melanoma and other cancer types , data about the role of rad6 in cancer pathogenesis are mostly limited to breast cancer . therefore , we hypothesized that comparison of rad6 and -catenin expressions in the same nevi and melanoma tumors would help determine whether these two signals collaborate to promote melanoma development and progression as they do in breast cancer [ 15 , 28 ] . accumulation of nuclear and cytoplasmic -catenin has been implicated in driving the development and progression of several cancer types ( e.g. , colon and ovarian cancers ) [ 2931 ] . however , our results show that the expression levels of -catenin do not contribute to melanoma initiation and progression since no difference in -catenin levels was found between nevi , primary melanoma , and metastatic melanoma ( 93%97% of all samples expressed -catenin in > 50% of the tumor cells ) . the high expression levels of -catenin are in line with the crucial role of -catenin in differentiation and proliferation of both normal melanocytes and metastatic melanoma cells . also , our findings are in agreement with previous reports of positive -catenin staining in nevi ( 100% ) and primary melanoma ( 95% , 94% ) but are higher than reported in metastatic melanoma ( 75% , 68% ) [ 9 , 21 ] . the variation in expression of -catenin levels in metastatic melanomas between the studies while we studied only melanoma metastases to the skin , other studies either obtained 58% of their specimens from lymph nodes , tonsil , and liver or did not identify the anatomical site of their metastases [ 9 , 21 ] . furthermore , different anatomical sites may regulate dissimilar antigen expressions in metastases that originate from the same primary tumor in the same patient [ 33 , 34 ] . previous studies have shown higher percentages of nuclear -catenin in nevi than in melanoma ( 84% versus 33% , and 44% versus 15% ) [ 9 , 22 ] . those observations provided the basis for the currently held concept that loss of nuclear and cytoplasmic -catenin suggest poor prognosis and decreased overall survival of melanoma patients [ 12 , 22 ] . in light of these data , the absence of nuclear -catenin in all the nevi and melanomas analyzed in our study was surprising . usage of different anti--catenin antibodies may explain in part the discrepancy in nuclear -catenin expression observed between the studies . however , our results are consistent with the lack of nuclear -catenin reported in four studies which comprised 57 nevi , more than 55 primary melanomas , and 20 metastatic melanomas [ 21 , 3537 ] . moreover , nuclear -catenin was not found in either the nevus portion or the melanoma portion of 15 cutaneous lesions and was absent in additional 42 primary melanomas . in another study of 70 primary melanomas , nuclear -catenin was reported in only 6.4% of the melanomas . finally , in a study of 230 primary and metastatic melanomas , nuclear -catenin was reported in only 13 cases ( 5.6% ) and therefore those cases were excluded from analysis . taken together , the absence or negligible amount of nuclear -catenin detected in the aforementioned studies as well as ours suggests possible extranuclear roles for -catenin in nevi and melanoma . this notion is supported by a role for cytoplasmic -catenin to execute functions that do not require nuclear translocation ( e.g. , activation of map kinase p38 and nf - kb ) [ 37 , 40 ] . a major finding of this study is the association between melanoma development and intracellular redistribution of -catenin . the percentage of cases that expressed -catenin on the cell membrane increased dramatically from 10% in nevi to 83% and 93% in primary and metastatic melanomas , respectively . concurrently , the percentage of cases that expressed cytoplasmic -catenin decreased from 90% in nevi to 45% and 39% in primary and metastatic melanomas , respectively ( figure 4 ) . we hypothesize that the relocation of -catenin from the cytoplasm to the cell membrane may serve as a deactivating mechanism of canonical wnt/-catenin signaling and that the resulting reduction in cytoplasmic -catenin level may contribute to the malignant transformation of melanocytic nevi . the proposed hypothesis is supported by the following observations : ( i ) as in our study , bachmann et al . also reported an association between nevus to melanoma development and relocation of -catenin to the cell membrane . nevertheless , the authors of that study did not offer an explanation for their observation ; ( ii ) our analysis of the data of kagashita et al . showed -catenin decrease in the cytoplasm and increase at the cell membrane and that these changes in -catenin distribution corresponded with the malignant transition of nevi ; ( iii ) wnt4 signal has been identified as a mechanism that can drive -catenin relocation from cytoplasm to cell membrane ; and ( iv ) -catenin relocation from cytoplasm to cell membrane has been reported to block -catenin signaling in a human embryonic kidney ( hek293 ) cell line . of note , this hypothesis can explain how despite the abundant -catenin expression in melanoma [ 1 , 7 ] , cytoplasmic -catenin is selectively decreased , a phenomenon that has been associated with unfavorable melanoma prognosis [ 9 , 21 , 22 ] . our current efforts are directed towards determining if the increases in membranous -catenin observed in primary and metastatic melanomas result from relocation of existing molecules in the cytoplasm or deposition of newly generated -catenin at the membranous site . rad6 has been implicated in early breast cancer development since an increase in rad6 levels is observed in adenosis and benign hyperplasias as compared to normal tissue . in contrast , our findings do not support rad6 to play a similar role in nevus formation as in benign breast neoplasia , since 63% of the nevi were negative for rad6 . rad6 has also been implicated in breast cancer progression because rad6 levels increase with progression from ductal carcinoma in situ to invasive primary carcinoma and metastatic cancer [ 15 , 28 ] . in accordance with the upregulation of rad6 in early stages of breast cancer development as compared to benign hyperplasia [ 15 , 17 ] , we observed a striking increase in rad6 expression in primary melanoma when compared to nevi . while all primary melanomas displayed strong rad6 staining ( > 50% of the tumor cells ) , rad6 was negative in 63% of the nevi . these findings suggest that rad6 may play a role in malignant transformation of nevi as in breast cancer . progression of melanoma from primary to metastatic disease was not significantly associated with further changes in the percentage of tumors expressing rad6 or rad expression intensity as > 50% of tumor cells stained positively in 100% and 96% of primary melanomas and metastatic melanomas , respectively . these findings suggest that rad6 may play a sustained role in melanoma metastasis as it does in melanoma development . in benign and malignant breast tumors , rad6 stabilizes -catenin , and , in turn , -catenin positively upregulates rad6 transcription [ 1517 ] . however , this direct positive correlation between -catenin and rad6 expression does not appear to be conserved in melanoma as the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin compared to only 27% of nevi that expressed rad6 in more than 50% of their cells ( figure 2 ) . these observations suggest that the high -catenin expression in nevi is likely driven by regulators other than rad6 . at first glance , it would appear that -catenin and rad6 expressions are correlated in primary melanoma because these proteins were coexpressed in approximately all primary melanomas . also , these findings correspond to the 80% correlation between rad6 and -catenin expressions in primary breast cancer . however , it is unlikely that the high rad6 expression in primary melanoma is driven by the concurrent high -catenin expression , because rad6 expression is low in nevi despite the presence of high cytoplasmic -catenin expression that is comparable to primary melanoma . this notion is confirmed by lack of statistical correlation between rad6 and -catenin expressions in primary melanoma . -catenin is not the only activator of rad6 ; for instance , rad6 is activated by nerve growth factor in nervous tissue . therefore , it is conceivable that , in primary melanoma , rad6 expression is regulated by yet unidentified activators . we also demonstrated that further progression of melanoma from primary to metastatic disease is not associated with a correlation between the -catenin and rad6 expressions . taken together , our study does not support a direct positive interaction between -catenin and rad6 in either benign or malignant melanocytic tumors . we characterized for the first time rad6 expression in cutaneous benign and malignant melanocytic tumors . we are showing a striking upregulation of rad6 from a negative expression in most benign melanocytic tumors to 100% of primary and metastatic melanomas . these findings strongly suggest a role for rad6 in the development of primary melanoma and metastatic disease . we show that in contrast to rad6 , -catenin is expressed in more than 50% of the tumor cells in almost all nevi and melanoma tumors . taken together , in contrast to the rad6 -catenin positive relationship in breast cancer [ 1517 ] , our study does not support a similar positive interaction between -catenin and rad6 in benign or malignant melanocytic tumors . finally , our findings suggest a role for the cytoplasmic to membrane translocation of -catenin in the development of primary melanoma . future studies will determine whether newly generated -catenin at the membranous site coincide with -catenin translocation from the cytoplasm . the guanine heterocyclic ring is rich in chemical reactivity toward oxidants and adduct - forming species . in dna , 2-deoxyguanosine ( dg , mass = m ) is the chief site for base oxidation due to its low redox potential leading to many products that are characterized by their mass signatures . the principal products characterized with one - electron oxidants in aqueous solutions can be grouped based on the site of reaction on the radical intermediate resulting from one - electron oxidation of dg . the 2-deoxyribonucleoside products arising from initial reactivity at c5 of dg include a four - electron oxidation product , an imidazolone ( diz , m-39 ) that hydrolyzes to an oxazolone ( dz , m-21 ) , and the two - electron oxidation product 5-carboxamido-5-formamido-2-iminohydantoin ( d2ih , m+34 ; scheme 1 ) . when the initial reaction occurs at c8 of dg , either 2,6-diamino-4-hydroxy-5-formamidopyrimidine ( fapy - dg , m + 18 ) is observed under reducing conditions or 8-oxo-7,8-dihydroguanine ( dog , m+16 ) is observed under oxidizing conditions , in which the latter compound is a key marker for monitoring oxidative stress in cells ( scheme 1 ) . the two - electron oxidation product dog is stable but is highly susceptible to further oxidation leading to two hydantoin compounds . the yield for the thermodynamically preferred product spiroiminodihydantoin ( dsp , m + 32 ) is greatest in nucleoside reactions at ph > 6 or in unencumbered reaction contexts ( i.e. , single - stranded dna or g - quadruplexes ) , while the yield of 5-guanidinohydantoin ( dgh , m + 6 ) is greatest in nucleoside reactions at ph < 6 or in sterically demanding duplex contexts ( scheme 1 ) . the hydantoins are also readily formed from direct four - electron oxidation with o2 . this list of products represents those that are consistently observed from many oxidant systems conducted by several laboratories ; however , other compounds have been reported in lower yield . in the cellular context , oxidation reactions with dg can have other nucleophilic participants such as amines or phenols . in most cases , when amines or phenols participate in the reaction , products are observed with similar backbone structures as characterized with water serving as the nucleophile . for example , when dg is oxidized in the presence of lysine , products retaining the dg heterocycle and those with a core similar to dgh or dsp have been observed . the adducts characterized by our laboratory and others show lysine competing with water for sites of covalent bond formation , where a single lysine is observed at c8 analogous to dog , at either c5 or c8 of a spirocyclic core similar to dsp , and last a bis - adduct at c5 and c8 with a spirocyclic ring structure has been identified ( figure 1 ) . the lysine adducts are all stable allowing their quantification and characterization . as a last example , the polyamine spermine has been adducted to dg and dog under oxidative conditions . in the dg oxidation studies , a spermine adduct at c8 is observed retaining the dg heterocyclic core , while dog oxidations yield an adduct at c5 that does not undergo acyl migration to create a spirocycle , but instead the polyamine can generate an unstable hemiaminal intermediate that decomposes leaving a ribosylurea lesion at the adduct formation site . moreover , the facile formation of spermine adducts to dog oxidation intermediates has been harnessed for quantification of dog from dna samples . in contrast to amines , phenols ( i.e. , tyrosine ) are more redox active than amines and better able to compete with dg for oxidant during reactions . the products observed when phenols participate with dg during oxidation have more variability in their structures compared to amine adducts . these model studies aid in understanding the chemical nature of dna protein cross - links that are detrimental to cellular processes . in the current work , oxidations that allowed dg to react with nh3 were conducted while the full product and diastereomer distributions were monitored . after careful characterization of the nucleoside reactions , similar oxidations were conducted in the single - stranded and duplex oligodeoxynucleotide ( odn ) contexts . the purified ammonia adducts were also studied with respect to their decomposition pathways leading to end products that have not been previously determined . lastly , polymerase insertion studies were conducted to test the hypothesis that amine adducts might have altered base - pairing preferences ; every substitution of an oxo group on a base for an amino group converts a hydrogen bond acceptor into a potential hydrogen bond donor . these results provide a fundamental understanding of the stability and base - pairing properties of amine adducts to dg that result during oxidation , which may occur in vivo due to the high concentration of nucleophilic amines in the vicinity of the genome . adducts observed when dg and lysine are allowed to react in the presence of oxidant . the unencumbered nucleoside dg ( 1 mm ) was chosen for the initial oxidation reactions where nh4cl ( 20 mm ) provided the source of nucleophilic nh3 . all reactions were conducted with 75 mm napi buffer at ph 7.4 at 22 c . the oxidants chosen include the photooxidants riboflavin and rose bengal and the one - electron oxidant na2ircl6 . reactions were conducted in triplicate to achieve 70% conversion to products in each reaction , and the products ( scheme 2 ) were analyzed by a dual hplc method . the first round of hplc analysis utilized a reversed - phase column to identify dog ( m+16 ) and 8-amino - dg ( m+15 ) , while all other products eluted in the void volume . the void volume from the previous run was collected and analyzed on a hypercarb hplc column that allowed analysis of the hydantoins , ammonia adducts , and dz . however , diz hydrolyzes to dz that was detected on the hypercarb column ; thus , formation of diz is inferred from quantification of dz . moreover , the hypercarb hplc column also allowed separation and quantification of all spirocyclic diastereomeric pairs of products . the dgh diastereomers are interconvertible , and thus , their diastereomer ratios are not reported ( see the supporting information for complete experimental details ) . lastly , a test reaction with na2ircl6 was conducted in which half was directly analyzed by the hypercarb hplc column and the other half was analyzed by the dual hplc method outlined above . the product distributions observed from these comparative studies were within 3% of one another . the photooxidant riboflavin led to the largest number of different products and was used to obtain suitable amounts of material for characterization . esi - ms ( figure s1 , supporting information ) , and the adducts observed that did not involve participation of nh3 during product formation included dz ( m - 21 ) , dog ( m + 16 ) , dgh ( m + 6 ) , and dsp ( m + 32 , scheme 2 ) . confirmation of their structures was achieved by esi - ms / ms fragmentation of the free bases while monitoring the daughter fragments . the esi - ms / ms experiments were conducted on hplc purified nucleosides in which the n - glycosyl bond was cleaved in the ionization source to liberate free bases that were further fragmented in the cid chamber to generate the daughter fragments used in identification of the structures . the ms / ms fragmentation spectra were compared to literature values for the two dsp diastereomers ( figure 2 ) and dz to further confirm their structural assignments ( figures s2 and s3 , supporting information ) . the structure for dsp has been further established by x - ray crystallography and nmr . ammonia adducts observed included two pairs of chromatographic peaks for the diastereomers of spirodi(iminohydantoin)-2-deoxyribonucleosides ( dsi ) , whose names are based on the site of nh3 attachment to guanine , thus 5-dsi ( m + 31 ) and 8-dsi ( m + 31 ) ( scheme 2 , see reference cited for the correct dsi iupac nomenclature ) . to confirm the identities of the 5- and 8-dsi constitutional isomers and their respective diastereomers , esi - ms / ms of the free bases were conducted ( figure 2 ) . comparison of the esi - ms / ms spectrum for 5-dsi with dsp gave a pair of peaks for 5-dsi that established c5 as the site of nh3 attachment ( m / z [ m + h ] = 140 and 96 , figure 2 ) . as for 8-dsi , comparison of its esi - ms / ms spectrum with those obtained from dsp and 5-dsi identified new masses that are best explained by nh3 attachment at c8 ( m / z [ m + h ] = 141 and 97 , figure 2 ) . on the basis of the proposed mechanism ( scheme 2 ) , a product that includes bis - addition of nh3 at c5 and c8 of an oxidized guanine to yield a spirocycle ( 5,8-dsi , m+30 ) is possible ; however , this adduct was not observed for reasons that will be elaborated on below . additionally , nh3 adducts with a dgh core were not observed most likely due to the fact that the reactions were performed at ph 7.4 where yields of dgh are minimal . lastly , the yields of dog ( m + 16 ) and 8-amino - dg ( m + 15 ) were very low ( < 1% ) as determined by lc - esi - ms ( figure s1 , supporting information ) ; hence , their quantities are not reported . the nucleosides dog and c8 amine - adducted dg have redox potentials that are 600 mv below the parent nucleoside dg causing them to be much more susceptible to further oxidation . further , one - electron oxidized dog was observed to have an even lower redox potential than the parent compound dog , which means that once oxidation of dog , and likely 8-amino - dg , occurs product formation is inevitable due to the 70% conversion to product that yielded the spirocyclic compounds dsp , 5-dsi , or 8-dsi . esi - ms / ms spectra for dsp ( top ) , 5-dsi ( middle ) , and 8-dsi ( bottom ) . the data provided was collected on the first eluting diastereomer of each spirocycle from a hypercarb hplc column . data for the second eluting diastereomer for dsp , 5-dsi , and 8-dsi can be found in the supporting information ( figures s2 , s4 , and s5 ) . the heavy lines represent the fragment observed and the thin lines represent the portion of the molecule lost upon fragmentation . relative product distributions observed with each oxidant system were determined by integration of the hplc peak areas measured at 240 nm followed by normalization via each molecule s unique extinction coefficient ( 240 nm ) . extinction coefficients for 5-dsi and 8-dsi are not known but were determined from experiments that are reported below . in the riboflavin - mediated oxidations , this result was anticipated because riboflavin is a type i photooxidant and effects oxidation by electron transfer from dg yielding o2 under aerobic reaction conditions . oxidation of dg by one - electron and proton transfer yields an intermediate radical ( dg ) that only couples with o2 to initially yield diz that is prone to hydration leading to dz ( scheme 2 ) , the species quantified . the adducts derived from the nucleophile trapping of electrophilic dg oxidation intermediates show nh3 participation through the products 5-dsi ( 29% ) that was the highest yielding nh3 adduct followed by nearly 3-fold less 8-dsi ( 11% ) . the participation of h2o as the trapping nucleophile was identified by the lower yields of dsp ( 11% ) and dgh ( 4% ) . the nucleophilicity of nh3 is much greater than h2o ; therefore , as expected , adducts resulting from nh3 participation dominated over those derived from h2o . relative product distributions observed when dg reacted with nucleophilic h2o or nh3 in oxidation reactions . the oxidants include the photooxidants riboflavin and rose bengal , as well as na2ircl6 . reactions were conducted with 1 mm dg , 20 mm nh4cl , in 75 mm napi buffer ( ph 7.4 ) at 22 c . ( 1 ) photoactivation of riboflavin ( 200 m ) was achieved with 350 nm light for 3 h , ( 2 ) photoactivation of rose bengal ( 100 m ) was achieved with 350 nm light for 3 h , and ( 3 ) oxidation with na2ircl6 ( 10 mm ) was achieved by bolus addition of the salt . results represent the average of triplicate trials and the error is 10% of each reported value . the second oxidant studied was rose bengal , a type ii photooxidant ( o2 ) that furnished 5-dsi as the major product ( 62% ) followed by a 3-fold lower amount of dsp ( 31% ) , and low yields of dz ( 5% ) and dgh ( 2% ) were observed to complete the mass balance ( figure 3 ) . as expected , this oxidant did not yield 8-dsi based on its mechanism of oxidation . oxidation of dg by o2 proceeds by 4 + 2 cycloaddition to the imidazole ring , followed by ring opening to yield 8-hoo - g that eliminates water giving the proposed electrophile dog . next , dog is trapped by nucleophiles at c5 leading to dsp with h2o or 5-dsi with nh3 ( scheme 3 ) . support for exclusive nucleophilic attack at c5 under o2 oxidations was derived from h2o studies followed by mapping the labeled site by esi - ms / ms ; the current observation that the only nh3 adducts were the 5-dsi diastereomers further support this previously proposed mechanism , and further confirms our structural assignments for these peaks . comparisons of the product distributions from the photooxidants riboflavin and rose bengal show a dramatic difference with respect to the major product observed ( figure 3 ) . in the riboflavin oxidation , dz was the major product while in the rose bengal oxidation , 5-dsi was the major product , and these observations can be ascribed to each oxidant s unique mechanism of oxidation ( schemes 2 and 3 ) . product distributions from this oxidant included nearly equivalent distributions of dsp ( 50% ) and 5-dsi ( 42% ) with the mass balance completed by dgh ( 8% , figure 3 ) . in this oxidation reaction , the distribution did not yield nh3 adducts as the major products , which was unexpected on the basis of nh3 being the better nucleophile compared to h2o . comparison of the product distributions observed with the one - electron oxidants riboflavin and na2ircl6 identified both nh3- and h2o - adducted compounds . the current results differ from those previously reported by our laboratory when lysine was adducted to dg in analogous oxidation reactions . previously , in riboflavin oxidations spirodihydantoins with lysine at c8 were 2-fold greater than those with lysine at c5 ( figure 1 ) , and in na2ircl6 oxidations a c8 lysine adduct was observed . in contrast to these results , riboflavin oxidations in the presence of nh4cl gave more c5 adducts ( 29% ) than c8 adducts ( 11% ) , and na2ircl6 oxidations did not yield detectable amounts of c8 adducts . we propose the difference in the current ammonia results compared to the lysine data can be attributed to a difference in the mechanism of product formation for the c8 adduct in the nucleoside context . the former work from our laboratory proposed that c8 amine adducts result from oxidation of the amine to the aminyl radical that adds at the c8 carbon of dg followed by oxidation leading to product formation ( scheme 4 ) . in nucleoside dg , this proposed mechanism best supports the observation of ammonia adducts at c8 , because upon one - electron oxidation of dg the initial radical cation ( dg ) formed is very acidic ( pka 3.9 ) and rapidly deprotonates to the neutral radical ( dg ) that is not susceptible to nucleophilic attack . thus , dg reacts with o2 yielding diz / dz and not with amines or h2o . formation of amine adducts at c8 must result from a difference in the amine reactivity . the key difference between lysine and ammonia resides in their standard reduction potentials . in general , primary amines ( 1.0 v vs nhe , ph 10 ) have a lower redox potential than ammonia ( > 1.3 v vs nhe , ph 9 ) ; this trend should scale down to ph 7 , in which the oxidations were conducted . thus , oxidation of lysine to an aminyl radical that adds to c8 of dg is possible with na2ircl6 ( 0.9 v vs nhe , ph 7 ) and riboflavin ( 1.7 v vs nhe , ph 7 ) ; in contrast , the analogous reaction does not readily occur for nh3 because at ph 7 dg is the dominant site of oxidation due to its lower redox potential leading to products other than 8-dsi . in summary , one - electron oxidant driven oxidations of dg in the presence of nh3 lead to spirocyclic adducts at c5 and c8 that are in competition with h2o adducts of the same core structure . as the next step , we set out to determine the decomposition products and pathways through which the ammonia adducts proceed . hplc was used to provide diastereomerically pure 5-dsi and 8-dsi samples that were subjected to conditions of ph 3 ( 0.1% formic acid ) or ph 10 ( 20 mm napi ) at 22 c for 30 min or 10 h. not only could the decomposition products be determined , but having diastereomerically pure starting material also allowed us to probe the mechanism of decomposition , specifically for 5-dsi . first , both 5- and 8-dsi were stable at ph 10 during this time frame , while 5-dsi readily hydrolyzed to dsp at ph 3 in 30 min and 8-dsi hydrolyzed to dsp after 10 h. for 5-dsi , deamination of the amine group can occur by two possible mechanisms : ( 1 ) a retro - acyl migration can occur followed by loss of nh3 to yield an electrophilic intermediate ( dog ) susceptible to h2o attack followed by a second acyl migration back to dsp or ( 2 ) acid - catalyzed deamination can occur directly to furnish dsp ( scheme 5 ) . if the retro - acyl migration proceeds on the diastereomerically pure sample , it is expected that a mixture of dsp diastereomers would be observed , and if direct deamination occurs , then only one dsp diastereomer would be predicted . upon incubation of one diastereomer of 5-dsi at ph 3 , only one diastereomer of dsp was observed , supporting the direct deamination mechanism . moreover , the early eluting 5- and 8-dsi nh3 adducts decomposed to the early eluting dsp isomer and the late eluting adducts deaminated to the later eluting dsp isomer ( figure s6 , supporting information ) . these results aid in establishing the absolute configuration of the 5-dsi and 8-dsi diastereomers that will be discussed below . moreover , these results also allude to an explanation as to why the bis - ammonium adduct was not observed in any of the reactions . based on these data , the bis - ammonium adduct , if formed , could rapidly deaminate to yield either 5-dsi or 8-dsi , although this hypothesis could not be further validated . utilization of the hypercarb hplc column for analyzing the product distributions of these spirocycles allowed determination of the diastereomer ratios . it has previously been determined that ( r)-dsp elutes first from this column and ( s)-dsp elutes second . the absolute stereochemistry for the diastereomers of the ammonia adducts had not yet been determined . the results of these studies identified ( r)-dsp and ( s)-dsp yields to be nearly equal . also , for the 5-dsi and 8-dsi ammonia adducts nearly equal yields for the diastereomers were observed . on the basis of these results , the defining point of the reaction to determine product stereochemistry must not be very sterically impeded in order to lead to such a small diastereomer preference . as stated above , the diastereomerically pure ammonia adduct samples were determined to decompose to give a single diastereomer of dsp ; therefore , on the basis of hplc elution order , the absolute configuration for the diastereomers of the two dsi constitutional isomers can be determined . the first - eluting 5-dsi and 8-dsi diastereomers decomposed to the first dsp diastereomer ( r ) , and the late - eluting diastereomer decomposed to the late - eluting dsp diastereomer ( s ) ; therefore , for 5-dsi the r diastereomer elutes first and the s diastereomer elutes second , because the r and s assignments are the same between dsp and 5-dsi . in contrast , the r and s assignments for the 8-dsi diastereomers are opposite those of dsp ; thus , ( s)-8-dsi elutes first and ( r)-8-dsi elutes second and from the hypercarb hplc column ( figure 4 ) . these examples provide a fascinating case outlining how the movement of ring substituents affects the r and s stereochemical assignments . assignment of absolute configurations for the diastereomers of dsp , 5-dsi , and 8-dsi based on their elution profile from a hypercarb hplc column and their ecd spectra . the r and s assignments for the diastereomers of 8-dsi are the opposite of those for the dsp and 5-dsi isomers with the same geometric configuration of the spirocyclic ring , due to a change in the cahn ingold the diastereotopic ammonia adducts were then probed by electronic circular dichroism spectroscopy ( ecd ) . previously , ecd was used in tandem with vibrational circular dichrosim spectroscopy , nmr , and x - ray crystallography to establish the absolute configuration for dsp . the ecd spectra for ( r)- and ( s)-dsp isomers gave three lobes that were mirror images of one another , as expected ( figure 4 ) . critical for assigning the absolute configuration for dsp was the low energy lobe , in which the s isomer gave a positive rotation at 258 nm , and the r isomer gave a negative rotation at 259 nm . in comparison to these results , the 5-dsi diastereomers gave ecd spectra that were different than dsp but showed a similarity in the low energy lobe ( figure 4 ) . for ( r)-5-dsp , the lobe at 258 nm gave a negative rotation and ( s)-5-dsi gave a positive rotation at 258 nm . the higher energy lobes observed in the ecd for the 5-dsi diastereomers were different than those measured for dsp ( figure 4 ) . as a last comparison , the 8-dsi diastereomer ecd spectra were compared to those recorded for dsp ( note : 8-dsi and dsp isomers with the same geometric configuration of the spirocyclic ring give opposite r and s assignments due to a difference in the cahn the low energy lobe gave a negative rotation at 262 nm similar to ( r)-dsp , and ( r)-8-dsi gave a positive rotation similar to ( s)-dsp . furthermore , the 8-dsi diastereomers also gave similar rotations and energies as their analogous dsp diastereomers at the 238 nm lobe , and 8-dsi and dsp gave similar rotations at the highest energy lobe , but the absolute energy was different ( i.e. , 202 nm for 8-dsi and 211 nm for dsp ; figure 4 ) . the similarity in the critical low energy lobe further supports the absolute configuration assignments that were made from the deamination studies monitored by hplc . future computational studies to model these ecd spectra will help solidify these conclusions , and may address some of the challenges that occurred during modeling of the ecd spectra for the dsp diastereomers . the ability to deaminate the dsi adducts to dsp allowed the determination of the extinction coefficients for 5- and 8-dsi relative to dsp , in which these were the values used to determine the relative yields . peak areas for identically pure 5-dsi samples were measured before and after incubation in formic acid and the change in peak area measured was used to determine the relative 240 nm compared to the value established for dsp . a similar experiment was conducted for the 8-dsi diastereomers . from these experiments , the 240 nm for the 5-dsi and 8-dsi diastereomers were determined to be 3800 and 3500 lmolcm , respectively , which are both slightly greater than that determined for the dsp diastereomers ( 3300 lmolcm ) . the next step from the nucleoside studies was to explore the context dependence of nh3 adduct formation in single- and double - stranded odns ( ssodn and dsodn ) . the photooxidant riboflavin was chosen for the studies because it gave the most diverse distribution of products in the nucleoside reactions and might provide the most insight into context effects . for these studies , the ssodns selected for analysis were the 18-mers odn-1 and odn-2 , and the dsodn context was studied via the duplex formed from these two single strands ( odn-12 ) . after oxidation of these odn systems , they were digested with a suite of nucleases and phosphatase to the nucleosides followed by analysis using the previously described hplc methods . previously , our laboratory demonstrated that the digestion method used provides complete degradation of dsp - containing odns to nucleosides ; it is assumed that the dsi adducts are equally digested to completion . the nuclease digestion conditions were modified to include ammonium salts for buffer to prevent loss of 5-dsi via deamination to dsp ( scheme 5 ) , controls conducted with sodium salts for buffers did not detect 5-dsi due to deamination to dsp during the nuclease digestion ( 18 h at ph 5.4 , scheme 5 ) . a comparison between the contexts studied when dg was allowed to react with nh3 in the presence of the photooxidant riboflavin is provided in figure 5 . the first observation from these data was with respect to dz distributions that dramatically decreased when proceeding from the nucleoside to odn contexts ; specifically , the dz distribution in the dsodn context ( 2% ) was 10-fold less than the ssodn context ( 20% ) and 20-fold less than that observed in the nucleoside context ( 40% ) . this observation has already been reported in the literature and is proposed to result from the odn context increasing the lifetime of the dg that reacts with nucleophilic h2o to give the c8 product dog while decreasing the lifetime of dg that reacts with o2 to yield dz ( schemes 2 and 6 ) . with respect to the yields of the hydantoins , more dsp relative to dgh was observed in the nucleoside context ( 11% vs 4% , respectively ) versus the dsodn context ( 9% vs 21% , respectively ) . again , this trend follows those in the literature in which the duplex context favors the less sterically demanding product dgh . the nh3 adducts 5-dsi and 8-dsi provided some interesting context - dependent product distributions . the yield of 5-dsi was greatest for the nucleoside ( 29% ) and decreased by half in all odn contexts studied ( 14% ) . in contrast , the yield of 8-dsi was smallest for the nucleoside ( 11% ) and increased by more than 3-fold in the ssodn context ( 39% ) , and over half the products observed in the dsodn context were 8-dsi ( 54% ) . as previously stated , the lifetime of the electrophilic dg is longer in the duplex context and the superior nucleophilicity of nh3 compared to h2o greatly increased the yield of 8-dsi in dsodn oxidations ( scheme 6 ) . furthermore , the increase in 8-dsi yield from the dsodn oxidations further supports this product resulting from nucleophilic addition of nh3 at c8 leading to 8-dsi , and not the nh3 aminyl radical adding at c8 to yield the same product . these results clearly demonstrate a dependence on the reaction context in formation of dg oxidation products and nh3 adducts . a look at the distribution of diastereomers resulting from oxidation of the odn contexts gave nearly equal amounts of both r and s diastereomers . this observation parallels our previous work looking at dsp diastereomer formation in single - stranded and duplex contexts . coupled together , these observations support a state of disorder in the duplex at the time of nucleophilic attack at c5 of oxidized dog or 8-amino - dg ( likely intermediates leading to the spirocycles ) that leads to nearly equal covalent bond formation from both the re and si faces of these electrophiles . relative product distributions measured when dg was allowed to react with nh3 in the presence of photoexcited riboflavin in various contexts . reactions were conducted in 75 mm napi buffer ( ph 7.4 ) at 22 c , and 20 mm nh4cl . in the nucleoside studies , 1 mm dg was studied , and in the odn contexts 20 m ssodn or 10 m dsodn was studied . ( 1 ) photoactivation of riboflavin ( 200 m ) was achieved with 350 nm light for 3 h in the nucleoside studies and 30 min in the odn studies . these conditions achieved 70% conversion to product in the nucleoside reactions and 50% in the odn studies . it must first be noted that the bis - ammonium adduct could not be quantified due to its instability , but if formed , the adduct is most likely deaminated to either 5-dsi or 8-dsi . keeping this limitation in mind , the oxidation of dg by riboflavin initially yields dg that rapidly deprotonates to dg in the nucleoside contexts ( pka 3.9 ) , dominating the product - forming step of the reaction . in contrast , dg in the dsodn context retains more cationic character because the acidic proton is h - bonded with 2-deoxycytosine in a base pair ; therefore , the product - forming steps of the reaction are determined by the radical cation intermediate . as shown in scheme 2 , dg could couple with radicals ( e.g. , o2 ) to ultimately yield dz , while dg reacts with nucleophiles ( e.g. , h2o and nh3 ) at c8 , ultimately yielding a spirocyclic product after completion of the four - electron oxidation . thus , the yield of 8-dsi was anticipated to be greatest in the duplex context , as indeed it is . furthermore , these results support the original hypothesis by steenken that the duplex context would favor the radical cation nature of one - electron oxidized dg , which is still the focus of many current research efforts . because of the limitation imposed by the inability to detect the bis - ammonium adduct , or to understand its decomposition pathway , any comparison between the ammonia adduct distributions would be based on poorly defined assumptions ; consequently , further discussion of these results is not provided . for the last set of studies , we were inspired by the observation that the a ring of dsp mimics the h - bonding pattern of thymidine , while the a ring of 5-dsi mimics the h - bonding pattern of 2-deoxycytidine ( figure 6 ) . on the basis of this observation , polymerase insertion assays opposite dsp vs 5-dsi were conducted to determine if these h - bonding schemes applied during the selection of a nucleotide to insert opposite these spirocycles . comparison of h - bonding patterns between dsp vs t and 5-dsi vs dc . site - specific synthesis of dsp or 5-dsi was achieved by synthesizing dog into an odn by solid - phase synthesis and then oxidizing this strand with or without nh4cl present with the one - electron oxidant na2ircl6 to furnish the desired products . upon ion - exchange hplc purification , the absolute stereochemical assignments for the dsp isomers have been established for the odns , and those for 5-dsi are assigned in this work to be analogous based on the mechanism of deamination discussed above ( see figure s7 , supporting information , for more details ) . standing start polymerase studies were conducted while providing the enzyme only one type of nucleotide per reaction . on the basis of previous studies , insertion of dttp opposite dsp was not observed ; therefore , to ensure that only one nucleotide was inserted opposite the lesion , and no extension past the lesions occurs , the sequence had an a placed 5 to the spirocycle ( figure 7 ) . klenow fragment exo- was selected as the polymerase , and after the extension reactions , polyacrylamide gel electrophoresis ( page ) was conducted to determine the amount of datp , dttp , dgtp , or dctp incorporated opposite the dsp or 5-dsi diastereomers . the polymerase did not select either pyrimidine for insertion opposite the two lesions ( figure s7 , supporting information ) ; thus , only data for purine insertion opposite the lesions is provided in figure 7 . with respect to insertion opposite the dsp diastereomers , ( r)-dsp was observed to give a slight preference for insertion of datp opposite , and ( s)-dsp gave nearly equal insertion of datp and dgtp . in comparison to the dsp results , the 5-dsi isomers showed that ( s)-5-dsi gives similar amounts of datp and dgtp insertion , while ( r)-5-dsi yields a slight preference for datp insertion . unfortunately , these results do not support the hypothesis that h - bonding in the a ring of the spirocycle is the key parameter for nucleotide selection , but do show that stereochemistry may be important for selecting a base pairing partner by this polymerase . sequence studied for polymerase dntp insertion studies and the percent dntp insertions opposite the diastereomers of dsp and 5-dsi . ( a ) sequence for the dsodn construct used in the polymerase insertion assays and ( b ) comparison of the percent purine dntp insertion opposite the lesions dsp and 5-dsi by the dna polymerase klenow fragment exo- . the present studies mapped the pathways and quantified the products observed when dg was allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose bengal , as well as the one - electron oxidant na2ircl6 . on the basis of the analysis of the products in the nucleoside context , the major products were oxidant dependent . the major product for riboflavin was dz , rose bengal yielded the 5-dsi diastereomers , and na2ircl6 gave the dsp diastereomers as the major products ( figure 3 ) . further analyses determined that 5-dsi and 8-dsi decompose via an acid - catalyzed deamination pathway leading to dsp as an end product ( scheme 5 ) . oxidations of dg in the nucleoside , ss- and dsodn contexts with photochemically activated riboflavin demonstrated three major context effects : ( 1 ) the yield of dz was highest in the nucleoside studies and decreased dramatically in dsodn contexts ; ( 2 ) when nh3 participated as the nucleophile , 5-dsi was obtained in the highest yield in nucleoside studies , while the yield of 8-dsi was highest in dsodn contexts ; and ( 3 ) when h2o was the nucleophile in nucleoside reactions , dsp presented in the highest yield , while in dsodn contexts dgh was obtained in the highest yield ( figure 4 ) . furthermore , a combination of mapping the decomposition pathways of the dsi compounds to dsp , in which the absolute configuration has been assigned , allowed the determination of the absolute configurations for the diastereomers of 5- and 8-dsi . these studies provide fundamental chemical insight into formation of amine adducts to dg and their stability . further , these studies insert a cautionary note to researchers who purify their odns using ammonium salts and then conduct oxidation reactions after purification . ammonia is a better nucleophile and out - competes water for the electrophilic intermediates derived from dg or dog oxidation , resulting in a new mass of dg + 31 or dog + 15 , respectively . a similar observation has been highlighted for oxidations occurring in tris buffer that generate tris adducts to dg . oxidations were conducted with 2-deoxyguanosine ( dg ) at 1 mm concentration in 75 mm napi buffer ( ph 7.4 ) and 22 c . reactions with 20 mm nh4cl and without this salt allowed product profile comparisons . the oxidants and specific reaction conditions were achieved as follows : ( 1 ) riboflavin oxidations were initiated by adding 200 m riboflavin while exposing the samples to 350 nm light for 3 h. ( 2 ) rose bengal oxidations were achieved by adding 100 m rose bengal and exposing these samples to 350 nm for 3 h. the light source for the riboflavin and rose bengal reactions came from a sun lamp that was placed 7 cm above the reaction eppendorf tubes . the tube lids were left open to allow all wavelengths of light to pass into the reaction samples . ( 3 ) the na2ircl6 oxidations were initiated by a bolus addition of the oxidizing salt with a final concentration of 10 mm , after a 30 min reaction the samples were quenched with 50 mm edta ( ph 8) . the reaction products and their distributions were determined by a dual hplc method following a previously reported set of protocols ( specific details can be found in the supporting information file ) . the first rp - hplc run allowed analysis of dog and 8-amino - dg ( both observed in < 1% yield ) , while all other products eluted in the void volume of this run . the void volume was collected , dried , and reinjected on a hypercarb hplc column to analyze the diastereomers of dgh , dsp , 5-dsi , and 8-dsi , as well as the product dz , while monitoring their absorbance at 240 nm . to determine product distributions the peak areas were integrated and normalized by each compound s 240 nm ( values provided below ) . esi - ms , and then each compound was hplc purified for further structural analysis . the dgh diastereomers were previously characterized by nmr , dz was also previously characterized by nmr , and the dsp diastereomers have been characterized by x - ray crystallography . structural characterization of 5-dsi and 8-dsi by nmr was not conducted because of the instability of these compounds toward deaminating to dsp , and the lack of nonexchangeable protons on the ring of either ammonia adducts makes them very challenging for structural analysis . furthermore , if deamination of either 5-dsi or 8-dsi to dsp occurs in the nmr tube during analysis , the peaks observed would be similar and challenging to interpret ; therefore , the best method for obtaining structural data on the diastereomers of 5-dsi and 8-dsi is via the esi - ms / ms that was conducted ( figure 2 ) , and provided satisfactory results to determine their structures . characterization for each molecule is as follows , the mixture of the dgh diastereomers ( not resolvable ) gave tr = 6 min ; lc esi - ms m / z [ m + h ] calcd 274.3 , found 274.1 ; hrms ( esi - tof ) m / z [ m + na ] calcd for c9h15n5o5na 296.0971 , found 296.0979 ; uv vis 240 = 2400 l molcm.(r)-dsp : tr = 11 min ; lc esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found 322.0761 ; esi - ms / ms m / z [ m + h ] lit . 184 , 156 , 141 , 114 , 113 , 99 , 86 , found 184 , 156 , 141 , 114 , 113 , 99 , and 86 ; uv vis 240 = 3,300 lmolcm ; cd ( c 1.24 10 m in ddh2o ) nm 259 ( 8.7 ) , 236 ( + 35.1 ) , and 211 ( 37.4 ) . ( s)-dsp : tr = 18 min ; lc esi - ms m / z [ m + h ] calcd 300.3 , found 300.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h13n5o6na calcd 322.0764 , found 322.0761 ; esi - ms / ms m / z [ m + h ] lit . 184 , 156 , 141 , 114 , 113 , 99 , 86 , found 184 , 156 , 141 , 114 , 113 , 99 , and 86 ; uv vis 240 = 3300 lmol cm ; cd ( c 1.30 10 m in ddh2o ) nm 258 ( + 8.7 ) , 234 ( 32.9 ) , and 212 ( + 40.5 ) . ( r)-5dsi : tr = 9 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , found 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0920 ; esi - ms / ms m / z [ m + h ] found values 183 , 166 , 140 , 123 , 113 , 96 , and 86 ; uv vis 240 = 3800 lmolcm ; cd ( c 1.50 10 m in ddh2o ) nm 258 ( 8.2 ) , 242 ( + 20.8 ) , and 216 ( + 61.8 ) . ( s)-5dsi : tr = 12 min ; lc - esi - ms m / z [ m + h ] calcd 299.3 , found 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0916 ; esi - ms / ms m / z [ m + h ] found 183 , 166 , 140 , 123 , 113 , 96 , and 86 ; uv vis 240 = 3800 lmolcm ; cd ( c 1.45 10 m in ddh2o ) nm 258 ( + 8.2 ) , 242 ( 20.1 ) , and 216 ( 62.2 ) . ( r)-8dsi : tr = 7 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0924 ; esi - ms / ms m / z [ m + h ] found 183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv vis 240 = 3500 lmolcm ; cd ( c 1.30 10 m in ddh2o ) nm 261 ( + 20.0 ) , 237 ( 43.2 ) , and 203 ( + 18.6 ) . ( s)-8dsi : tr = 14 min ; lc esi - ms m / z [ m + h ] calcd 299.3 , expt 299.3 ; hrms ( esi - tof ) m / z [ m + na ] for c10h14n6o5na calcd 321.0923 , found 321.0926 ; esi - ms / ms m / z [ m + h ] found 183 , 165 , 155 , 138 , 113 , 98 , and 86 ; uv vis 240 = 3500 lmolcm ; cd ( c 1.24 10 m in ddh2o ) nm 262 ( 13.7 ) , 239 ( + 46.0 ) , and 201 ( 33.1 ) . dz : tr = 27 min ; lc esi - ms m / z [ m + h ] calcd 247.3 , found 247.3 ; hrms ( esi - tof ) m / z [ m + na ] for c8h14n4o5na calcd 269.0862 , found 269.0870 ; esi - ms / ms m / z [ m + h ] lit . 131 and 117 , found 131 and 117 ; uv vis 240 = 1800 lmolcm . the odns were hplc purified using an ion - exchange hplc column , and the purification salts ( naoac ) were removed by dialysis prior to their oxidation following previously reported methods . the riboflavin oxidations were conducted similarly to those reported for the nucleoside studies , with the following exceptions : the ssodn oxidations were conducted on 20 m samples while the dsodn oxidations were conducted on 10 m samples . in addition , the reaction times were decreased for the odn reactions to 30 min . after the oxidations , the odns were digested with a suite of nucleases and phosphatases to nucleoside samples following a previously established protocol , with the exception that all buffers during the digestion process were comprised of ammonium salts . next , the digested mixture was analyzed by the hplc method used in the nucleoside studies . the polymerase insertion assays were conducted on duplex odn samples that had site specific incorporation of dsp or 5-dsi in the template strand . the site - specific synthesis was commenced on odns that had a dog phosphoramidite synthesized at the desired site of modification within the sequence 5-cgt tax ggc gca act gga aa-3 where x = dog . the modifications were synthesized by taking 1 nmol of the dog - containing odn and placing it in 100 l of reaction buffer ( 75 mm napi , ph 7.4 ) with or without 2 mm nh4cl . the reaction without nh4cl gave the dsp diastereomers and the reaction with nh4cl gave the 5-dsi diastereomers . the individual diastereomers were purified using an ion - exchange hplc column running naoac as the resolving salt and characterized via digestion of the odn to nucleosides followed by hplc analysis ( purification details can be found in the supporting information ) . the primer template duplex for the insertion studies was made by annealing 125 nm primer ( 5-tt tcc agt tgc gcc-3 ) with 156 nm lesion - containing template ( 5-cgt tax ggc gca act gga aa-3 where x = ( r)-dsp , ( s)-dsp , ( r)-5-dsi , or ( s)-5-dsi to obtain 100 nm duplex in klenow fragment exo- buffer ( 50 mm tris , 50 mm nacl , 5 mm mgcl2 , 1 mm dtt at ph 8) . to a 25 l reaction 20 l of the annealed duplex was added , 1 l of klenow fragment exo- ( 0.2 units/l ) , 0.5 l of dntp ( 500 m stock solution ) , and 8.5 l of klenow buffer to obtain a 100 nm duplex solution with 10 m dntp and 0.2 u of polymerase . the reaction was incubated at 37 c for 30 min , after which loading dye ( 95% dmf plus 0.025% bromophenol blue , and 0.025% xylene cylanol ) was added to the samples and they were heated at 95 c for 20 min to quench the reaction and denature the dna the denatured samples were loaded on a 20% page gel and electrophoresed for 2 h at 45 w. upon completion of the electrophoresis , the gel was placed in a phosphor screen overnight and imaged by storage phosphor autoradiography .

we have previously demonstrated that rad6 and -catenin enhance each other 's expression through a positive feedback loop to promote breast cancer development / progression . while -catenin has been implicated in melanoma pathogenesis , rad6 function has not been investigated . here , we examined the relationship between rad6 and -catenin in melanoma development and progression . eighty - eight cutaneous tumors , 30 nevi , 29 primary melanoma , and 29 metastatic melanomas , were immunostained with anti--catenin and anti - rad6 antibodies . strong expression of rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas . -catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas , and unlike rad6 , in 93% of nevi . none of the tumors expressed nuclear -catenin . -catenin was exclusively localized on the cell membrane of 55% of primary , 62% of metastatic melanomas , and only 10% of nevi . cytoplasmic -catenin was detected in 90% of nevi , 17% of primary , and 8% of metastatic melanoma , whereas 28% of primary and 30% of metastatic melanomas exhibited -catenin at both locations . these data suggest that melanoma development and progression are associated with rad6 upregulation and membranous redistribution of -catenin and that -catenin and rad6 play independent roles in melanoma development . upon oxidation of the heterocyclic ring in 2-deoxyguanosine ( dg ) , the initial electrophilic intermediate displays a wide range of reactivities with nucleophiles leading to many downstream products . in the present study , the product profiles were mapped when aqueous solutions of dg were allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose bengal as well as the diffusible one - electron oxidant na2ircl6 . product characterization identified the 2-deoxyribonucleosides of spiroiminodihydantoin , 5-guanidinohydantoin , and oxazolone resulting from h2o as the nucleophile . when nh3 was the nucleophile , a set of constitutional isomers that are diastereotopic were also observed , giving characteristic masses of dg + 31 . esi+-ms / ms of these nh3 adducts identified them to be spirocycles with substitution of either the c5 or c8 carbonyl with an amine . the nh3 adducts exhibit acid - catalyzed hydrolysis to spiroiminodihydantoin . quantification of the nh3 and h2o adducts resulting from oxidation of dg in the nucleoside , single - stranded , and duplex oligodeoxynucleotide contexts were monitored allowing mechanisms for product formation to be proposed . these data also provide a cautionary note to those who purify their oligonucleotide samples with ammonium salts before oxidation because this will lead to unwanted side reactions in which ammonia participates in product formation .

a review of the literature and an extensive medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility . he reported an inability to conceive with his wife after 18 months of unprotected intercourse . a semen analysis was performed that included spermatozoa count , liquefaction , morphology , motility , viscosity and volume . two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility . evidence for the effectiveness of guaifenesin is almost entirely anecdotal . given the mechanism of action of guaifenesin , it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . additional studies of the effects of guaifenesin on male fertility could yield information of the medication s effect on men with normal or decreased total sperm counts . there are currently anecdotal reports and popular news media stories on the use of guaifenesin , particularly the brand name product robitussin ( pfizer , inc . , new york , ny ) , for use in treating both male and female infertility.14 guaifenesin is an expectorant medication sold over the counter and usually taken by mouth to assist expectoration of phlegm from the airways in acute respiratory tract infections . its mode of action in treating infertility is not well understood , but it appears to decrease mucus viscosity . a 32-year - old male patient presented to his primary care provider for an infertility evaluation . the patient is a nonsmoker , who consumes little or no alcohol with no known allergies . a recent screening exam for pulmonary tuberculosis was negative and the patient had recently undergone a required military service physical exam . he reported an inability to conceive with his wife after 18 months of unprotected , regular intercourse . as part of a routine infertility evaluation a semen analysis was performed that included spermatozoa count , liquefaction , morphology , motility , viscosity and volume ( cpt code 89320 ) . initial results of the semen analysis demonstrated low sperm count and motility ( table 1 ) . this sample , as well as the follow - up sample , were obtained through masturbation and provided to the lab within 30 minutes of collection . the patient s primary care provider offered treatment with guaifenesin 600 mg extended release tablets twice daily . the repeat semen analysis demonstrated marked improvement in both total sperm count and motility ( table 1 ) . the patient made no other significant lifestyle changes during the treatment course with guaifenesin . at the time of writing this case report describes the semen analysis laboratory results in a male patient who was given guaifenesin . guaifenesin is a mucolytic agent usually taken orally to assist the expectoration of phlegm from the airways in acute respiratory tract infections . scientific evidence for the effectiveness of guaifenesin is almost entirely anecdotal ; a review of medical literature revealed very limited data on use of guaifenesin for infertility.5,6 there appeared to be some improvement in a small study without controls of female infertility related to hostile cervical mucus.5 check regards guaifenesin as the simplest but least effective method of improving cervical mucus.7 given the proposed mechanism of action of guaifenesin , it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . additional study of the effects of guaifenesin on male fertility suggests the need to conduct a more rigorous placebo - controlled clinical trial that could yield information of the medication s effects on men with normal or decreased total sperm counts . agriculture has been one of the primary economic avenues in the philippines contributing to about 20% to the gross domestic product ( gdp ) . crops comprise about 47.56% of the total agricultural sector and have contributed to about 510 billion pesos ( p510b ) to the country 's national income . benguet is a province in the northern portion of the philippines belonging to the cordillera administrative region . there are about 27.5 thousand farms covering 30 thousand hectares of agricultural land in benguet . it is also the largest producer of vegetables and fruits , supplying the capital cities in the philippines . the province is known as the salad bowl of the philippines as its major crops are tubers , roots , bulbs , leafy vegetables , stems , and flowers . in 2005 , benguet was the top producer of brocolli and carrots producing about 1.2 thousand and 13.7 metric tons contributing to 87.4% and 81.4% , respectively , to the national output . however , growing vegetables is considered a risk occupation in some areas in developing countries . soogarun et al . in 2003 found significantly low / abnormal mean blood cholinesterase levels among vegetable growers in thailand . health impacts of pesticide misuse on the other hand greatly affect the farming communities in the philippines questioning the economic advantages of its use . many researchers have correlated the extent of direct and indirect pesticide exposure and health hazards such as increased mortality , dermal contamination , depression in cholinesterase level , fetal abnormalities , and spontaneous abortion among pregnant women [ 36 ] . it is a discouraging fact though , that with knowledge of health risks , many filipino families still perceive that crop yield outweighs the health risks associated with pesticide use . pesticide poisoning is one of the most prevalent health problems in the philippines . in a study by the department of health ( doh ) from 19911995 , organophosphates accounted for the highest number of poisoning cases while organochlorines caused the most number of deaths . cheng in 1994 studied 2000 benguet vegetable farmers and found that the most common complaints were allergic reactions both in the skin and the eyes , abdominal pain , dizziness , chest pain , headache , and nose bleed . meanwhile , a study on pesticide poisoning in selected hospitals in four philippines regions in 2001 found that cases of acute poisoning were more prevalent than chronic cases . this study aimed to identify the pesticide exposure and risk factors among vegetable farmers . the data can be used as baseline data on the vegetable industry in the philippines . this was a cross - sectional study to investigate the prevalence of pesticide exposure and its risk factors . target population consisted of vegetable farmers in the largest vegetable producing community in the philippines . the inclusion criteria were farmers living in the community for at least one year from the time of interview , and practicing farmers who own or work a farm in the community . those who were involved in organic farming and the migrant farmers who have been in the area for less than one year were excluded . there were 211 respondents from the identified municipalities selected as the study population using cluster sampling . the sampling size calculated with p = .05 was 211 vegetable farmers and 37 farms . data gathering was done using the following : ( 1 ) questionnaire structured personal interview with farm workers / farmers was done by research assistants who were trained prior to the data collection . details included personal information , health history , pesticide usage , work practices , work conditions , risk factors associated with pesticide exposure , and health data ; ( 2 ) exposure assessment monitoring on work conditions , work practices , and pesticide concentration ; ( 3 ) work analysis in each farm was also done to validate work practices related to pesticide preparation and application . recall bias was dealt with by confining the health data questionnaire to the last one year from the time of interview . the health data were also collected by medical doctors who simultaneously conducted physical assessment of the farmers . this project study was collaborated with the local agencies coordinating with farmers in the vegetable industry in benguet . ethical clearance was given by the research , information and dissemination office of the proponent 's institute . the study included 127 males ( 60.2% ) and 84 females ( 39.8% ) with ages ranging from 16 to 72 ( mean = 45 12 ) showing a relatively adult population . seventy one percent ( 71% ) were married and majority were working as agricultural workers ( 82% ) , and the remaining were pesticide applicators , mixers , and loaders ( 18% ) . the respondents were living in their present address for an average of 34.76 years ( sd = 16.72 ) with a mean distance of 3163 meters ( sd = 36539.13 ) from the vegetable plantation or farm . few farmers reported history of smoking ( 16.2% ) , and 7% claimed they smoked and 2% had a history of chewing tobacco . the average number of cigarettes and tobacco consumed in a week were 12 sticks and < 1 tobacco , respectively . the farmers used pesticides in their farms in an average of 1.9 days per week . the mean total application time was 3.47 hours ( mean = 3.47 2.09 ) . the mean amount of pesticide used in an application was 21.35 l per application ( mean 21.35 48.17 ) . the farmers also reported that in an average year , there were 2.3 ( mean = 2.3 0.53 ) cropping seasons with a mean of 3.84 ( mean = 2.3 0.53 ) months per cropping season ( table 1 ) . 44.5% reported that they wiped their sweat with a contaminated piece of fabric , 41.7% re - entered recently sprayed area , 37.4% had exposure because of damaged backpack sprayer , and 31.8% were exposed when they sprayed against the wind ( table 2 ) . however , further analysis shows that they did not frequently use such equipment nor had adequate gear to fully protect themselves . the same pattern was seen among all kinds of personal protective equipment ( ppe ) with the exception of boots which was frequently used by 77.5% of farmers . 94% said that they have worked with or used pesticides in their lifetime , and 16.4% from this population used pesticides in their own households . the vegetables commonly grown in the area were potatoes ( 67.4% ) , cabbage ( 63.7% ) , and carrots ( 36.8% ) . majority ( 87% ) the most common route of pesticide entry in the study was respiratory ( 68.9% ) followed by dermal and ocular entry ( 60.5% and 38% , resp . ) . sumicidine was the most commonly used pyrethroid which contains fenvalerate as its active ingredient . table 3 shows that 37% and almost 14.7% of the study population used pesticides with active ingredients of fenvalerate and cypermethrin , respectively . although pyrethroid was the most frequently used pesticide , it is organophosphate that consisted the largest amount of exposure among farmers at 210.02 liters , followed by pyrethorid at 151.4 liters , and carbamates at 32.16 liters for the entire one year . the pesticide exposure of the farmers measured in table 4 as dependent variable was related to the amount of pesticide used in liters , frequency of use and duration of use . all the independent variables , except amount and years of pesticide use were categorical variables . those who used more pesticides over a longer period of time had higher total pesticide exposure . furthermore , a farmer who was a pesticide applicator , mixer , loader and who wiped sweat with contaminated piece of fabric , and who had not been given instructions through training association was at risk of having higher pesticide exposure . seventy four percent ( 74% ) of the respondents became ill because of work for the last 12 months preceding the study . the most common symptoms were headache ( 64.1% ) , muscle pain ( 61.1% ) , cough ( 45.5% ) , weakness ( 42.4% ) , eye pain ( 39.9% ) , chest pain ( 37.4% ) , and eye redness ( 33.8% ) . a subsequent study is recommended to focus on adverse health effects of these farmers and association with certain risk factors . the results of this study identified pesticide exposure and farming practices of farmers in the largest vegetable producing area in the philippines . this has also been documented in other countries . in the study of coble et al . in 2005 , and thompson et al . in 2003 , unsafe practices like re - entry of recently spayed area , use of damaged backpack sprayer and wiping sweat with a contaminated piece of fabric were identified in this study . re - entering a recently sprayed area , as mentioned in the study of tielemans et al . in 1999 is an important determinant of dermal exposure to specific chemicals such as captan and tolylfluanid . a pesticide 's formulation is a significant factor for human exposure , with greater risks present among aqueous and emulsifiable concentrates because it impairs the protective function of chemically protective gloves . according to wolfe in 1993 , pesticides may react through chemical and biotic processes . however , pesticides may undergo activation processes unexpectedly and may be broken down to equally or more potent and mobile toxic compounds posing a greater threat to nontarget organisms . it is then advised to limit or decrease the frequency or duration of staying in the contaminated crops right after pesticide application . organophosphates , carbamates and pyrethroid pesticides were the most commonly used type of pesticides among the farmers in this study . the same was seen in the study of clarke et al . in 1997 in ghanna where organophosphates consisted of the most commonly used pesticides followed by carbamates and organochlorines . the same trend was seen among farmers in sri lanka and in brazil [ 15 , 16 ] . the farmers in this study used pesticides in their farms with a mean application time of 3.47 hours ( mean = 3.47 2.09 ) . the mean amount of pesticide used in an application was 21.35 liters per application ( mean 21.35 48.17 ) . the number of spray operations per week has been proven to have significant association with the likelihood of experiencing neurobehavioral , respiratory , or intestinal symptoms in a study among indonesian farmers . in a study among north carolina growers and agents , it was found that the study population perceived that once the pesticide is diluted and reentry intervals are observed , the risk it poses becomes diminished . sumicidine , which was the most commonly used pesticides among the benguet farmers contains fenvalerate . fenvalerate induces numbness , itching , tingling , and burning sensations in exposed workers that developed after a latent period of approximately 30 minutes , peaked by 8 hours , and disappeared within 24 hours . additional data among chinese workers demonstrated that fenvalerate decreased the semen quality of occupational workers . on the other hand , other active ingredients like cypermethrin , mancozeb , and skin sensations were reported to occur among field workers and usually lasted only for a few hours and did not persist for more than one day after exposure to cypermethrin . for mancozeb , prolonged low - level exposure to mancozeb affected several aspects of immune functioning and moderate association existed between mancozeb and neural tube defects . most often , gloves were the most commonly used personal protective equipment because the hands were the most exposed areas [ 23 , 24 ] . many circumstances contributed to nonadherence to proper use of ppe like extreme heat during pesticide application , uncomfortable to use , few resources to afford new ppe , peer - related factors , and increasing age [ 18 , 2527 ] . the study also showed certain risk factors associated with pesticide exposure such as re - entering recently sprayed area , spraying against the wind , use of damaged backpack sprayer , spills on the back , spills while mixing pesticides , among others . aside from direct pesticide use , the different agricultural tasks mentioned above may also contribute as risk factors to pesticide exposure . re - entering a recently sprayed area , as mentioned in the study of tielemans et al . in 1999 is an important determinant of dermal exposure to specific chemicals such as captan and tolylfluanid . there is evidence that weight loss could be a possible health effect of chronic pesticide poisoning . decreased body mean mass accompanied by reduced cholinesterase activities among seven farm workers was documented . also , kackar et al . in 1999 found that when rats were administered orally with mancozeb ( ethylenebisdithiocarbamate ) , dose - dependent signs of poisoning , weight loss , and mortality developed . senthilselvan et al . found a significant association between carbamate exposure and prevalence of asthma among those non - asthmatic farmers and lower mean lung function variables among those with asthma . this study has shown the pesticide exposure of farmers in the largest vegetable producing area in the philippines . it is vital that a sequential exposure assessment be done in order to come up with a correlation study between pesticide exposure and health problems . the study showed that pesticide use is prevalent among farmers in benguet which is the largest vegetable producer in the philippines . it is also suggested that chronic effects of pesticide cited in certain studies [ 31 , 32 ] such as carcinogenic effects , poor reproductive outcomes , neurologic and respiratory disorders , impairments of the immune system and birth defects should also be investigated in future studies . this manuscript adds to existing literature on pesticide exposure in the philippines which are so far mainly descriptive in nature . this paper also identifies risk factors such as work practices and designs of containers / sprayers that may increase pesticide exposure among farmers . this also calls for a local level policy research for program intervention among vegetable farmers using pesticides .

backgrounda review of the literature and an extensive medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility.casea 32-year - old male presented for an infertility evaluation . he reported an inability to conceive with his wife after 18 months of unprotected intercourse . a semen analysis was performed that included spermatozoa count , liquefaction , morphology , motility , viscosity and volume . initial results of the semen analysis demonstrated low sperm count and motility . the provider offered treatment with guaifenesin 600 mg extended release tablets twice daily . two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility.conclusionevidence for the effectiveness of guaifenesin is almost entirely anecdotal . given the mechanism of action of guaifenesin , it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . additional studies of the effects of guaifenesin on male fertility could yield information of the medication s effect on men with normal or decreased total sperm counts . this was a cross - sectional study that investigated pesticide exposure and its risk factors targeting vegetable farmers selected through cluster sampling . the sampling size calculated with p = .05 was 211 vegetable farmers and 37 farms . the mean usage of pesticide was 21.35 liters . risk factors included damaged backpack sprayer ( 34.7% ) , spills on hands ( 31.8% ) , and spraying against the wind ( 58% ) . the top 3 pesticides used were pyrethroid ( 46.4% ) , organophosphates ( 24.2% ) , and carbamates ( 21.3% ) . those who were exposed to fungicides and insecticides also had higher total pesticide exposure . furthermore , a farmer who was a pesticide applicator , mixer , loader , and who had not been given instructions through training was at risk of having higher pesticide exposure . the most prevalent symptoms were headache ( 64.1% ) , muscle pain ( 61.1% ) , cough ( 45.5% ) , weakness ( 42.4% ) , eye pain ( 39.9% ) , chest pain ( 37.4% ) , and eye redness ( 33.8% ) . the data can be used for the formulation of an integrated program on safety and health in the vegetable industry .

evans , using a mouse mutant for the lim homeodomain transcription factor ( islet1 , isl1 ) , demonstrated that an isl1 positive ( isl1 + ) population is localised in the secondary heart field ( shf ) , contributing to the outflow tract , right ventricle , atria and ( to a small extent ) the left ventricle . isl1 is considered a marker of shf cells that contribute to the venous and arterial poles of the cardiac tube , while the other cells from the shf contribute only to the arterial pole . the shf or anterior heat field ( ahf ) was identified as a splanchnic mesoderm underlying the caudal pharynx , which provides myocardium to the outflow tract during tube looping . in 2005 , the same research group also identified isl1+/c - kit negative ( c - kit-)/stem cell antigen-1 negative ( sca-1- ) progenitors in the postnatal heart , demonstrating that these cells persist after birth and that these organ - specific progenitor cells may be isolated , purified , expanded and differentiated into mature cardiac myocytes for future cardiac applications . our research group identified isl1 + cells during gestation until the postnatal age , and demonstrated the contemporary expression of isl1 and c - kit in the interstitial cells in the ventricular myocardium in human foetal and postnatal hearts . this paper demonstrated , by confocal analysis , that all isl1 + cells were also c - kit+ , while c - kit+ cells were not necessarily isl1 + , as if isl1 + cells were a subpopulation of c - kit+ cells . it has been supposed that there may be a primordial cardiovascular precursor in the shf that expresses the three markers isl1 , nkx2.5 and fetal liver kinase 1 ( flk1 ) and can generate three lineages : cardiac muscle , smooth muscle and endothelium . the shf cooperates with the first heart field ( fhf ) and the cardiac neural crest cells during the development of the vertebrate heart . the fhf lineage forms the early cardiac tube and generates the left ventricle , while the shf contributes additional cells to the maturing heart and , along with the cardiac neural crest cells , generates the outflow tract vessels and valves . the presence of cardiovascular progenitor cells positive for isl1 and/or nkx2.5 in the shf has been well confirmed by a recent work published in science . the authors suggested that there are two distinct populations , one for the shf and the other for the fhf ; the first population is clearly positive for isl1 and expresses mirna 199a / b , while the second has no distinct markers and expresses mirna 200a / b . progenitor cells of the shf depend on the cardiac specific transcription factors isl1 and nkx2.5 , which play a key role at an early stage of cardiac progenitor formation . their expression is regulated by wnt / beta - catenin signalling , which in turn also regulates bmp4 signalling . this last signalling pathway activates gata4 and srf . despite the key role of isl1 , this transcription factor is not considered an indubitable marker of this subpopulation of shf progenitor cells . it has been demonstrated very recently that isl1 labels both shf precursors and some cardiac neural crest cells and that mef2c is a more efficient marker of the shf progenitor cells in labelling experiments . this paper indirectly suggests both a review of past literature in view of these new data and using a different marker in studies of the shf progenitor cells . moreover , considering heart development in different species , such as chicken , mouse and xenopus , it appears that isl1 is expressed in a common cardiac cell population that splits into fhf and shf lineages during heart development and that this common progenitor also contributes cells to the cardiovascular system . anversa s research group described rat cardiac stem cells for the first time , many research groups have claimed to have discovered new and important cardiac progenitor / stem cells in the adult heart .. since 2005 , the scientific community talked about adult c - kit+ and/or sca-1 + and/or mdr-1 + cells , and embryonic isl1 + cells . these two populations have always been considered different entities and are described separately in many research papers and reviews , although expression of the isl1 transcription factor by cardiac precursor cells has also been reported in adult hearts . di nardo , described for the first time the concomitant expression of the three markers , i.e. , c - kit , sca-1 and isl1 , in the same cardiac precursor cell . prof . di nardo s research group described the concomitant expression of the isl1 , c - kit and sca-1 markers in adult mouse cardiac progenitor cells in 2008 , while in 2009 our research group confirmed the expression of the three markers in adult rat cpcs . in a systematic work published in 2011 about the identification of isl1 + cells in the mouse heart from postnatal day 1 to young adulthood in different strains , the authors found clusters of positive cells in the cardiac ganglia of the studied strains , while found very a few clusters of isl1 + cardiac precursors only in 129svj or balb / c strains and in animals not older than 4 months . recently genead et al . demonstrated the contemporary expression of c - kit and isl1 markers in rat adult hearts in normal , pregnant and infarcted individuals . they reported the expression of both markers in the entire heart and in the right ventricle , the left ventricle , the outflow tract and the peri - infarct and peri - ischemia regions . unfortunately , the study was based on real - time pcr analysis , and whenever immunocytochemistry was shown , no double staining for both markers had been performed . other evidence supports the hypothesis that isl1 + cells are not a different population from sca-1 + cpcs . the contemporary expression of sca-1 and isl1 has been described both in a subpopulation of sca-1+/c - kit- cells identified and isolated from adult mouse hearts and in sca-1 + cardiosphere - derived cells obtained from cardiac explants from normal , sham - operated or post - myocardial infarct hearts . in this study , only sca-1+/cd45- cells were also positive for isl1 and increased in number only after an acute myocardial infarct . considering that other authors identified isl1 + cells in adult murine and rat hearts , weinberger et al . used heterozygous isl1-lacz mice , a more sensitive genetic approach , to investigate the presence and localisation of these precursor cells in 30 animals at different time points after birth ( 10 weeks to 18 months ) . they found four different populations of isl1 + cells : i ) clusters of isl1 + neurons were found in the cardiac parasympathetic ganglia of the posterior side of the heart and in the nervous plexus surrounding the pulmonary veins ; ii ) clusters of isl1 + smooth muscle cells were found in the muscular layer above the aortic and pulmonary valve in the proximal part of the aorta and the trunk of the pulmonary artery ( only a few positive cells were present in the aortic valve leaflets ) ; iii ) clusters of isl1 + cardiomyocytes were found in the left ventricular outflow tract region ; iv ) clusters of isl1 + sinoatrial node ( san ) cells were found in the muscular wall between right atrium and vena cava superior . these results support the hypothesis that in the adult heart many cell populations may derive from isl1 + embryonic precursors , as smooth muscle cells , parasympathetic neurons , san cells , but also that , even if reduced in number , isl1 + cardiomyogenic progenitors , may be present in the adult myocardium . many papers have been published on cpcs , and many research groups claimed to have discovered different populations of progenitor cells . it is strange to think that the cardiac tissue possesses a larger number of stem / progenitor cells compared to the other tissues found in our body , and it is hopeful that cpcs may vary in number with external stimuli or with age as it happens for other adult stem cells . the hypothesis that the myocardium is a non - regenerative tissue is not valid anymore , and much evidence supports the new idea that there is at least one cardiac progenitor cell . isl1 + and c - kit+/sca-1 + cells were considered to be two different populations until 2012 , when prof . sussmann , citing our recent paper published in the european journal of histochemistry in an editorial in circulation research , suggested considering the possibility of studying a population of cells positive for both isl1 and c - kit instead of choosing to study only one side of the coin . in the review and in the research paper published in 2009 , we reported the concomitant expression of isl1 , c - kit and sca-1 in a population of cpcs isolated without any sorting or selection and suggested for the first time that there could be a unique population of cells with several subpopulations and that the several markers identified could be only the effect of different cultivation conditions in the different laboratories . other research groups later described the co - expression of isl1 , sca-1 and/or c - kit and we further demonstrated that , in samples from foetal and postnatal human hearts , there was also an isl1 + subpopulation of c - kit+ cells . anversa among the authors , on a study of the presence of c - kit+ cardiac stem cells in embryonic , foetal and neonatal mouse hearts . they demonstrated that these cells can form cardiomyocytes and coronary vessels and , as we have demonstrated in our paper , they are more abundant than isl1 + cells . moreover , they stated that isl1 + cells are present only in the outflow tract , atria and part of the right ventricle and that they are a population of committed cells derived from an immature primitive cell . we can say that there is strong evidence suggesting that c - kit , sca-1 and isl1 may be markers of subpopulations of cpcs that are present in the embryonic , foetal and neonatal heart and persist from after birth until adulthood . the identification of the real immature cardiac precursor expressing the four cell markers ( c - kit , sca-1 , mdr-1 and isl1 ) and above all the identification of a unique isolation protocol is very important in the clinical practice . this immature common cardiac precursor may represent the perfect candidate for cardiac tissue engineering and , hopefully , may be driven pharmacologically to one phenotype or the other . to identify a standardised protocol is extremely important to go further in the studies on cardiac tissue regeneration , and common guidelines are needed in cardiac stem cell therapy . the isolation of stem cells by cell sorting or immunobead selection using surface markers such as c - kit , sca-1 or mdr-1 is important to identify a standardised protocol of isolation , however isl1 may be useful in basic research to follow the fate of cpcs in heart development and myocardium differentiation . moreover , it is extremely important to understand which is the predominant precursor population in the different stages of life and which population should be used in a paediatric patient instead of an adult one . if fate of immature cardiac precursors may be determined pharmacologically , the possibility to regenerate san cells is very important in curing arrhythmia s both in patients with ischemia and malformations . the identification of the different subpopulations in the developing heart may be a fundamental step in identifying the optimal cell line for cell - based therapies . primary diffuse leptomeningeal gliomatosis ( pdlg ) is a rare neoplasm with a short survival time of a few months . we report on a 53-year - old male patient who presented with epileptic seizures , gait disturbance , paraparesis and sensory deficits in the dermatomes t8 - 10 . magnetic resonance imaging ( mri ) revealing numerous spinal and cranial gadolinium - enhancing nodules in the meninges and histopathology led us to diagnose primary diffuse leptomeningeal gliomatosis with who grade iii astrocytic cells . consecutively , the patient underwent craniospinal radiotherapy ( 30 gy ) and 11 sequential cycles of temozolomide . thirteen months later , spinal mri revealed tumor progression . second - line chemotherapy with 5 cycles of irinotecan and bevacizumab did not prevent further clinical deterioration . the patient died twenty - two months after diagnosis , being the longest survival time described thus far with respect to pdlg consisting of astrocytic tumor cells . radiochemotherapy including temozolomide , as established standard therapy for brain malignant astrocytomas , might be valid as a basic therapeutic strategy for this pdlg subtype . primary diffuse leptomeningeal gliomatosis ( pdlg ) is diagnosed when a glioma is located in the subarachnoid space , while intraparenchymal tumor lesions are absent . pdlg must be distinguished from secondary meningeal gliomatosis resulting from a primary gliomatous cns tumor . pdlg , especially when caused by malignant astrocytic cells , is associated with very poor survival . we describe a patient with pdlg consisting of malignant astrocytic cells who underwent combined radio- and chemotherapy leading to the longest survival time described in the literature thus far . a 53-year - old man was referred to the dept . of neurology due to a generalized epileptic seizure and five months later he presented with bilateral sensory deficits at dermatome levels t8 - 10 , paraparesis and gait disturbance . cerebrospinal fluid ( csf ) analysis revealed a high cell count ( 300/3 cells/l ; reference range : 5/3 cells/l ) with atypical cells that could not be further characterized . t1-weighted images ( t1-wi ) revealed spinal and cranial gd - enhancing nodules in the leptomeninges ( figures 1a - e ) and on t2-weighted images ( t2-wi ) an intramedullary edema ( figure 1c ) . note gd - enhanced supra- and in - fratentorial meningeal thickenings of the brain in sagittal t1-w image ( a ) . prior to ra - diochemotherapy , t1-wi reveals lep - tomeningeal gd - enhancement ( b ) and t2-wi shows edema of the myelon on levels c7 , t1 - 2 and t5 - 6 due to minor tumor extensions in the adjacent spinal cord ( c ) . following radiochemotherapy , t1-wi displays reduced leptomeningeal gd - enhancement ( d ) while t2-wi shows less myelon spinal edema on those levels ( e ) . three weeks later , intradural biopsy from a gd - enhancing nodule at l2 - 3 level revealed fibrously - thickened meninges infiltrated by malignant astrocytic tumor cells ( figures 2a - c ) . the tumor was categorized as primary diffuse leptomeningeal gliomatosis ( pdlg ) confirmed by the reference center for brain tumors in dsseldorf , germany . intradural lumbar biopsy displays fibrously - thickened lep - tomeninges infiltrated by pleomorphic neoplastic astrocytic cells ( a ) . the astrocytic tumor cells reveal intensive intracytoplasmic staining with anti - glial fibrillary astrocytic protein . ki67 ( mib-1 ) staining reveals a high proliferation index of up to 20% ( c ) . prior to radiotherapy of the craniospinal axis ( 4 2.5 gy / week , total dose : 30 gy ) , the patient underwent 3 cycles of temozolomide ( tmz ) ( cycle 1 : 150 - 200 mg / mtzm , d1 - 5 , q28d ) . on re - staging , spinal t1-weighted images showed tumor - lesion regression on l5 and spinal t2-wi images demonstrated spinal - edema regression ( compare figures 1c - d with figures 1a - b ) . three weeks after the completion of radiotherapy the patient received eight additional cycles of tmz ( 200 mg / mtzm , d1 - 5 , q28d ) , during which meningeal thickening of the brain regressed . however , spinal mri thirteen months after starting tmz therapy revealed meningeal tumor progression at the lesions on spinal levels c1 - 2 , c7-t2 and t5 - 8 . five cycles of second - line chemotherapy with irinotecan and bevacizumab failed to halt further clinical deterioration . the patient died twenty - two months after the diagnosis of pdlg and exhibited until dead an encouraging karnovsky performance status of 60% . the diagnosis of pdlg is usually established by autopsy but rarely diagnosed prior to death . pdlg of the oligodendroglial and that of the well - differentiated astrocytic tumor type are associated with a considerably longer median survival time than that of the malignant astrocytic tumor type . pdlg was diagnosed in the aforementioned case , as we observed on histopathology leptomeningeally - encapsulated malignant astrocytic cells without primary attachment to the spinal cord or brain parenchyma and gd - enhancing leptomeningeal thickening at the base of the brain and spinal level in mri . various treatment modalities were used for 14 patients suffering from pdlg with malignant astrocytic cells reported in the literature so far ( cf . table 1 ) , demonstrating the lack of a standardized treatment regimen of pdlg with malignant astrocytic cells . although the number of cases is small , the data on their survival times ( cf . table 2 ) might suggest that radiotherapy and temozolomide ( tmz ) as established treatment for newly diagnosed recurrent anaplastic astrocytomas also seem to be valid for pdlg with malignant astrocytic cells . this concept is supported by the observation that radiotherapy alone can prolong the median survival time of five months for patients without any specific therapy to a median survival of 12 months and that integration of tmz in the chemotherapy alone may lead to a prolonged median survival of 15 months . the importance of tmz in the chemotherapy treatment of pdlg is supported by the observation that the median survival time fell to 3 months when the integration of tmz was omitted . clinico - pathological characteristics and treatment of adult patients suffering from pdlg with malignant astrocytic cells including our case 5-fu = 5-fluouracil , adr = adriamycin , auc 5 = carboplatine , bcnu = carmustine , brs = brainstem , bvz = bevacizumab , c = cycle , ca = cytarabine , ccnu = lomustine , cr = cranial , cyc = cyclophosphamide , ddp = cisplatin , eto = etoposide , inn = topotecan , int = irinotecan , irp = raised intracranial pressure , it = intrathecally , mcnu = ramustine , mtx = methotrexate , sc = spinal cord , tmz = temozolomide , top = thiotepa , vincristine , prednison summary of the literature : pdlg with malignant astrocytic cells the prolonged survival of our patient may be due to the addition of tmz to radiation therapy as it resulted in an extent of the median survival time for high grade glioma patients . in addition , the hypofractionated radiotherapy regimen used is supposed to be more effective than conventional fractionated irradiation . additionally , the patient 's good karnofsky performance status may have contributed to our patient 's prolonged survival . in conclusion , we report on partial regression and long survival in a patient with pdlg of the malignant astrocytic type following hypofractionated radiotherapy and tmz . an immediate radiochemotherapy seems crucial for a prolonged survival of pdlg patients in good general condition . the authors disclose no or potential conflict of interest including any financial , personal or other relationships with other people or organizations within the three years of beginning the submitted work that could inappropriately influence or be perceived to influence their work .

cardiac progenitor cells are multipotent stem cells isolated from both embryonic and adult hearts in several species and are able to differentiate at least into smooth muscle cells , endothelial cells and cardiomyocytes . the embryonic origin of these cells has not yet been demonstrated , but it has been suggested that these cells may derive from the first and secondary heart fields and from the neural crest . in the last decade , two different populations of cardiac progenitor or stem cells have been identified and isolated , i.e. , the islet1 positive ( isl1 + ) and c - kit positive ( c - kit+)/stem cell antigen-1 positive ( sca-1 + ) cells . until 2012 , these two populations have been considered two separate entities with different roles and a different origin , but new evidence now suggests a connection between the two populations and that the two populations may represent two subpopulations of a unique pool of cardiac stem cells , derived from a common immature primitive cell . to find a common consensus on this concept is very important in furthering the application of stem cells to cardiac tissue engineering . objectiveprimary diffuse leptomeningeal gliomatosis ( pdlg ) is a rare neoplasm with a short survival time of a few months . there is currently no standardized therapeutic approach for pdlg.materials and methodswe report on a 53-year - old male patient who presented with epileptic seizures , gait disturbance , paraparesis and sensory deficits in the dermatomes t8-10.resultsmagnetic resonance imaging ( mri ) revealing numerous spinal and cranial gadolinium - enhancing nodules in the meninges and histopathology led us to diagnose primary diffuse leptomeningeal gliomatosis with who grade iii astrocytic cells . consecutively , the patient underwent craniospinal radiotherapy ( 30 gy ) and 11 sequential cycles of temozolomide . this regimen led to partial tumor regression . thirteen months later , spinal mri revealed tumor progression . second - line chemotherapy with 5 cycles of irinotecan and bevacizumab did not prevent further clinical deterioration . the patient died twenty - two months after diagnosis , being the longest survival time described thus far with respect to pdlg consisting of astrocytic tumor cells.conclusionsradiochemotherapy including temozolomide , as established standard therapy for brain malignant astrocytomas , might be valid as a basic therapeutic strategy for this pdlg subtype .

in past years , numerous studies have described the role of microalbuminuria ( mau ) as a predictor of cardiovascular disease ( cvd ) and death among subjects with type 2 diabetes ( t2d).13 mau is one of the earliest clinical signs for diabetic nephropathy and a significant risk factor for progression to proteinuria.1 additionally , hypertensive t2d individuals with mau have an increased risk of developing end - stage renal disease ( esrd).4 risk factors known to be associated with cvd and diabetic nephropathy are high blood pressure ( bp ) and elevated glycosylated hemoglobin ( a1c).5,6 achieving adequate bp and glycemic control ( gc ) plays an essential role in preventing renal and cvd events in individuals with t2d . a current secondary analysis from the hispanic health and nutritional examination survey ( hhanes)7 indicated that cuban americans have higher serum cholesterol and systolic bp than puerto ricans and mexican americans . furthermore , compared with other hispanic subgroups , cuban americans have the highest proportion of hypertension ( htn ) and mean serum creatinine levels.8 smith and barnett9 examined the national center for health statistics ( nchs ) from 1996 to 1997 and concluded that cuban americans 35 years of age and older have the highest percentage of diabetes - related deaths compared with other hispanics . although previous studies have shown significant differences and diversity within hispanics , further studies conducted in the cuban american population are scarce . over the past decade , the prevalence of t2d has increased , especially among cuban americans who have a higher incidence of diabetes ( 8.2% ) compared with 6.6% of non - hispanic whites.10 the high incidence of t2d combined with an increased risk for developing diabetes complications warrants further examination of the cuban american population . screening for mau can detect individuals at risk for renal dysfunction and cvd events and possibly reduce the burden associated with diabetes complications . therefore , the purpose of this study was to investigate to what degree the coexistence of htn and poor gc influences the likelihood of having mau among cuban americans with t2d . it was hypothesized that individuals with t2d , htn , and poor gc will have an increased likelihood to test positive for mau . it was further hypothesized that this association will be stronger after controlling for confounding variables . this was a cross - sectional study conducted in cuban americans with and without t2d . data from a complete sample set of cuban americans with and without t2d were used in the present study . recruitment of participants was conducted in alternate phases of potential subjects with and without t2d , age matching subjects by age groups . during a 1-year period , approximately 10,000 letters outlining the study were mailed to subjects aged 30 years or older with and without diabetes . letters were sent in english and spanish and included an invitation flyer to which interested participants could respond . the participants were initially recruited by random selection ( every tenth address ) from a randomly generated mailing list . this company provided a mailing list of cuban americans identified as with and without t2d from miami - dade and broward counties , florida . three percent ( n = 300 ) of the letters were returned due to unknown addresses . from interested participants were initially interviewed on the phone , at which time the study purpose was explained and the age and gender of the responders were determined . to ascertain t2d status , only 18 subjects did not qualify for the study for not being cuban americans ( n = 2 ) , age younger than 30 years ( n = 9 ) , and having other chronic illnesses ( n = 7 ) . if a subject was determined to be eligible , then their participation was requested at the human nutrition laboratory at florida international university ( fiu ) . participants were instructed to refrain from smoking , consuming any food and beverages except water , and doing any unusual exercise for at least 8 hours prior to their blood collection . the purpose and protocol of the study were explained to the subjects , and their written consent , either in spanish or english , was obtained prior to the commencement of the study . seven participants reported not having diabetes but were reclassified because their lab results classified them as having t2d according to american diabetes association ( ada ) standards . for the data analysis , subjects with caloric intakes > 5000 kcals ( n = 2 ) and missing a1c levels ( n = 2 ) were excluded . for two participants , we were unable to perform a1c analysis . in total , we included only the data from subjects with t2d ( n = 179 ) who were aged 30 years and older . a sociodemographic questionnaire was given to each participant to complete , which included questions related to age , gender , smoking status , medications for diabetes , htn , and cholesterol . height and weight were measured using a seca balance scale ( seca corp , columbia , md , usa ) . body mass index ( bmi ) was calculated as weight in kg / height in m. bp was measured twice then averaged in participants in a sitting position after a 15-minute rest using a random zero sphygmomanometer ( tycos 5090 - 02 welch allyn pocket aneroid sphygmomanometer , arden , nc , usa ) and a stethoscope ( littmann cardiology , 3 m , st paul , mn , usa ) . htn was defined as follows : systolic bp 140 mm hg systolic or diastolic bp 90 mm hg or using antihypertensive treatment.11 dietary intake was measured using a validated semiquantitative food frequency questionnaire ( ffq ) developed by willett et al.12 this ffq has also been validated by nath and huffman13 exclusively for the cuban american population . participants self - reported average consumption of specified amounts of various foods over the past year and chose from frequency responses ranging from never to six or more servings per day . in addition to food items , the ffq included questions about type and duration of vitamin / mineral supplement use , alcohol consumption , and specific details about fat , salt , and sugar used in cooking and as condiments . macro- and micronutrient intake was calculated by multiplying frequency of consumption by the nutrient value of the food item obtained from the harvard university food composition database ( boston , ma , usa ) . venous blood ( 20 ml ) was collected from each subject after an overnight fast ( at least 8 hours ) by a certified phlebotomist using standard laboratory techniques . blood samples were collected into a vacutainer serum separator tube ( sst ) ( becton , dickinson and company , franklin lakes , nj ) for analysis of lipids and another tube containing ethylenediamine tetraacetic acid to analyze a1c . after coagulation was completed ( 3045 minutes ) , the sst was centrifuged at 2500 rpm for 30 minutes . lipid panel was assayed by enzymatic methods , and a1c percentages were measured from whole blood samples with the roche tina - quant method by laboratory corporation of america , miami , fl , usa ( labcorp ) . poor gc was defined according to the ada standards ( a1c > 7%).14 fresh , single - voided , first morning urine samples were collected from each participant to determine mau by a semi - quantitative assay ( immunodip , diagnostic chemicals limited , oxford , ct , usa ) . immunodip urinary albumin test uses a monoclonal antibody against human serum albumin to detect mau . the study conducted by davidson et al15 was designed to evaluate the clinical performance of the immunodip dipstick compared with a reference measure recommended by the ada for detecting mau ( albumin : creatinine ratio < 30 ug / mg [ negative ] ; > 30 ug / mg [ positive ] ) determined by laboratory techniques ( quest hitachi 717 autoanalyzer ) . additionally , results from the immunodip were compared with quantitatively measured albumin concentrations as a secondary outcome . urinary albumin concentrations were considered < 18 mg / l ( negative ) and > 18 mg / l ( positive ) . screening for mau with immunodip exhibited a sensitivity of 96% and specificity of 80% when compared with albumin : creatinine ratio > 30 ug / mg . when immunodip was examined against quantitatively measured albumin concentrations , the dipstick yielded a sensitivity of 95% and specificity of 94% . recommendations from the national academy of clinical biochemistry ( nacb ) for the diagnosis and management of diabetes indicated that a useful semiquantitative screening test for mau should have a sensitivity > 95%.16 the immunodip dipstick fulfilled the requirements from the nacb as a screening tool to detect mau . in our study , this cut - off value was established by the manufacturer ( immunodip , diagnostic chemicals ltd ) and corresponded to albumin : creatinine ratio > 30 ug / mg values for mau detection.15 all analyses were performed using spss version 17 ( spss inc . , chicago , il , usa ) . t - tests and chi - square tests were performed to compare means and proportion differences between subjects with and without mau . unadjusted odds ratios and logistic regression analysis were conducted to investigate the extent to which htn and gc are associated with an increased likelihood of having mau . controlled variables included in the logistic regression analysis were age , gender , bmi , known duration of diabetes , total cholesterol levels , diabetes and cholesterol medications , smoking , total kcal and protein intake , and intake of potassium , phosphorous , and sodium . this was a cross - sectional study conducted in cuban americans with and without t2d . data from a complete sample set of cuban americans with and without t2d were used in the present study . recruitment of participants was conducted in alternate phases of potential subjects with and without t2d , age matching subjects by age groups . during a 1-year period , approximately 10,000 letters outlining the study were mailed to subjects aged 30 years or older with and without diabetes . letters were sent in english and spanish and included an invitation flyer to which interested participants could respond . the participants were initially recruited by random selection ( every tenth address ) from a randomly generated mailing list . this company provided a mailing list of cuban americans identified as with and without t2d from miami - dade and broward counties , florida . three percent ( n = 300 ) of the letters were returned due to unknown addresses . from the remaining delivered mail , 4% ( n = 388 ) responded . interested participants were initially interviewed on the phone , at which time the study purpose was explained and the age and gender of the responders were determined . to ascertain t2d status , only 18 subjects did not qualify for the study for not being cuban americans ( n = 2 ) , age younger than 30 years ( n = 9 ) , and having other chronic illnesses ( n = 7 ) . if a subject was determined to be eligible , then their participation was requested at the human nutrition laboratory at florida international university ( fiu ) . participants were instructed to refrain from smoking , consuming any food and beverages except water , and doing any unusual exercise for at least 8 hours prior to their blood collection . the purpose and protocol of the study were explained to the subjects , and their written consent , either in spanish or english , was obtained prior to the commencement of the study . seven participants reported not having diabetes but were reclassified because their lab results classified them as having t2d according to american diabetes association ( ada ) standards . for the data analysis , subjects with caloric intakes > 5000 kcals ( n = 2 ) and missing a1c levels ( n = 2 ) were excluded . for two participants , in total , we included only the data from subjects with t2d ( n = 179 ) who were aged 30 years and older . a sociodemographic questionnaire was given to each participant to complete , which included questions related to age , gender , smoking status , medications for diabetes , htn , and cholesterol . height and weight were measured using a seca balance scale ( seca corp , columbia , md , usa ) . body mass index ( bmi ) was calculated as weight in kg / height in m. bp was measured twice then averaged in participants in a sitting position after a 15-minute rest using a random zero sphygmomanometer ( tycos 5090 - 02 welch allyn pocket aneroid sphygmomanometer , arden , nc , usa ) and a stethoscope ( littmann cardiology , 3 m , st paul , mn , usa ) . htn was defined as follows : systolic bp 140 mm hg systolic or diastolic bp 90 mm hg or using antihypertensive treatment.11 dietary intake was measured using a validated semiquantitative food frequency questionnaire ( ffq ) developed by willett et al.12 this ffq has also been validated by nath and huffman13 exclusively for the cuban american population . participants self - reported average consumption of specified amounts of various foods over the past year and chose from frequency responses ranging from never to six or more servings per day . in addition to food items , the ffq included questions about type and duration of vitamin / mineral supplement use , alcohol consumption , and specific details about fat , salt , and sugar used in cooking and as condiments . macro- and micronutrient intake was calculated by multiplying frequency of consumption by the nutrient value of the food item obtained from the harvard university food composition database ( boston , ma , usa ) . venous blood ( 20 ml ) was collected from each subject after an overnight fast ( at least 8 hours ) by a certified phlebotomist using standard laboratory techniques . blood samples were collected into a vacutainer serum separator tube ( sst ) ( becton , dickinson and company , franklin lakes , nj ) for analysis of lipids and another tube containing ethylenediamine tetraacetic acid to analyze a1c . after coagulation was completed ( 3045 minutes ) , the sst was centrifuged at 2500 rpm for 30 minutes . lipid panel was assayed by enzymatic methods , and a1c percentages were measured from whole blood samples with the roche tina - quant method by laboratory corporation of america , miami , fl , usa ( labcorp ) . fresh , single - voided , first morning urine samples were collected from each participant to determine mau by a semi - quantitative assay ( immunodip , diagnostic chemicals limited , oxford , ct , usa ) . immunodip urinary albumin test uses a monoclonal antibody against human serum albumin to detect mau . the study conducted by davidson et al15 was designed to evaluate the clinical performance of the immunodip dipstick compared with a reference measure recommended by the ada for detecting mau ( albumin : creatinine ratio < 30 ug / mg [ negative ] ; > 30 ug / mg [ positive ] ) determined by laboratory techniques ( quest hitachi 717 autoanalyzer ) . additionally , results from the immunodip were compared with quantitatively measured albumin concentrations as a secondary outcome . urinary albumin concentrations were considered < 18 mg / l ( negative ) and > 18 mg / l ( positive ) . screening for mau with immunodip exhibited a sensitivity of 96% and specificity of 80% when compared with albumin : creatinine ratio > 30 ug / mg . when immunodip was examined against quantitatively measured albumin concentrations , the dipstick yielded a sensitivity of 95% and specificity of 94% . recommendations from the national academy of clinical biochemistry ( nacb ) for the diagnosis and management of diabetes indicated that a useful semiquantitative screening test for mau should have a sensitivity > 95%.16 the immunodip dipstick fulfilled the requirements from the nacb as a screening tool to detect mau . in our study , this cut - off value was established by the manufacturer ( immunodip , diagnostic chemicals ltd ) and corresponded to albumin : creatinine ratio > 30 ug / mg values for mau detection.15 t - tests and chi - square tests were performed to compare means and proportion differences between subjects with and without mau . unadjusted odds ratios and logistic regression analysis were conducted to investigate the extent to which htn and gc are associated with an increased likelihood of having mau . controlled variables included in the logistic regression analysis were age , gender , bmi , known duration of diabetes , total cholesterol levels , diabetes and cholesterol medications , smoking , total kcal and protein intake , and intake of potassium , phosphorous , and sodium . mau was present in 26% ( n = 47 ) of cuban americans with t2d . there was a significantly higher percentage of subjects with mau classified as hypertensive ( p = 0.038 ) and taking diabetes medication ( p = 0.039 ) compared with those without mau . additionally , subjects who tested positive for mau had significantly higher a1c levels ( p = 0.002 ) than those with normoalbuminuria ( table 1 ) . unadjusted odds ratios indicated that subjects with poor gc were 3.96 times more likely to have positive mau if they had htn compared with those without htn ( p = 0.014 ; 95% confidence interval [ ci ] 1.25 , 12.5 ) ( figure 1 ) . logistic regression analysis showed that after controlling for covariates , subjects with poor gc were 6.76 times more likely to have mau if they had htn compared with those without htn ( p = 0.004 ; 95% ci 1.83 , 23.05 ) ( table 2 ) . the results of this study showed that htn and poor gc are major contributors to increasing the likelihood of having mau among cuban americans with t2d . the combination and/or interaction of these factors over time might increase the risk for progression to proteinuria / esrd and cvd in this population . our findings are supported by the multicenter study conducted in europe that found that the likelihood of having mau increases in patients when htn and poor gc are present along with other coexisting risk factors for cvd.17 ravid et al5 reported from their longitudinal study that a combination of risk factors , including abnormal bp , plasma cholesterol , a1c levels , high bmi , and male gender , identifies a group of individuals for poor renal and cardiovascular outcomes . maintaining adequate the uk prospective diabetes study ( ukpds),18 a longitudinal study with a 10-year median follow - up , showed that exposure over time to hyperglycemia was associated with diabetes complications in subjects with t2d . this study also indicated that for every 1% reduction in a1c level , the risk for microvascular complications decreased by 37% and diabetes - related death by 21% . a study by thomaseth et al19 showed that in hypertensive t2d subjects with incipient diabetic nephropathy , both tight bp control and optimal gc delay the progression of glomerular filtration rate deterioration . additionally , hypertensive individuals with t2d under tight bp control experience a reduction in risk for microvascular and macrovascular complications.20 the mechanism and pathways involving mau with diabetic nephropathy and cvd are not fully understood . they may be interrelated with endothelial dysfunction and inflammation.21 stehouwer et al22 examined the relationship between endothelial dysfunction and inflammation with mau and risk of death in a prospective study among subjects with t2d . the results of that study showed that those participants with mau , endothelial dysfunction , and inflammation had an increased risk in mortality ; however , the associations of these variables with risk of mortality were independent from each other . hyperglycemia and obesity were associated with an increase in markers of endothelial dysfunction and inflammation activity,22 possibly indicating that the combinations of these factors may be interrelated in increasing the risk of death . the usual course of mau is progressive ; however , not all t2d individuals with mau will develop macroalbuminuria . several authors have documented remission and/or regression of mau in subjects with t2d.2327 antihypertensive therapy has been shown to reduce or slow the progression of diabetic nephropathy . in a 6-year prospective study , remission and regression to normoalbuminuria were observed in about 50% of t2d individuals.23 other factors independently linked with remission and/or regression of mau were proper bp and gc and short duration of mau . the study by chan et al24 with a 5-year mean follow - up of t2d patients with mau treated with angiotensin - converting enzyme ( ace ) inhibitor medications showed a 13% reduction in urinary albumin excretion ( uae ) . in addition , the authors pointed out the main role that bp and gc play in renal function . t2d individuals treated with angiotensin receptor blockers ( arbs ) ( eg , irbesartan and valsatran ) experienced a 38% and 44% reduction in uae over 2-year and 6-month follow - ups , respectively.25,26 evidence from another prospective study with a 7.8-year follow - up indicated that 30% of the participants on antihypertensive therapy achieved remission to normoalbuminuria.27 additionally , the odds for remission to normoalbuminuria increased in these participants with every 1% decrease in a1c level . furthermore , in a study conducted by mogensen et al,28 urinary albumin : creatinine ratio was decreased by 50% after a 3-month combination treatment with arb and ace inhibitors in hypertensive t2d individuals with mau . remission and/or regression of mau may not only conserve renal function but also reduce the risk of cvd . testing for mau has been recommended by the ada in individuals with t2d to be performed at diagnosis of diabetes and every year afterwards.29 the immunodip dipstick is a rapid and easy screening test to perform , does not involve equipment and/or skilled personnel , exhibits a high sensitivity , requires only a random urine sample ( first morning urine is recommended ) , and has a relatively low cost . however , this test does not quantify urinary albumin values and requires confirmation with a secondary analysis . this method can be used in a physician s office and/or research setting as a first screening tool to detect the presence of mau . the clinical significance of early screening and monitoring for mau is possibly to improve individuals prognosis for microvascular and macrovascular complications , especially among t2d individuals with other concomitant conditions . first , due to the cross - sectional design of the study , single - voided urine was collected to measure mau , and we were not able to determine the cause second , due to the relatively small sample size in this study , our sample of cuban americans with t2d is not representative of the entire cuban american population living in the usa . nevertheless , to our knowledge , this is the first and only study that has examined the relationship between mau , htn , and gc in this hispanic subgroup , which has an increased risk for t2d and cvd . first , due to the cross - sectional design of the study , single - voided urine was collected to measure mau , and we were not able to determine the cause second , due to the relatively small sample size in this study , our sample of cuban americans with t2d is not representative of the entire cuban american population living in the usa . nevertheless , to our knowledge , this is the first and only study that has examined the relationship between mau , htn , and gc in this hispanic subgroup , which has an increased risk for t2d and cvd . early detection of mau in this population may provide more valuable treatment and improve individuals renal and cvd outcomes . the therapeutic goals and strategies for mau should focus on preventing long - term complications associated with t2d , such as kidney and heart diseases . further investigations need to be carried out to fully understand the mechanism and absolute cvd risk that an individual with t2d has when mau coexists with other comorbidities . cancer is the second leading cause of mortality and morbidity in both developed and developing countries . in india , cancer prevalence is estimated around 2.5 million , with over 0.8 million new cases and 0.5 million deaths occurring each year . there is an increase in the incidence of breast cancer and found to be gradually overtaking cancer of the cervix . breast self - examination ( bse ) is an important screening measure for detecting breast cancer . there is evidence that women who correctly practice bse monthly are more likely to detect a lump in the early stage of its development , and early diagnosis has been reported to influence early treatment , to yield a better survival rate . thus the present study aimed at identifying the level of knowledge and practice of bse among degree female students who are the citizen of the future and they can teach their family members , neighbors , friends and the community which helps the people to detect breast cancer in early stage . thus the morbidity or mortality can be reduced . in the current study only one participant was practicing bse occasionally so incorporating the bse concept in the degree education curriculum is very useful and helpful . the present study aimed at assessing the level of knowledge and the effectiveness of planned teaching program among degree female students on bse . it is a patient - centred , inexpensive and noninvasive method of screening for breast cancer . based on increased incidence of breast cancer and unawareness of bse among young women , researcher felt a need to provide awareness of bse among young women and can be reduced the incidence and prevalence of breast cancer in future . the objectives of the study were to : to assess the level of knowledge of degree college female students on bse.to determine the effectiveness of planned teaching program among degree college female students on bse.to find the association between pretest knowledge and selected demographical variables . to assess the level of knowledge of degree college female students on bse . to determine the effectiveness of planned teaching program among degree college female students on bse . the study attempted to test following hypotheses all the hypotheses were tested at 0.05 level of significance . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students.there will be a significant association between pretest knowledge score and selected demographical variables . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students . the study assumed that : the degree college female students will have some knowledge on bse.feel free to express their attitude toward bse.bse helps in early detection of breast cancer . the degree college female students will have some knowledge on bse . feel free to express their attitude toward bse . independent variables : teaching program on bse . selected variables : age , education , parent 's education and exposure to mass media . the present study aimed at assessing the level of knowledge and the effectiveness of planned teaching program among degree female students on bse . it is a patient - centred , inexpensive and noninvasive method of screening for breast cancer . based on increased incidence of breast cancer and unawareness of bse among young women , researcher felt a need to provide awareness of bse among young women and can be reduced the incidence and prevalence of breast cancer in future . the objectives of the study were to : to assess the level of knowledge of degree college female students on bse.to determine the effectiveness of planned teaching program among degree college female students on bse.to find the association between pretest knowledge and selected demographical variables . to assess the level of knowledge of degree college female students on bse . to determine the effectiveness of planned teaching program among degree college female students on bse . the study attempted to test following hypotheses all the hypotheses were tested at 0.05 level of significance . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students.there will be a significant association between pretest knowledge score and selected demographical variables . there will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students . the study assumed that : the degree college female students will have some knowledge on bse.feel free to express their attitude toward bse.bse helps in early detection of breast cancer . the degree college female students will have some knowledge on bse . feel free to express their attitude toward bse . independent variables : teaching program on bse . selected variables : age , education , parent 's education and exposure to mass media . a pre - experimental one group pretestpost - test study was conducted among degree female students from selected colleges of udupi district . a cluster sampling technique was used to select the college and 40 students selected by convenient method from all the streams of study . demographic proforma consisted of age , stream of study , education and occupation of parents , income of parents and sources of information on bse . a structured questionnaire consisted of 25 multiple choice questions ( mcq ) was developed to assess the knowledge on bse . knowledge scores were categorized into poor ( 0 - 8 ) , average ( 9 - 16 ) and good ( 17 - 25 ) . planned teaching program . lesson plan with power point presentation given for validation contained basic anatomy and physiology , risk factors for breast cancer and steps of bse . validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . data was collected by administering knowledge questionnaire on bse and planned teaching program was introduced for them . on 8 day post - test was done by administering the same tool . the data was analyzed using descriptive ( frequency and percentage ) and inferential statistics based on the objectives and hypotheses . demographic proforma consisted of age , stream of study , education and occupation of parents , income of parents and sources of information on bse . a structured questionnaire consisted of 25 multiple choice questions ( mcq ) was developed to assess the knowledge on bse . knowledge scores were categorized into poor ( 0 - 8 ) , average ( 9 - 16 ) and good ( 17 - 25 ) . planned teaching program . lesson plan with power point presentation given for validation contained basic anatomy and physiology , risk factors for breast cancer and steps of bse . validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . demographic proforma consisted of age , stream of study , education and occupation of parents , income of parents and sources of information on bse . a structured questionnaire consisted of 25 multiple choice questions ( mcq ) was developed to assess the knowledge on bse . knowledge scores were categorized into poor ( 0 - 8 ) , average ( 9 - 16 ) and good ( 17 - 25 ) . planned teaching program . lesson plan with power point presentation given for validation contained basic anatomy and physiology , risk factors for breast cancer and steps of bse . validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . administrative permission was obtained from the principal of selected colleges . written consent was obtained from the study participants . data was collected by administering knowledge questionnaire on bse and planned teaching program was introduced for them . on 8 day post - test was done by administering the same tool . the data was analyzed using descriptive ( frequency and percentage ) and inferential statistics based on the objectives and hypotheses . the data presented in table 1 shows that among 40 samples , the majority ( 52.5% ) of the samples were in the age group of 18 - 19 years and majority ( 90% ) of them were studying in basic science group . sample characteristics only 35% of them were heard about bse through mass media and 8.5% of participant ( only one participant ) were practiced bse only one time at the time of study . the description of knowledge scores shows that 72.5% of students had average knowledge on bse in pre test and 85% of students had good knowledge score in post - test [ figure 1 , table 2 ] . t test computed to test the effectiveness of planned teaching program on bse ( t=12.46 ) shown in table 3 . percentage distribution of sample based on knowledge score frequency and percentage of knowledge score computation of effectiveness of planned teaching program the computed to find the association between knowledge and selected variables shows no significant association between knowledge and selected variables . hence the null hypotheses was accepted and research hypotheses was rejected shown in table 4 . a survey conducted among nurses and midwives in turkey found that among 80 samples only 52% of samples performed bse , no significant relation was found between sociodemographic factor and bse . a study conducted in chennai by s. aruna supported this study as no significant association found between demographic variables and level of knowledge of breast cancer and bse among working women . study conducted among resettlement colony women , shows that they have poor knowledge on breast cancer and risk factors , warning signs and early detection procedure . the world health organization stresses on promoting awareness in the community and encouraging early diagnosis of breast cancer , especially for women aged 40 - 69 years who are attending primary health care centres of hospitals for other reason , by offering clinical breast examination . there are other many methods to detect the breast cancer but those are expensive for the community . breast - related matters are sensitive issues for few females ; unless the breast lesions starts bothering them they may not seek medical attention . bse is the cheapest and convenient method to detect the breast cancer in the early stage . in this study majority of the samples acquired good knowledge on bse . bse plays a major role in early detection and prevention or prompt treatment of breast cancer . by giving teaching to the young girls , they can teach their mother and siblings so that the incidence of the breast cancer may be reduced . it is essential task of each and every woman to do bse and protect herself and her family . the nurse has to play an important role in health promotion and it is only possible when she teaches or educates her client . however , the teaching of bse can help women to be alert to any abnormal changes in their breasts and seek medical advice immediately .

purpose : to investigate to what degree the presence of hypertension ( htn ) and poor glycemic control ( gc ) influences the likelihood of having microalbuminuria ( mau ) among cuban americans with type 2 diabetes ( t2d).methods : a cross - sectional study conducted in cuban americans ( n = 179 ) with t2d . participants were recruited from a randomly generated mailing list purchased from knowledge - base marketing , inc . blood pressure ( bp ) was measured twice and averaged using an adult size cuff . glycosylated hemoglobin ( a1c ) levels were measured from whole blood samples with the roche tina - quant method . first morning urine samples were collected from each participant to determine mau by a semiquantitative assay ( immunodip).results : mau was present in 26% of cuban americans with t2d . a significantly higher percentage of subjects with ma had htn ( p = 0.038 ) and elevated a1c ( p = 0.002 ) than those with normoalbuminuria . logistic regression analysis showed that after controlling for covariates , subjects with poor gc were 6.76 times more likely to have mau if they had hypertension compared with those without hypertension ( p = 0.004 ; 95% confidence interval [ ci ] : 1.83 , 23.05).conclusion : the clinical significance of these findings emphasizes the early detection of mau in this hispanic subgroup combined with bp and good gc , which are fundamentals in preventing and treating diabetes complications and improving individuals renal and cardiovascular outcomes . introduction : breast cancer accounts for 19 - 34% of all cancer cases among women in india . there is high mortality due to late stage diagnosis as patients usually present at an advanced stage because of lack of awareness and nonexistent breast cancer screening programs . early detection and prompt treatment offer the greatest chance of long - term survival and breast self - examination ( bse ) seems to be a important viable optional substitute for early detection of cancer.objectives:1 ) to assess the level of knowledge of degree college female students on bse . 2 ) to determine the effectiveness of planned teaching program among degree college female students on bse . 3 ) to find the association between pretest knowledge and selected demographic variables.materials and methods : pre - experimental one group pretestpost - test design was carried out among 40 degree female students by using cluster sampling method from selected colleges of udupi district.results:the data analyzed showed that majority ( 52% ) of them was in the age group of 18 - 19 years and 72% of them were had average knowledge on bse in the pretest score . out of 40 participants only one student was performing bse occasionally.conclusions:awareness regarding breast self examination among young generations is useful and it is the most important viable tool for early detection .

der p 1 was isolated from house dust mite fecal pellets ( allergon ) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti der p 1 mab ( clone 4c1 ; indoor biotechnologies ) , removal of contaminating serine proteases on immobilized soybean trypsin inhibitor ( sigma chemical co. ) , and finally fast protein liquid chromatography ( fplc ) to remove low - molecular - mass contaminants . the purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer ( applied biosystems , inc . ) , sds - page analysis ( 15% gel ) , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 ( l - trans - epoxysuccinyl - leucylamido [ 4-guanidino]butane ) . protein concentration was determined using a bicinchoninic acid ( bca ) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e ( 280 nm ) = 16.4 . before use , der p 1 was preactivated with 5 mm cysteine ( sigma chemical co. ) to regenerate its thiol group , which becomes oxidized during purification . the catalytic activity of der p 1 was ascertained in a continuous rate ( kinetic ) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl ( boc)-gln - ala - arg7-amino-4-methyl - coumarin ( amc ; reference 6 ) . to block the proteolytic activity of cysteine - activated der p 1 , 1,000-fold molar excess of e-64 ( sigma chemical co. ) was used ; a similar molar ratio of the cysteine protease inhibitor iodoacetamide ( sigma chemical co. ) was used as another sulfhydryl reactive agent . the cells ( 2 10 ) were suspended in rpmi ( gibco life technologies ) and stimulated for 3 d at 37c with con a ( 5 g / ml final concentration ) in the presence of il-2 ( 100 u / ml ) in a humidified atmosphere of 5% co2 . cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 ( preactivated with 5 mm cysteine ) for 1 h at 37c in a total volume of 200 l serum - free aim v medium ( gibco life technologies ) . the cells were then resuspended in rpmi containing 2% fcs , stained for 45 min at room temperature in the dark with fitc - labeled anti mouse cd25 mab ( clone amt-13 ; sigma chemical co. ) , and fixed with 5% formaldehyde . the expression of other t cell surface markers , namely cd3 , cd4 , and cd8 , was monitored in the same way using appropriate pe- or fitc - labeled antibodies ( clone kt3 , beckman coulter ; and clones yts191.1 and kt15 [ serotec ltd . five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1 , 10 g of e-64blocked der p 1 , 10 g of iodoacetamide - blocked der p 1 , 10 g of ova ( as proteolytically inactive antigen ; sigma chemical co. ) , or 10 g of ova with e-64 , respectively . a tail bleed was obtained 1 wk before the start of immunization ( prebleed ) , and a total bleed was obtained by cardiac puncture 1 wk after the last injection ( final bleed ) . the proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above . serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass . the optimal dilutions used here were 1/10 for detecting total ige , der p 1specific ige , and ova - specific ige and 1/20,000 , 1/40,000 , and 1/250 for detecting der p 1specific igg , igg1 , and igg2b , respectively . mouse ige ( clone r35 - 72 ; pharmingen ) as capture antibody and a second biotinylated monoclonal anti der p 1specific ige , ova - specific ige ( measured using samples that have been depleted of igg on a protein g column [ pharmacia ] ) , and der p 1specific igg , igg1 , and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated ( for ige clone r35 - 118 and igg1 clone a85 - 1 [ pharmingen ] and for igg2b clone ab275 [ the binding site ] ) or alkaline phosphatase conjugated isotype - specific antibodies ( for igg ; sigma chemical co. ) . alkaline phosphatase conjugated extravidine ( sigma chemical co. ) was used in conjunction with biotinylated antibodies . unpaired student 's t test was used to compare levels of antibody responses between the different immunization groups ; p < 0.05 was considered significant . der p 1 was isolated from house dust mite fecal pellets ( allergon ) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti der p 1 mab ( clone 4c1 ; indoor biotechnologies ) , removal of contaminating serine proteases on immobilized soybean trypsin inhibitor ( sigma chemical co. ) , and finally fast protein liquid chromatography ( fplc ) to remove low - molecular - mass contaminants . the purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer ( applied biosystems , inc . ) , sds - page analysis ( 15% gel ) , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 ( l - trans - epoxysuccinyl - leucylamido [ 4-guanidino]butane ) . protein concentration was determined using a bicinchoninic acid ( bca ) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e ( 280 nm ) = 16.4 . before use , der p 1 was preactivated with 5 mm cysteine ( sigma chemical co. ) to regenerate its thiol group , which becomes oxidized during purification . the catalytic activity of der p 1 was ascertained in a continuous rate ( kinetic ) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl ( boc)-gln - ala - arg7-amino-4-methyl - coumarin ( amc ; reference 6 ) . to block the proteolytic activity of cysteine - activated der p 1 , 1,000-fold molar excess of e-64 ( sigma chemical co. ) was used ; a similar molar ratio of the cysteine protease inhibitor iodoacetamide ( sigma chemical co. ) was used as another sulfhydryl reactive agent . spleen t cells were obtained from c57bl/6j mice using standard procedures . the cells ( 2 10 ) were suspended in rpmi ( gibco life technologies ) and stimulated for 3 d at 37c with con a ( 5 g / ml final concentration ) in the presence of il-2 ( 100 u / ml ) in a humidified atmosphere of 5% co2 . cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 ( preactivated with 5 mm cysteine ) for 1 h at 37c in a total volume of 200 l serum - free aim v medium ( gibco life technologies ) . the cells were then resuspended in rpmi containing 2% fcs , stained for 45 min at room temperature in the dark with fitc - labeled anti mouse cd25 mab ( clone amt-13 ; sigma chemical co. ) , and fixed with 5% formaldehyde . the expression of other t cell surface markers , namely cd3 , cd4 , and cd8 , was monitored in the same way using appropriate pe- or fitc - labeled antibodies ( clone kt3 , beckman coulter ; and clones yts191.1 and kt15 [ serotec ltd . five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1 , 10 g of e-64blocked der p 1 , 10 g of iodoacetamide - blocked der p 1 , 10 g of ova ( as proteolytically inactive antigen ; sigma chemical co. ) , or 10 g of ova with e-64 , respectively . a tail bleed was obtained 1 wk before the start of immunization ( prebleed ) , and a total bleed was obtained by cardiac puncture 1 wk after the last injection ( final bleed ) . the proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above . serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass . the optimal dilutions used here were 1/10 for detecting total ige , der p 1specific ige , and ova - specific ige and 1/20,000 , 1/40,000 , and 1/250 for detecting der p 1specific igg , igg1 , and igg2b , respectively . mouse ige ( clone r35 - 72 ; pharmingen ) as capture antibody and a second biotinylated monoclonal anti der p 1specific ige , ova - specific ige ( measured using samples that have been depleted of igg on a protein g column [ pharmacia ] ) , and der p 1specific igg , igg1 , and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated ( for ige clone r35 - 118 and igg1 clone a85 - 1 [ pharmingen ] and for igg2b clone ab275 [ the binding site ] ) or alkaline phosphatase conjugated isotype - specific antibodies ( for igg ; sigma chemical co. ) . alkaline phosphatase conjugated extravidine ( sigma chemical co. ) was used in conjunction with biotinylated antibodies . unpaired student 's t test was used to compare levels of antibody responses between the different immunization groups ; p < 0.05 was considered significant . der p 1 is a 25-kd cysteine protease whose structure has been modeled 7 on the crystal structure of papain , with which it shows considerable sequence similarities , most notably for residues involved in the enzyme active site 8 . the proteolytic activity of der p 1 can be inhibited by e-64 , the class - specific inhibitor of microbial origin 9 . this inhibition is brought about when cysteine within the der p 1 active site forms a thioether covalent bond with the epoxy group of e-64 . this is an irreversible process that does not lead to significant structural changes , as evidenced by crystallographic studies of a papain e-64 complex 10 . we have purified der p 1 from fecal pellets using a multistep procedure and confirmed its purity by nh2-terminal sequencing , sds - page analysis , and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and inhibited by e-64 and iodoacetamide ( fig . 1 ) . we have recently shown that der p 1 selectively cleaves human cd25 from the surfaces of peripheral blood t cells 4 . here we demonstrate that der p 1 also selectively cleaves cd25 from cultured mouse spleen t cells ( fig . 2 ) , which is not surprising given the high degree of sequence homology that exists between human 11 and mouse 12 cd25 . this observation has therefore provided the justification for using this animal species for testing our hypothesis , namely that the proteolytic activity of der p 1 is a major contributor to its allergenicity . intraperitoneal immunization of groups of 10 cba / j mice with either proteolytically active or inactive ( e-64blocked ) der p 1 over a 6-wk period showed a statistically significant enhancement in total ige ( p < 0.01 ) and der p 1specific ige ( p < 0.02 ) responses in animals immunized with proteolytically active der p 1 . this effect was ige specific , as der p 1specific igg , igg1 , and igg2b responses increased to the same extent with proteolytically active or inactive der p 1 ( fig . we are not sure why the igg1 response did not follow that of ige , as these two isotypes are considered to be coregulated in the mouse . however , the ige - restricted enhancement seen in response to immunization with proteolytically active der p 1 does suggest a mechanism that is unique to ige isotype switching / synthesis . furthermore , our control experiments clearly show that the ige - specific effect observed here is not due to e-64 exerting a suppressive influence on ige production by a mechanism that is independent of its binding to the der p 1 enzyme active site ( fig . , suppression of total ige ( p < 0.04 ) and der p 1specific ige ( p < 0.05 ) productions was also obtained when the proteolytic activity of der p 1 was blocked with iodoacetamide , another sulfhydryl reactive agent . second , the ige antibody response to ova , a proteolytically inactive antigen , was not suppressed when the animals were immunized with ova plus e-64 . our results are direct evidence that the cysteine protease activity of der p 1 induces a significant increase in ige responses . such an effect is clearly consistent with the ability of der p 1 to proteolytically cleave mouse cd25 and induce a th2 response by modulating the balance between il-4 and ifn- 13 . the recent demonstration in mice that leishmania mexicana cysteine proteinase deficient mutants potentiate a th1 response , compared with the th2 response normally seen in response to infection with wild - type parasite 14 , is also of great relevance here . allergic individuals could potentially be achieved by administering der p 1 in a catalytically inactive ( mutant ) form . on the other hand , exploring the potential th2 adjuvant property of the proteolytic activity of der p 1 would have important implications in defining principles for modulation of th1-mediated pathological conditions . our demonstration that the cysteine protease activity of der p 1 enhances total ige production , apart from increasing der p 1-specific ige , suggests that this allergen may play a central role in destabilizing the microenvironment within target tissues to one that is proallergic and thus aids in the initiation and propagation of the allergic cascade . in other words , the proteolytic activity of der p 1 may exert an ige - specific adjuvant effect . the in vivo relevance of the proteolytic activity of der p 1 is further highlighted by reports demonstrating that it increases the permeability of the human respiratory epithelium to macromolecules 1516 . such observations , together with our current findings showing a direct effect on the immune system , indicate that the proteolytic activity of der p 1 is a major contributor to its allergenicity . given the risk of paralysis associated with cervical transforaminal injection , is it time to reconsider transforaminal injections of the lumbar spine ? arguments for discontinuing lumbar injections have been discussed in the anesthesia literature , raising concern about the risks of epidural steroid injections ( esis ) . in a 47-year - old man , paraplegia of the lower extremities developed , specifically conus medullaris syndrome , after he underwent an esi for recurrent pain . the patient felt his legs going dead ; paraplegia of the lower extremities was noted . an initial magnetic resonance imaging study performed after the patient was transferred to the emergency department was unremarkable . however , a later neurosurgical evaluation showed conus medullaris syndrome , and a second magnetic resonance imaging study showed the conus infarct . we conducted a search of the pubmed database of articles from 2002 to 2011 containing the following keywords : complications , lumbar epidural steroid injection(s ) , cauda equina syndrome , conus medullaris infarction , spinal cord infarction , spinal cord injury , paralysis , paresis , plegia , paresthesia , and anesthesia . summarizing this case and 5 similar cases , we weigh the potential benefits and risks of esi . although one can safely assume that this severe , devastating complication is rare , we speculate that its true incidence remains unknown , possibly because of medicolegal implications . we believe that the rarity of this complication should not preclude the continued use of transforaminal esi ; rather , it should be emphasized for discussion with patients during the consent process . a 47-year - old man who had undergone an l4/l5 laminectomy and discectomy 5 years earlier presented with recurrent pain in the left buttock and posterior leg . after receiving an esi by a local anesthesiology group within the cincinnati , ohio , area , the patient was transferred from the outpatient pain clinic to our institution . information regarding his medical history and prior treatment , as well as details of the procedure , was limited to that provided by the patient himself or the accompanying procedure report . the patient reported that he had previously received esis on the left side of his lower back ; this was the second injection in a series of 3 . correct needle placement in the left l5-s1 neural foramen was verified with epidurography immediately after the injection , the patient felt his legs going dead ; paraplegia of the lower extremities was noted . because of the concern for intrathecal injection with resultant motor blockade , the patient was monitored for 4 hours . when no clinical improvement was observed , he was transferred to a nearby community hospital emergency department for neurologic examination . although the initial magnetic resonance imaging ( mri ) study performed in the emergency department was unremarkable ( fig . however , a second mri study to evaluate the possibility of vascular complications obtained 48 hours after the injection showed a conus infarct ( fig . 2 ) . five hours after esi in a 47-year - old man , magnetic resonance imaging scans performed at the emergency department were unremarkable , showing a normal appearing conus . ( a ) ( b ) t1-weighted sagittal image ( repetition time , 416.7 ; echo time , 15.0 ) . magnetic resonance images ( about 48 hours after the procedure ) showing extensive signal abnormalities within the lower thoracic spinal cord and conus compatible with the clinical diagnosis of conus infarct . ( a ) t1-weighted sagittal image ( repetition time , 675.0 ; echo time , 9.6 ) . ( b ) t2-weighted sagittal image ( repetition time , 3640.0 ; echo time , 102.0 ) . ( c ) short tau inversion recovery ( stir ) sagittal image ( repetition time , 4000.0 ; echo time , 58.0 ) . at 1-month follow - up , the patient could walk without assistance with a slow and calculated gait , had symmetric lower - extremity strength , and had dorsiflexion strength of 4 of 5 bilaterally . his urinary urge sensation had returned , and occasional episodes of fecal incontinence occurred in relation to bladder overdistention . our search of the pubmed database ( 20022011 ) included the keywords complications , lumbar epidural steroid injection(s ) , cauda equina syndrome , conus medullaris infarction , spinal cord infarction , spinal cord injury , paralysis , paresis , plegia , paresthesia , and anesthesia . esi is regarded as an effective and conservative means of treating low - back pain resulting from nerve root inflammation . recent case reports of post - procedural conus medullaris syndrome after spinal vascular compromise raise the question regarding safety . our case represents a sixth patient in whom conus medullaris syndrome developed after sustaining an acute spinal cord infarct during esi . in 5 other case reports ( table 1 ) , this complication affected patients ( aged 4271 years ) who had previous lumbar surgery and then underwent a transforaminal steroid injection . of note , findings of spinal cord injury were not detectable on the initial mri study within the first 24 hours in 1 case . injections are typically evaluated first by aspiration and injection of contrast . a nonvascular penetrating injection of contrast media verifies safe needle placement . however , in a series of 761 lumbosacral transforaminal injections performed , furman et al . reported that the sensitivity of positive flash or actual aspiration was only 44.7% , because only 38 of 85 patients had shown either flash or actual aspiration before injection . specifically , they showed that there was an 11.2% rate of vascular injection in their series . in evaluating correct placement of 316 caudal - approach esis , implicated aspiration as an errorprone method for verifying needle placement , reporting a 9.2% incidence of vascular violation despite negative findings on aspiration . blood is supplied to the caudal - most portion of the spinal cord by the anterior spinal artery , 2 posterior spinal arteries , segmental radiculomedullary arterial branches , and most importantly , the artery of adamkiewicz . the location of the artery of adamkiewicz , which is the primary blood supply to the conus medullaris , is fairly unpredictable . it travels with the nerve through the foramen at or near the level of its origin from the thoracolumbar segmental arteries . . showed that the artery of adamkiewicz originated from the left t9 - 12 posterior intercostal arteries in 75% of cases and from the l1 - 2 lumbar arteries in 10% of cases . in 31 cadavers biglioli et al . located this artery between t12 and l3 in 26 cases ( 83.9% ) . in a 2002 review of more than 4000 spinal angiograms , lo et al . examined the variability of the artery of adamkiewicz , noting that it originated at l2 in 1% of cases and l4 in 0.075% of cases . of the proposed mechanisms by which spinal cord medullary infarction may occur , one explanation may be the combined effect of an undetected direct arterial injection into a low - lying artery of adamkiewicz and the resultant embolic incident from the injectate . houten and errico proposed that the collaterals surrounding the cord at the level of this artery were proximal to the injection site and thus allowed direct passage of the injected material into the conus . however , as discussed by lo et al . considering this observation , we believe that it is unlikely that our patient had this low - lying artery that could have then been injected with an epidural steroid at this precise location . another plausible explanation is that an inadvertent sacral radicular artery injection carried injection material distally to the spinal cord . reported that material injected into the abdominal aorta below the level of the artery of adamkiewicz would appear in the conus through collateralization . the most likely cause is injection of steroid particulate through either the artery of adamkiewicz or collateral radiculomedullary arterial branches . prior lumbar surgery may lead to compromise of normal vascular supply to the spinal cord , thus making it more susceptible to vascular injury . subsequent thrombus formation would result in an embolic infarct in the spinal cord supplied by the affected artery . florey noted that such vasoconstriction was a local effect lasting 5 seconds to 10 minutes at the site of injury only . found that fine needle ( 30-gauge ) arterial puncture in rhesus monkeys induced intense vasospasm , typically lasting anywhere from 4 hours to 4 days . many studies have examined mechanically induced vasospasm of the cerebral vasculature and may be indirectly suggestive that a similar phenomenon can occur in the spinal cord and conus medullaris . of the multiple recommendations made to avoid such devastating complications as paralysis , first and foremost is the strict adherence to the standard and widely accepted techniques of transforaminal esis . these guidelines include the use of multiplanar fluoroscopy or computed tomography guidance together with contrast material to prevent complications . if recovery of neurologic function fails to occur in a patient within a 2- to 3-hour time period after esi , an initial mri study may be obtained to exclude an epidural hematoma . repeat mri after 24 hours should be performed as well . in our patient , as well as 1 other reported case , a delayed effect was observed with mri signal changes occurring after 24 hours . given the risk of paralysis associated with cervical transforaminal injection , is it time to reconsider transforaminal injections of the lumbar spine ? although the risk of permanent neurologic deficit is negligible , arguments for discontinuing lumbar injections have been presented in the anesthesia literature . however , when one considers the potential benefit of this noninvasive treatment modality , abandoning such injections may be premature . however , we speculate that more than 6 cases of conus medullaris syndrome after esi have occurred , and medicolegal considerations may explain why they may go unreported . at the very least , patients should be clearly informed regarding the potential risks of esi , including paralysis . although one can safely assume that this severe , devastating complication is rare , its true incidence remains unknown . in our opinion , the rarity of this complication should , at this time , not preclude the continued use of transforaminal esi for relief of pain in select patients but should be included in the consent process .

the house dust mite dermatophagoides pteronyssinus allergen der p 1 is the most immunodominant allergen involved in the expression of dust mite specific immunoglobulin ( ig)e mediated hypersensitivity . the reason for this potent ige - eliciting property of der p 1 remains unknown , but there is mounting in vitro evidence linking the allergenicity of der p 1 to its cysteine protease activity . here we demonstrate for the first time that immunization of mice with proteolytically active der p 1 results in a significant enhancement in total ige and der p 1specific ige synthesis compared with animals immunized with der p 1 that was irreversibly blocked with the cysteine protease inhibitor e-64 . we conclude that the proteolytic activity of der p 1 is a major contributor to its allergenicity . backgroundgiven the risk of paralysis associated with cervical transforaminal injection , is it time to reconsider transforaminal injections of the lumbar spine ? arguments for discontinuing lumbar injections have been discussed in the anesthesia literature , raising concern about the risks of epidural steroid injections ( esis).methodsin a 47-year - old man , paraplegia of the lower extremities developed , specifically conus medullaris syndrome , after he underwent an esi for recurrent pain . correct needle placement was verified with epidurography . immediately after the injection , the patient felt his legs going dead ; paraplegia of the lower extremities was noted.resultsan initial magnetic resonance imaging study performed after the patient was transferred to the emergency department was unremarkable . however , a later neurosurgical evaluation showed conus medullaris syndrome , and a second magnetic resonance imaging study showed the conus infarct . we conducted a search of the pubmed database of articles from 2002 to 2011 containing the following keywords : complications , lumbar epidural steroid injection(s ) , cauda equina syndrome , conus medullaris infarction , spinal cord infarction , spinal cord injury , paralysis , paresis , plegia , paresthesia , and anesthesia.conclusionssummarizing this case and 5 similar cases , we weigh the potential benefits and risks of esi . although one can safely assume that this severe , devastating complication is rare , we speculate that its true incidence remains unknown , possibly because of medicolegal implications . we believe that the rarity of this complication should not preclude the continued use of transforaminal esi ; rather , it should be emphasized for discussion with patients during the consent process .

alzheimer 's disease ( ad ) is a neurodegenerative disorder that is clinically characterized by progressive mental decline and histopathologically defined by highly abundant amyloid deposits and neurofibrillary tangles in the brain parenchyma . the identification of mutations within the amyloid precursor protein ( app ) and presenilin ( ps ) genes that cause autosomal dominantly inherited ad and that result in increased production of amyloid - prone forms of a established beyond doubt that the processing of app and the production of a peptides are intimately involved in the disease process and led to the proposal and the reinforcement of the alzheimer amyloid cascade hypothesis [ 1 , 2 ] . the role of amyloid in neuronal dysfunction has recently been extended by the discovery of small , soluble , oligomers of the a peptide , some forms of which have been termed addls ( a-derived diffusible ligands ) , protofibrils , or a*56 [ 36 ] . these a oligomers are not only potential intermediates in the formation of amyloid filaments , but they also have been shown to be neurotoxic themselves and to inhibit long - term potentiation ( ltp ) , a cellular model of memory , in hippocampal slices [ 4 , 7 , 8 ] . thus , the amyloid cascade hypothesis now includes the essential role of a oligomers in the neurodegeneration process . despite its strength , the amyloid cascade hypothesis is incomplete without including the essential role of amyloid - associated inflammatory proteins . for example , biochemical and histological studies first showed that , in addition to a , amyloid deposits also contained the inflammation / acute phase protein 1-antichymotrypsin ( act ) and , later , apolipoprotein e ( apoe ) [ 10 , 11 ] , which were both hypothesized to serve as catalysts or pathological chaperones of amyloid formation [ 9 , 11 , 12 ] . these and other results also indicated that alzheimer 's disease and its manifestation in middle - aged down syndrome may include an inflammatory process , for both act and apoe are inflammatory and/or acute phase proteins in other contexts , and both are overexpressed in affected regions of the ad brain ( for reviews see [ 1315 ] ) . indeed , alzheimer himself first identified the inflammatory component of alzheimer 's disease when he described reactive astrocytes and microglia in affected brain regions of his first patient . however , until inflammatory proteins such as act , il-1 , hla , and apoe were found to be overexpressed in ad and ds brains , the term inflammation was explicitly excluded from the clinical and pathological description of ad because of the lack of edema and lymphocyte infiltration [ 911 , 17 , 18 ] . the significance of these biochemical results instigated and was reinforced by parallel genetic discoveries implicating a role for inflammation in ad . in particular , inheritance of the apoe 4 allele was found to be the strongest known risk factor for ad besides age , with one copy increasing ad risk 35-fold and two copies over 10-fold [ 1921 ] . because of apoe 's essential genetic , and therefore presumably biochemical , contribution to ad pathology and cognitive decline , it is critical that its role in the ad pathogenic pathway / amyloid cascade be elucidated in order for therapeutics based on apoe to be designed . while recent excellent and encyclopedic literature reviews describe the many potential roles that apoe plays in ad [ 2326 ] , this focused review will concentrate on the interaction between a and apoe and other inflammatory proteins , on the effects of such interactions , and on their implications for designing apoe - based ad therapies . the central question we try to answer is whether increasing or decreasing apoe level and/or function will serve best to reduce ad / ds pathology and cognitive decline . lack of a clear answer may lead to the development of drugs that , rather than serving as an ad therapy , instead potentially exacerbate the disease . to determine whether inflammation contributes to alzheimer 's disease rather than being merely a correlative pathological feature in the ad brain , we and others tested the hypothesis that act and/or apoe serve as amyloid catalysts or pathological chaperones . numerous in vitro and in vivo studies showed that mature amyloid deposition and the associated cognitive decline is strongly stimulated by apoe and act in a dose - dependent and isoform - specific manner , with apoe4 being the strongest promoter of a polymerization and apoe2 being an inhibitor , paralleling the effect of these two isoforms in humans [ 2738 ] . indeed , without one or the other of these amyloid catalysts expressed in the brain , amyloid deposition is profoundly delayed in app transgenic mice and does not become filamentous . such app+/apoe ko animals also exhibit normal cognition despite levels of a expression equal to the apoe - expressing app animals . elegant work by manelli and colleagues also showed that native lipidated apoe4 from transgene replacement astrocytes increases a neurotoxicity compared to apoe3 or e2 , indicating that apoe4 provides a negative gain of function . finally , jones and colleagues recently showed that apoe4 also promotes the conversion and enhanced synaptic localization of a as oligomers , the most neurotoxic form of the alzheimer amyloid peptide [ 40 , 41 ] . these recent studies extended prior work showing that apoe copurifies with a during biochemical isolation of amyloid from human brains , and that apoe preferentially interacts with a peptides in a -sheet structure [ 4245 ] . together these results show that inflammatory proteins , particularly apoe , are integral parts of the amyloid cascade , and that without them the cascade would be arrested at the level of the harmless a monomer , and no ad would ensue . the view of apoe as an integral and pathological part of the amyloid cascade has been shaken by experiments that suggest that apoe , far from being an amyloid catalyst , serves to clear a from the brain . under this view , apoe is protective , with human apoe4 being less protective than apoe3 or e2 ( for the most recent discussion , see and commentary at http://www.alzforum.com/ ) . the first experiments that suggested apoe 's role as a neuroprotector examined the pathology and cognition of app transgenic mice carrying a second transgene expressing one or another human apoe isoform . contrary to expectations , amyloid deposition in these mice was inhibited by the human apoe transgene , as though human apoe was protective . ultimately , the mice did develop amyloid , with the apoe4-expressing strain accumulating earlier and more extensive pathology [ 33 , 34 , 48 , 49 ] . it was proposed that human apoe might serve to inhibit a clearance from the brain compared to mouse apoe , with apoe4 inhibiting clearance the most . other experiments showed that indeed , clearance of a species was inhibited by complexing with apoe , especially apoe4 [ 46 , 50 ] . the possibility that interaction with apoe modulated an a clearance mechanism appeared to be supported by the finding that introduction of anti - a antibodies or other a-binding proteins such as gelsolin , led to a reduced amyloid load in the brain and rapidly improved cognition , with little evidence of a-binding agents invading the brain parenchyma [ 4154 ] . we also introduced apoe itself into the circulation via parabiosis and found that it induced amyloid clearance without entering the brain in ad model mice . thus the peripheral sink hypothesis became a viable alternative or addition to the amyloid cascade hypothesis , with apoe potentially playing an additional role as an a-binding peripheral protein . most recently , an approach to therapy has been investigated in ad mice that is based on activating the liver x receptor ( lxr ) , which also exists on other cells including microglia [ 5557 ] . activation of lxr results in increased expression of many proteins including apoe and its lipidating enzyme , atp - binding cassette transporter a1 ( abca1 ) . the results indicate that activating lxr with the ligand gw3965 or the fda - approved antiskin cancer drug bexarotene reduces soluble and insoluble a and improves cognition in app tg mice , while knocking out the abca1 gene in app mice showed a tendency to reduced amyloid load . because apoe expression and lipidation is stimulated by lxr activation , the results were interpreted as proof that increased apoe levels help microglia clear a and amyloid , as indeed some earlier cell culture experiments had suggested . however , it has also been shown that genetic overexpression of abca1 reduces amyloid deposition in mice where the apoe levels are unchanged . hence , because lxr stimulation influences the levels of many proteins , it is problematic to definitively link its in vivo action to the altered level of one particular protein . furthermore , the increased levels of abca1 induced by bexarotene enhance apoe lipidation , a change that is known to alter apoe / a interactions . hence , it is important to consider the lipidation state of apoe , which affects its function , in addition to the absolute levels of apoe . when trying to distinguish and weigh the value of two hypotheses , it is instructive to consider their testable predictions . if apoe is an amyloid catalyst , then reducing apoe levels or function in the brain should result in reduced amyloid deposition and reduced cognitive decline . if on the other hand , apoe is involved in a clearance with human apoe4 being a greater inhibitor of clearance ( or poorer clearer ) , then reducing apoe levels or apoe binding to a should increase amyloid deposition and cognitive decline . all experiments carried out so far in vitro or in transgenic mice indicate that the ability of a to form neurotoxic filaments or oligomers and cause cognitive decline are increased in the presence of apoe , particularly mouse apoe and human apoe4 , with apoe2 being protective . in contrast , in the complete absence of apoe , the mutant app gene and its product a are harmless , generating neither amyloid deposits , synaptic disfunction , or cognitive decline , with one copy of apoe having an intermediate effect , as discussed above . the in vitro experiments in particular indicate that apoe likely acts catalytically to promote a polymerization , as the molar ratio of a to apoe of about 200/1 was appropriate for the formation of neurotoxic products [ 2730 ] . most recently , earlier work showing that mice expressing only one apoe gene accumulated less amyloid than those with two apoe genes ( 32 ) was repeated in two different laboratories using human apoe knock - in mice , and the same result was found , that is , lower doses of apoe3 or apoe4 led to reduced amyloid deposition [ 59 , 60 ] . the simplest interpretation of the in vitro , cell culture , and transgenic mouse data is that apoe is necessary for a to polymerize into neurotoxic oliogomers / filaments , probably by binding to a and thus altering its structure more toward the -sheet and more easily allowing successive a peptides to add on to the growing chain . the recent finding that apoe promotes a oligomer formation in vivo reinforces this interpretation [ 40 , 41 ] . whether apoe is only needed to initiate the polymerization or also to prepare each peptide for addition to the growing filament is not yet known . even though the key predictions of the polymerization hypothesis , that is apoe serving as an a filament catalyst , have been borne out , the compelling experiments demonstrating that human apoe inhibits filament formation in a mouse background require explanation . furthermore , data from ladu and colleagues and by others have shown that lipidated apoe , presumably the prevalent form in vivo , binds a with an affinity of e2 > e3 finally , the elegant and thorough experiments of castellano and colleagues show very convincingly that expression of a human apoe4 transgene ( in the absence of mouse apoe ) leads to a longer half - life , ( i.e. , slower clearance ) of a in the brain interstitial fluid compared to e2 or e3 . the apoe - a binding studies might be interpreted as support for apoe functioning in a clearance because apoe2 , for example , would bind a tightly and could thereby promote its removal from the interstitial fluid via lrp receptors [ 50 , 6164 ] . however , an important feature of any catalyst is that it must bind its substrate only tightly enough to convert it to the transition state structure and then release it as the reaction is completed [ 65 , 66 ] . if a mutation leads to an overly tight substrate binding , then no further reaction can occur . thus apoe2 could indeed bind a most tightly , and thereby not only prevent apoe4 from binding and promoting a oligo / polymerization , but also prevent the spontaneous polymerization of the peptide . the ability of different apoe isoforms to bind a with different strengths can also explain why human apoe isoforms slow amyloid deposition in the presence of the endogenous mouse apoe , for they may bind a more tightly or differently than mouse apoe and slow the catalytic conversion of a into oligomers / polymers in the mouse background . the data showing that human apoe inhibits a clearance can also be interpreted as reflecting apoe 's role in catalyzing a oligo / polymerization . pathologic macromolecular structures are often resistant to various clearance mechanisms designed for monomeric species , whether by intracellular proteasome degradation or cross - membrane / bbb transfer , thus allowing their accumulation . only when oligo / polymeric structures are anticipated and physiological clearance mechanisms are in place to handle them , as for antibody - antigen complexes , will clearance be facilitated by conversion to larger structures . because apoe clearly has the ability to catalyze the conversion of a into oligomeric and polymeric structures , it is reasonable to assume that those structures will be more difficult to clear , and that such difficulty will be detected as clearance inhibition in the brain , for instance , by apoe4 , in pulse chase type experiments , while the higher apoe levels in blood may aid the clearance of a from the circulations ( figure 1 ) . finally , the ability of gw3965 and bexarotene to reduce soluble and insoluble a in the brain of tg app mice and improve cognition is most easily understood as resulting from a general activation of the phagocytic activity of microglia . previous work showed that activation of microglia by acute intracerebral treatment of app mice with lps or with granulocyte - macrophage stimulating factor can similarly reduce amyloid load and improve cognition [ 6769 ] but that long - term peripheral treatment with lps exacerbated amyloid deposition in an apoe - dependent manner . stimulation of microglial activity via induction of toll - like receptor 9 ( tlr9 ) has also been shown to greatly reduce amyloid load and improve cognition . clearly the interaction of neuroinflammation , microglia , and amyloid load is complex , and the fact that bexarotene cures ad in mice is more likely to be despite , rather than because it stimulates expression of apoe . a good test of any hypothesis about the pathogenesis of a disease is whether it successfully predicts how the pathogenesis can be inhibited or reversed . for example , small fragments of a corresponding to the amino acid sequences to which act ( a1 - 12 ) and apoe ( a12 - 28 ) bind can serve as decoy peptides that prevent the binding of apoe to a and its catalysis of a into neurotoxic species . this early in vitro work has recently been repeated and confirmed in other laboratories [ 72 , 73 ] . the decoy principal was extended in vivo by preparing a version of a12 - 28 that has a better plasma 1/2 life and is nonfibrillogenic / nontoxic . it was shown that this peptide could be peripherally introduced into a transgenic app mouse , where it effectively entered the brain and prevented / reversed oligomer formation , amyloid deposition , and cognitive decline [ 7476 ] . similarly , amyloid plaques in app mice contain mouse act and injecting a1 - 11 into one side of the app mouse brain to block act 's binding site with endogenous a rapidly reduced amyloid load compared to the other , vehicle - injected side of the brain . furthermore the inflammatory cytokine il-1 that is overexpressed in ad brain induces astrocyte expression of act , and blocking il-1 expression in app transgenic mice by ibuprofen treatment , thereby reducing mouse act expression , lowers amyloid formation and restores cognition . evidently , blocking act or apoe expression or function , both in vitro or in vivo , successfully prevents a pathology and neurotoxicity . apolipoprotein j also binds a and can be shown to aid its passage across the blood brain barrier [ 7983 ] . interestingly , knocking out either apoj or apoe reduces amyloid deposition in app transgenic animals , yet knocking out both leads to robust amyloid deposition at an even earlier age than arises in nonmanipulated app animals . this result may reflect the ability of mouse act to promote amyloid formation , but that in the presence of the stronger binding apoe and apoj proteins mouse act is prevented from exhibiting its catalytic activity . the role of apoe and act in the alzheimer pathogenic pathway has potentially general implications . one of the most studied classes of a binding proteins are specific anti - a antibodies , which form the basis of both passive and active immunization therapies for alzheimer 's disease ( for review see ) . the finding that apoe and act can catalyze a oligo / polymerization begs the question of whether a antibodies might also promote or inhibit a polymerization . indeed we found that two a antibodies , 6e10 which is directed to the same the n - terminal sequence bound by act , and 13 m , which binds to the c - terminus , function very differently in the in vitro a polymerization assay . 6e10 inhibits act - catalyzed polymerization of a while 13 m inhibits act catalysis much less and even promotes some polymerization itself . interestingly , the n - terminus of a is also the target of many attempts at ad immunotherapy with the aim of inducing microglial phagocytosis of neurotoxic a species . yet removing the microglial - binding fc portion of 3d6 antibodies to a1 - 5 to generate fab'2 fragments does not reduce the antibody 's ability to remove diffuse amyloid in app mice . evidently , only its a-binding feature is required to allow the antibody to remove amyloid . a possible explanation for this result is that the antibody functions by blocking a interaction with mouse act . the consequent suppression of act - catalyzed oligo / polymerization could thus tilt the dynamic process of plaque development toward depolymerization . these results illustrate the fact that a-binding proteins can have multiple effects on polymerization and that their full range of activities must be considered when using them as potential targets or tools for therapeutic intervention . although a oligomers have been shown to be highly neurotoxic in vitro and in vivo , and their formation is promoted by apoe4 , the mechanism of their toxicity is still being elucidated . the data reviewed above coupled to other recent findings suggest a novel mechanism for a toxicity that encompasses the essential role of apoe . specifically , a oligomers bind to and inhibit certain microtubule motors that are essential for the function and stability of the mitotic spindle similar motors , including kinesin 5 , are also present in mature neurons [ 88 , 89 ] . we have found recently that inhibition of mt motor function by a or by the specific kinesin 5 inhibitor monastrol prevents the efficient transport of receptors such as the ldlr , the nmda neurotransmitter receptor , and the p75 neurotrophin receptor to the cell surface , resulting in reduced function (; in preparation ) . similarly , apoe , particularly apoe4 , has been shown to reduce the cell surface levels and function of nmda , ampa , and apoer2 receptors in neurons . this latter finding can now be understood as potentially reflecting the ability of apoe4 to promote the conversion of endogenous neuronal a into oligomers , which then inhibit mt - based transport of key cellular components such as receptors to their functional location . in sum , it appears that the preponderance of the data can be most consistently interpreted as showing that the brain inflammatory protein apoe plays a catalytic role in the ad / ds amyloid cascade and consequent cognitive decline , with binding and clearance differences between the apoe isoforms reflecting their differing abilities to bind to a and catalyze its conversion into neurotoxic macromolecular species ( figure 2 ) . this conclusion , and the in vivo demonstration that blocking apoe - a interaction prevents ad in a mouse model , suggests that this decoy approach should be translatable into human patients and serve as an effective new approach to ad therapy . other a-binding proteins may be similarly manipulated by a decoy approach to reduce oligomerization and polymerization of a into neurotoxic species . however , the finding that different antibodies to a can both inhibit act - catalyzed a polymerization and promote polymerization of a itself , argues that immunotherapy must be approached with care to avoid the use or induction of antibodies that can catalyze further oligo / polymerization of a , instead of inducing its phagocytosis and removal . furthermore , human and mouse intracerebral environments may differ in important ways with respect to the pattern and activities of a-binding proteins and may also respond differently to intervention or inflammation . such differences may explain why so many treatments that were successful in reducing amyloid - dependent cognitive decline in transgenic mice have failed to translate into human ad patients . finally , the ability of a oligomers to inhibit key microtubule motors and prevent the transport of neurotrophin , neurotransmitter , and other receptors to the cell surface may underlie their neuronal toxicity . it is apparently the apoe- , especially e4-dependent formation of such a oligomers , that constitutes the key catalytic step in the ad pathogenic pathway .

because of rampant concern that estrogenic chemicals in the environment may be adversely affecting the health of humans and wildlife , reliable methods for detecting and characterizing estrogenic chemicals are needed . it is important that general agreement be reached on which tests to use and that these tests then be applied to the testing of both man - made and naturally occurring chemicals . as a step toward developing a comprehensive approach to screening chemicals for estrogenic activity , three assays for detecting estrogenicity were conducted on 10 chemicals with known or suspected estrogenic activity . the assays were 1 ) competitive binding with the mouse uterine estrogen receptor , 2 ) transcriptional activation in hela cells transfected with plasmids containing an estrogen receptor and a response element , and 3 ) the uterotropic assay in mice . the chemicals studied were 17 beta - estradiol , diethylstilbestrol , tamoxifen , 4-hydroxytamoxifen , methoxychlor , the methoxychlor metabolite 2,2-bis(p - hydroxyphenyl)-1,1,1-trichloroethane ( hpte ) , endosulfan , nonylphenol , o , p'-ddt , and kepone . these studies were conducted to assess the utility of this three - assay combination in the routine screening of chemicals , or combinations of chemicals , for estrogenic activity . results were consistent among the three assays with respect to what is known about the estrogenic activities of the chemicals tested and their requirements for metabolic activation . by providing information on three levels of hormonal activity ( receptor binding , transcriptional activation , and an in vivo effect in an estrogen - responsive tissue ) , an informative profile of estrogenic activity is obtained with a reasonable investment of resources.imagesp1296-afigure 1.figure 2.figure 3.figure 4.figure 5 . the amyloid cascade hypothesis remains a robust model of ad neurodegeneration . however , amyloid deposits contain proteins besides a , such as apolipoprotein e ( apoe ) . inheritance of the apoe4 allele is the strongest genetic risk factor for late - onset ad . however , there is no consensus on how different apoe isotypes contribute to ad pathogenesis . it has been hypothesized that apoe and apoe4 in particular is an amyloid catalyst or pathological chaperone . alternatively it has been posited that apoe regulates a clearance , with apoe4 been worse at this function compared to apoe3 . these views seem fundamentally opposed . the former would indicate that removing apoe will reduce ad pathology , while the latter suggests increasing brain apoe levels may be beneficial . here we consider the scientific basis of these different models of apoe function and suggest that these seemingly opposing views can be reconciled . the optimal therapeutic target may be to inhibit the interaction of apoe with a rather than altering apoe levels . such an approach will not have detrimental effects on the many beneficial roles apoe plays in neurobiology . furthermore , other a binding proteins , including act and apo j can inhibit or promote a oligomerization / polymerization depending on conditions and might be manipulated to effect ad treatment .

musculoskeletal tissues show increased bone fragility , loss of cartilage resilience , reduced ligament elasticity , loss of muscular strength , and fat redistribution decreasing the ability of the tissues to carry out their normal functions . the loss of mobility and physical independence resulting from arthropathies and fractures can be particularly devastating in this population , not just physically and psychologically , but also in terms of increased mortality rates . the aim of this article is to present some of the most frequent musculoskeletal disorders of the elderly , including some misleading presentations . we used the following terms and some of its combinations : elderly , aged , epidemiology , fracture , osteoporosis , vertebral , hip , pelvis , arthritis , neoplasm , malignancy , myeloma , paget , gout , infection , microcrystal , and radiology . articles without english abstracts were excluded . besides , certain relevant rheumatological , orthopedic , and radiological books were also used . fractures are frequent in the elderly and result mainly from the effects of falls and osteoporosis . low - impact falls , even from standing height , are the most common cause of injury in geriatric patients . falling is a multi - factorial problem due to both extrinsic ( e.g. , environmental and housing conditions ) and intrinsic risk factors ( e.g. , impaired mobility , loss of muscular strength , poor visual acuity , and medication , for instance corticoids).[46 ] osteoporosis , which is characterized by qualitatively normal , but quantitatively deficient bone , leads to bone fragility and increased risk of fractures . the general prevalence of osteoporosis in women is approximately 50% at the age of 85 years , while in men the prevalence is about 20% at that age . ethnic differences also exist : mineral bone density is higher in black women and lower in asian women , while in white women evidence shows an intermediate value . due to its precision , the most widely used quantitative technique is dual energy x - ray absorptiometry , which makes it possible to diagnose osteoporosis early , predict the risk of fracture , determine therapeutic intervention , and monitor response to treatment . this method grades bone loss according to standard deviations ( sd ) from the mean young adult value . a value less than 2.5 times the sd below the young adult mean , is considered to indicate osteoporosis . the main radiographic features of osteoporosis are increased radiolucency and cortical thinning , mainly in the spine , giving rise to a well - demarcated outline of the vertebral body , which has been described as , picture framing. besides , in the spine , an increased biconcavity of the vertebral end plates , with a protrusion of the intervertebral disk into the vertebral body can be observed . however , early radiographic changes are subtle , as a bone loss of approximately 30% must occur before it can be detected . vertebral compression fractures are the most common osteoporotic fractures , with a greater incidence in women over 60 years of age . they can be observed in up to 25% of women over 70 years of age and 40% of those over 80 years . after one episode , the risk of subsequent vertebral fracture increases at least four - fold , with substantially increased rates during the first year . these fractures are associated with higher mortality and significant morbidity , even though they are afforded little clinical attention . they often appear as multiple fractures with anterior wedging , and may be associated with significant deformity , leading to kyphosis . they are usually well demonstrated on radiographs , but can be better assessed with computed tomography ( ct ) or magnetic resonance ( mr ) imaging , when necessary . the latter imaging modality may be indicated in case of associated neurological symptoms a rare event or when a vertebroplasty is discussed , in order to better localize the recently fractured vertebra . vertebral compression fractures in the elderly may involve several problems : first is differentiating between osteoporotic and malignant vertebral collapse ( vc ) , particularly metastatic vc , as the latter is by far the most common malignant tumor affecting the skeleton in the elderly . in most patients , multiple , relatively symmetrical thoracolumbar vcs , associated with diffuse increased radiolucency are suggestive of osteoporosis [ figure 1a ] . the whole vertebral endplate is impacted , sometimes in association with increased or decreased radiolucency of the subjacent trabecular bone ( band - like distribution of the abnormalities under the fractured vertebral end plate ) . gas can also be seen under the fracture and is nearly pathognomonic of the benign nature of the collapse . there is no cortical osteolysis , although fractures can be identified with retropulsion of a bone fragment . conversely , the following are a cause for concern : a single vc , involvement of the cervical spine , heterogeneous increased or decreased radiolucency that can not be explained by an underlying end plate fracture , a focal collapse ( on the ap or lateral view ) , or a bulging of the vertebral body or cortical osteolysis [ figure 1b ] . if in doubt or for a better assessment of the spinal canal and soft tissue , ct , or better yet , an mri can be performed , in order to differentiate between these two disorders . in benign fractures , an mri may show a band distribution of abnormal signal intensities on t1 and t2 weighted images , spared normal bone marrow signal intensity , retropulsion of a posterior bone fragment , and multiple compression fractures ; on the contrary , signs such as a convex posterior border of the vertebral body , abnormal signal intensity of the posterior elements , an epidural mass or a focal paraspinal mass are suggestive of spinal metastasis.[1921 ] second , vertebral fractures can be missed following moderate trauma , because the fracture may be difficult to identify in an osteoporotic radiolucent bone and / or because the pain is attributed to another painful condition , such as degenerative changes . this is observed in particular in the cervical spine , particularly at c2 , and may lead to secondary neurological complications [ figure 2 ] . ankylosis may be related to ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis , which especially affect males and they have an estimated frequency of 5 to 15% in the elderly.[2225 ] these ankylosed spines become increasingly susceptible to injury , even in the event of low - energy impacts , and are prone to unstable vertebral fractures with an increased frequency of neurological complications , either primary or secondary , after unprotected transfers and manipulation . these lesions may be missed on plain films when non - displaced or mildly displaced , especially in hyperextension , whereas , they are well demonstrated on ct or mr imaging . however , the mri features can sometimes be misleading and mimic infectious spondylitis when signal abnormalities of the disco - vertebral junction are present . caution is required when an elderly patient presents with an ankylosed spine , minor trauma or acute pain , especially if signal abnormalities of the anterior and posterior spine are observed [ figure 3 ] . usual benign vertebral compression ( a ) multiple levels , diffuse radiolucency , and whole vertebral endplate involvement ( arrows ) ; usual malignant vertebral compression characteristics ( b ) single vertebral involvement , focal and bulging of the posterior wall ( arrowheads ) . missed fracture of the odontoid process and vertebral body of c2 ( arrows ) in a patient with a history of fall one month before . transverse fracture of thoracic vertebral body ( arrow ) and posterior arch ( arrowhead ) with posterior epidural hematoma ( curved arrow ) in an ankylosed spine depicted on ( a ) t1- and ( b ) t2-weighted sequences . other important sites of fractures are the hip and the pelvis , which are associated with increased mortality and specific diagnostic problems in the elderly population . they are common and frequently result from lateral falls directly on the greater trochanter , in the case of hip fractures , and forward or backward falls in the case of pelvic fractures . most fractures are easily diagnosed by conventional radiography , particularly fractures of the proximal femur . however , non - displaced fractures may be negative or equivocal , particularly if the bone is osteoporotic . if a clinical suspicion of a fracture persists , ct or better still mr imaging is advocated , as this modality can show the fracture line surrounded by bone marrow edema . they may occur everywhere , but are particularly common in the hip and pelvis , the majority being located in the sacrum and pubic ramus [ figure 4 ] . radiographic findings are often subtle or unremarkable , and in suspected cases mr imaging is indicated due to its superior accuracy as compared to other radiological methods . all the images should be carefully analyzed , as multiple insufficiency fractures are frequent [ figure 5 ] . subchondral fractures are particularly difficult to identify on radiographs , but the thin hypointense line that represents the fracture , surrounded by a variable degree of edema , is well demonstrated on an mri [ figure 6 ] . this kind of fracture can be complicated by a necrosis of the subchondral bone , which is hypointense on t2 weighted images , and is not enhanced following gadolinium administration . insufficiency fractures ( arrows ) located in the sacrum with adjacent edema ( arrowheads ) , better depicted on ( a ) t1- and ( b ) t2- weighted sequences , respectively . multiple fractures ( white arrows ) in ( a ) the proximal femur and ( b ) pubic ramus on coronal t1-weighted images subchondral fracture of the femoral head : ( a ) conventional radiography and ( b ) fat saturated sagittal t2-weighted image ( white arrow ) . osteoarthritis ( oa ) can be defined as a group of distinct , but overlapping diseases , which may have different etiologies , but similar biological , morphological , and clinical outcomes affecting the articular cartilage , subchondral bone , ligaments , joint capsule , synovial membrane , and periarticular muscles . pathological changes can include fibrillation of cartilage , disruption of collagen fibers , changes in proteoglycan staining , chondrocyte proliferation , necrosis , and neovascularization . oa is the most common joint disease in persons 65 years of age and above , with a radiographic prevalence as high as approximately 90% in women and 80% in men.[3335 ] its etiology is not fully understood , although there are several related factors , such as female gender , genetics , metabolism , and excessive mechanical stress . it frequently leads to decreased function and loss of independence . although the joints of the hand are the most commonly affected , they are less likely to be symptomatic than the knee or hip [ figure 7 ] . the diagnosis of oa is primarily based on clinical history and physical examination . plain radiographs can help confirm both the diagnosis and grade the severity of the condition . the cardinal radiographic features of oa are focal / non - uniform narrowing of the joint space in the areas subjected to the most pressure , subchondral cysts , subchondral sclerosis , and osteophytes . however , the severity of the spondylolisthesis of the radiographic changes does not correlate with the clinical symptoms . shoulder oa and rotator cuff disease are also fairly common in the elderly and are associated with significant shoulder pain and disability related to decreased shoulder movement . direct signs of oa and indirect signs of large rotator cuff tears can be seen on plain films , including superior humeral head migration and remodeling of the acromion and the greater tuberosity . ultrasound is another valuable tool for easy assessment of the rotator cuff tendons due to its good sensitivity and specificity [ figure 8 ] . however , when surgical treatment is considered , arthro - ct , or mri may be preferred , in order to improve its planning . the lumbar and cervical spine is also frequently affected , with degenerative changes of the disk spaces , facet joints , and spinous processes , and sometimes with progressive scoliosis . although the intervertebral disks are not synovial joints , the pathological changes are similar to those seen in the articular cartilage . ct and mri are routinely used for the evaluation of narrowing of the central canal , lateral recesses , and neural foramina , when such an assessment is required ( percutaneous or surgical treatment ) . besides , mri can be useful in evaluating marrow changes in the vertebral plate , especially inflammatory changes ( type i modic ) , which present a positive correlation with low back pain . typical osteoarthritis of ( a ) the distal and proximal interphalangeal joints and ( b ) hip joint : joint space narrowing ( arrows ) , subchondral cysts ( arrowheads ) , osteophytes ( curved arrows ) , and subchondral sclerosis ( thick arrow ) . ultrasound images in ( a ) the long and ( b ) short axis of the supraspinatus tendon demonstrating a complete tear ( arrowheads ) . the frequency of microcrystal disorders also increases with age ; this is mainly true of gout and calcium pyrophosphate dihydrate deposition ( cpdd ) arthropathy . gout is the most common inflammatory arthritis in the elderly and is characterized by a disturbance of purine metabolism , with deposits in the joints , cartilage , and kidneys . the first metatarsophalangeal joint is the most common site of involvement in gouty arthritis [ figure 9 ] , but any joint can be involved . the typical radiographic features include eccentric nodular soft tissue masses ( particularly suggestive of tophus when they are dense ) , close erosions ( also suggestive when located at a distance from the joint and when associated with an elevated bony margin ) , preservation of the joint space , exuberant bony proliferation , and lack of periarticular osteoporosis . when the clinical , biological , and radiographic findings are uncertain , an ultrasound may be particularly helpful , as this technique may demonstrate more tophi [ figure 10 ] and erosions than plain films , more synovitis than a clinical examination , suggestive hyperechoic aggregates in the synovium or in the joint fluid , which may be aspirated , and the double contour sign ( an irregular hyperechoic band over the superficial margin of the articular cartilage , related to crystal deposition).[4244 ] calcium pyrophosphate dihydrate deposition ( cpdd ) disease is characterized by articular and periarticular tissue deposits ( hyaline cartilage , fibrocartilage , and other soft - tissue structures ) , even in the spine [ figure 11 ] . unlike gout , in which the increase in serum urates leads to supersaturation and deposits in the joints , in cpdd , the calcium deposits usually appear in the cartilage in the absence of any serum abnormality . even though such deposits may be seen in asymptomatic patients , they may be associated with the development of severe arthropathies or with acute pain related to the migration of the crystals in the synovial fluid , mimicking septic arthritis . gout of the first mtp joint in conventional radiography : excentric dense nodular soft tissue mass ( arrows ) , large erosions ( arrowhead ) , lack of periarticular osteoporosis , and exuberant bony proliferation ( curved arrow ) . gout involvement of the mtp ( a ) shows the tophus ( arrow ) with hyperechogenic microcrystal deposits ( arrowheads ) and a thickened synovium ( curved arrows ) . another patient with tophus deposition in the dorsal midfoot ( b ) demonstrates microcrystal deposits ( arrowhead ) with acoustic shadows ( thick arrows ) . calcium pyrophosphate dihydrate deposition disease demonstrated by deposits of microcrystals in the menisci ( arrow ) , articular cartilage ( arrowhead ) , and periarticular soft tissue ( curved arrow ) . elderly patients are more prone to infection because of an increased incidence of predisposing disorders ( diabetes mellitus , peripheral vascular disease , poor dentition , ) , immunosuppression and surgical procedures in this population ( dental extractions , open heart surgery , prosthetic joint replacement , ) . the infectious agents affecting elderly patients are similar to those found in the younger population ; although in the elderly , evidence shows an increased incidence of infection by nosocomial organisms ( due to institutionalization and hospitalization ) . these infections can have a relatively benign course , as in the case of cellulitis , or serious consequences , as with necrotizing fasciitis . in patients over 80 years of age , the knee , shoulder , and hip are the most frequently affected joints . another peculiarity of the elderly , uncommon in adults , is acute hematogenous osteomyelitis that mainly affects the spine . attention should , moreover , be paid to the diagnosis of tuberculosis , as early manifestations are subtle and advanced disease mimics other infections , granulomatous diseases , and malignancy . tuberculosis may affect the spine ( about 50% of cases ) , with potential neurological consequences , or any joint , causing deformity [ figure 12 ] . it should be noted that elderly men may present with bone lesions , as a manifestation of reactivation of the disease . as there are no pathognomonic imaging signs of musculoskeletal tuberculosis , the most conclusive means of reaching a diagnosis is by biopsy and culture , as chest radiographs and skin tests may not be positive in the elderly . as regards imaging techniques , magnetic resonance imaging is the modality of choice for musculoskeletal infection , as it best delineates the extension of soft tissue infections . computed tomography , ultrasound , radiography , and nuclear medicine studies are considered to be ancillary . in some circumstances ultrasound can be very useful , as it allows the aspiration of fluid collected in the joints or soft tissue , the evaluation of structures adjacent to orthopedic hardware , or the assessment of small peripheral joints . besides , in the case of debilitated elderly patients , a mobile ultrasound device can be easily moved close to their beds . ( a ) on conventional radiography there is mild osteoporosis , extensive soft tissue swelling , and large marginal erosion ( arrowhead ) , ( b ) on doppler ultrasound the synovitis ( arrows ) and bone erosion ( arrowhead ) are well - depicted . paget 's disease is a bone disorder characterized by an increase in osteoclast - mediated bone resorption accompanied by osteoblast - mediated formation of new bone of inferior quality . the frequency of this condition is decreasing , but it still exists in 1 5% of persons over 50 years of age , varying according to geographic areas.[5356 ] the radiographic signs include an advancing wedge of bone resorption , an accentuation and coarsening of the bone trabeculae along lines of stress , cortical thickening , and enlargement of the bone [ figure 13 ] . as a result , secondary osteoarthritis , insufficiency fractures , bowing of the bones , and even spinal cord or nerve root compression can be observed . in a minority of cases , sarcomatous degeneration may develop , mainly in the humerus , pelvis , and proximal femur , with a frequency believed to range from 0.9 to 2% . paget 's disease affecting the iliac bone demonstrated with coarsening of the bone trabeculae and cortical thickening ( arrows ) . tumors : tumors can be revealed by bone pain , pathological fractures , or disability , or be discovered as an incidental finding . primary bone tumors in elderly patients are not common , but , due to the risk of metastases , any newly discovered bone lesion or one developing within a known pre - existing lesion must be assumed to be malignant until proven otherwise . metastases : bone metastases are a common complication of malignant disease , especially of the breast , prostate , and lung . although they are often multiple , a solitary metastasis is still more common than a primary neoplasm . plain films are relatively insensitive for the detection of bone metastases , especially subtle lesions . mr imaging is superior to ct in the detection of malignant marrow infiltration , and it has a better contrast resolution for visualizing soft tissue and spinal cord lesions . even with the advances in mri , especially for bone marrow screening , bone scintigraphy continues to be used as an effective method for initial evaluation of the whole body for bone metastases . sarcomas : in the elderly population , many sarcomas are secondary to a pre - existing disorder of the bone , such as paget 's disease , or lesions treated by radiation . chondrosarcoma must also be kept in mind , especially when isolated osteolytic lesions of the pelvis are seen , as this is the most common primary sarcoma of bone in adults . the cartilaginous nature of the lesion may be suggested by the presence of calcifications or by a lobulated hyperintense lesion in an mri [ figure 14 ] . chondrosarcoma of the pelvis in ( a ) conventional radiography appears as an isolated osteolytic lesion ( arrows ) , ( b ) coronal t2-weighted image demonstrates a lobulated hyperintense lesion ( arrowheads ) . myeloma : myeloma is a characteristic disease in the elderly population , with a peak incidence in the eighth decade that results from an unregulated , progressive proliferation of neoplastic monoclonal plasma cells that accumulate in the bone marrow , leading to anemia and marrow failure . osteolytic lesions may be present on plain films , but differentiating osteoporotic vertebral compression fractures from those associated with myeloma is a common diagnostic dilemma , as bone loss may be the first sign revealing the latter disease . indeed , widespread osteoporosis , due to cytokine - mediated osteoclast activation , is common in patients with myeloma . this possible association must be kept in mind , particularly if other features are present , including unexplained back or bone pain . we used the following terms and some of its combinations : elderly , aged , epidemiology , fracture , osteoporosis , vertebral , hip , pelvis , arthritis , neoplasm , malignancy , myeloma , paget , gout , infection , microcrystal , and radiology . articles without english abstracts were excluded . besides , certain relevant rheumatological , orthopedic , and radiological books were also used . fractures are frequent in the elderly and result mainly from the effects of falls and osteoporosis . low - impact falls , even from standing height , are the most common cause of injury in geriatric patients . falling is a multi - factorial problem due to both extrinsic ( e.g. , environmental and housing conditions ) and intrinsic risk factors ( e.g. , impaired mobility , loss of muscular strength , poor visual acuity , and medication , for instance corticoids).[46 ] osteoporosis , which is characterized by qualitatively normal , but quantitatively deficient bone , leads to bone fragility and increased risk of fractures . the general prevalence of osteoporosis in women is approximately 50% at the age of 85 years , while in men the prevalence is about 20% at that age . ethnic differences also exist : mineral bone density is higher in black women and lower in asian women , while in white women evidence shows an intermediate value . due to its precision , the most widely used quantitative technique is dual energy x - ray absorptiometry , which makes it possible to diagnose osteoporosis early , predict the risk of fracture , determine therapeutic intervention , and monitor response to treatment . this method grades bone loss according to standard deviations ( sd ) from the mean young adult value . a value less than 2.5 times the sd below the young adult mean , is considered to indicate osteoporosis . the main radiographic features of osteoporosis are increased radiolucency and cortical thinning , mainly in the spine , giving rise to a well - demarcated outline of the vertebral body , which has been described as , picture framing. besides , in the spine , an increased biconcavity of the vertebral end plates , with a protrusion of the intervertebral disk into the vertebral body can be observed . however , early radiographic changes are subtle , as a bone loss of approximately 30% must occur before it can be detected . vertebral compression fractures are the most common osteoporotic fractures , with a greater incidence in women over 60 years of age . they can be observed in up to 25% of women over 70 years of age and 40% of those over 80 years . after one episode , the risk of subsequent vertebral fracture increases at least four - fold , with substantially increased rates during the first year . these fractures are associated with higher mortality and significant morbidity , even though they are afforded little clinical attention . they often appear as multiple fractures with anterior wedging , and may be associated with significant deformity , leading to kyphosis . they are usually well demonstrated on radiographs , but can be better assessed with computed tomography ( ct ) or magnetic resonance ( mr ) imaging , when necessary . the latter imaging modality may be indicated in case of associated neurological symptoms a rare event or when a vertebroplasty is discussed , in order to better localize the recently fractured vertebra . vertebral compression fractures in the elderly may involve several problems : first is differentiating between osteoporotic and malignant vertebral collapse ( vc ) , particularly metastatic vc , as the latter is by far the most common malignant tumor affecting the skeleton in the elderly . in most patients , multiple , relatively symmetrical thoracolumbar vcs , associated with diffuse increased radiolucency are suggestive of osteoporosis [ figure 1a ] . the whole vertebral endplate is impacted , sometimes in association with increased or decreased radiolucency of the subjacent trabecular bone ( band - like distribution of the abnormalities under the fractured vertebral end plate ) . gas can also be seen under the fracture and is nearly pathognomonic of the benign nature of the collapse . there is no cortical osteolysis , although fractures can be identified with retropulsion of a bone fragment . conversely , the following are a cause for concern : a single vc , involvement of the cervical spine , heterogeneous increased or decreased radiolucency that can not be explained by an underlying end plate fracture , a focal collapse ( on the ap or lateral view ) , or a bulging of the vertebral body or cortical osteolysis [ figure 1b ] . if in doubt or for a better assessment of the spinal canal and soft tissue , ct , or better yet , an mri can be performed , in order to differentiate between these two disorders . in benign fractures , an mri may show a band distribution of abnormal signal intensities on t1 and t2 weighted images , spared normal bone marrow signal intensity , retropulsion of a posterior bone fragment , and multiple compression fractures ; on the contrary , signs such as a convex posterior border of the vertebral body , abnormal signal intensity of the posterior elements , an epidural mass or a focal paraspinal mass are suggestive of spinal metastasis.[1921 ] second , vertebral fractures can be missed following moderate trauma , because the fracture may be difficult to identify in an osteoporotic radiolucent bone and / or because the pain is attributed to another painful condition , such as degenerative changes . this is observed in particular in the cervical spine , particularly at c2 , and may lead to secondary neurological complications [ figure 2 ] . ankylosis may be related to ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis , which especially affect males and they have an estimated frequency of 5 to 15% in the elderly.[2225 ] these ankylosed spines become increasingly susceptible to injury , even in the event of low - energy impacts , and are prone to unstable vertebral fractures with an increased frequency of neurological complications , either primary or secondary , after unprotected transfers and manipulation . these lesions may be missed on plain films when non - displaced or mildly displaced , especially in hyperextension , whereas , they are well demonstrated on ct or mr imaging . however , the mri features can sometimes be misleading and mimic infectious spondylitis when signal abnormalities of the disco - vertebral junction are present . caution is required when an elderly patient presents with an ankylosed spine , minor trauma or acute pain , especially if signal abnormalities of the anterior and posterior spine are observed [ figure 3 ] . usual benign vertebral compression ( a ) multiple levels , diffuse radiolucency , and whole vertebral endplate involvement ( arrows ) ; usual malignant vertebral compression characteristics ( b ) single vertebral involvement , focal and bulging of the posterior wall ( arrowheads ) . missed fracture of the odontoid process and vertebral body of c2 ( arrows ) in a patient with a history of fall one month before . transverse fracture of thoracic vertebral body ( arrow ) and posterior arch ( arrowhead ) with posterior epidural hematoma ( curved arrow ) in an ankylosed spine depicted on ( a ) t1- and ( b ) t2-weighted sequences . other important sites of fractures are the hip and the pelvis , which are associated with increased mortality and specific diagnostic problems in the elderly population . they are common and frequently result from lateral falls directly on the greater trochanter , in the case of hip fractures , and forward or backward falls in the case of pelvic fractures . most fractures are easily diagnosed by conventional radiography , particularly fractures of the proximal femur . however , non - displaced fractures may be negative or equivocal , particularly if the bone is osteoporotic . if a clinical suspicion of a fracture persists , ct or better still mr imaging is advocated , as this modality can show the fracture line surrounded by bone marrow edema . they may occur everywhere , but are particularly common in the hip and pelvis , the majority being located in the sacrum and pubic ramus [ figure 4 ] . radiographic findings are often subtle or unremarkable , and in suspected cases mr imaging is indicated due to its superior accuracy as compared to other radiological methods . all the images should be carefully analyzed , as multiple insufficiency fractures are frequent [ figure 5 ] . subchondral fractures are particularly difficult to identify on radiographs , but the thin hypointense line that represents the fracture , surrounded by a variable degree of edema , is well demonstrated on an mri [ figure 6 ] . this kind of fracture can be complicated by a necrosis of the subchondral bone , which is hypointense on t2 weighted images , and is not enhanced following gadolinium administration . insufficiency fractures ( arrows ) located in the sacrum with adjacent edema ( arrowheads ) , better depicted on ( a ) t1- and ( b ) t2- weighted sequences , respectively . multiple fractures ( white arrows ) in ( a ) the proximal femur and ( b ) pubic ramus on coronal t1-weighted images subchondral fracture of the femoral head : ( a ) conventional radiography and ( b ) fat saturated sagittal t2-weighted image ( white arrow ) . osteoarthritis ( oa ) can be defined as a group of distinct , but overlapping diseases , which may have different etiologies , but similar biological , morphological , and clinical outcomes affecting the articular cartilage , subchondral bone , ligaments , joint capsule , synovial membrane , and periarticular muscles . pathological changes can include fibrillation of cartilage , disruption of collagen fibers , changes in proteoglycan staining , chondrocyte proliferation , necrosis , and neovascularization . oa is the most common joint disease in persons 65 years of age and above , with a radiographic prevalence as high as approximately 90% in women and 80% in men.[3335 ] its etiology is not fully understood , although there are several related factors , such as female gender , genetics , metabolism , and excessive mechanical stress . although the joints of the hand are the most commonly affected , they are less likely to be symptomatic than the knee or hip [ figure 7 ] . the diagnosis of oa is primarily based on clinical history and physical examination . plain radiographs can help confirm both the diagnosis and grade the severity of the condition . the cardinal radiographic features of oa are focal / non - uniform narrowing of the joint space in the areas subjected to the most pressure , subchondral cysts , subchondral sclerosis , and osteophytes . however , the severity of the spondylolisthesis of the radiographic changes does not correlate with the clinical symptoms . shoulder oa and rotator cuff disease are also fairly common in the elderly and are associated with significant shoulder pain and disability related to decreased shoulder movement . direct signs of oa and indirect signs of large rotator cuff tears can be seen on plain films , including superior humeral head migration and remodeling of the acromion and the greater tuberosity . ultrasound is another valuable tool for easy assessment of the rotator cuff tendons due to its good sensitivity and specificity [ figure 8 ] . however , when surgical treatment is considered , arthro - ct , or mri may be preferred , in order to improve its planning . the lumbar and cervical spine is also frequently affected , with degenerative changes of the disk spaces , facet joints , and spinous processes , and sometimes with progressive scoliosis . although the intervertebral disks are not synovial joints , the pathological changes are similar to those seen in the articular cartilage . ct and mri are routinely used for the evaluation of narrowing of the central canal , lateral recesses , and neural foramina , when such an assessment is required ( percutaneous or surgical treatment ) . besides , mri can be useful in evaluating marrow changes in the vertebral plate , especially inflammatory changes ( type i modic ) , which present a positive correlation with low back pain . typical osteoarthritis of ( a ) the distal and proximal interphalangeal joints and ( b ) hip joint : joint space narrowing ( arrows ) , subchondral cysts ( arrowheads ) , osteophytes ( curved arrows ) , and subchondral sclerosis ( thick arrow ) . ultrasound images in ( a ) the long and ( b ) short axis of the supraspinatus tendon demonstrating a complete tear ( arrowheads ) . the frequency of microcrystal disorders also increases with age ; this is mainly true of gout and calcium pyrophosphate dihydrate deposition ( cpdd ) arthropathy . gout is the most common inflammatory arthritis in the elderly and is characterized by a disturbance of purine metabolism , with deposits in the joints , cartilage , and kidneys . the first metatarsophalangeal joint is the most common site of involvement in gouty arthritis [ figure 9 ] , but any joint can be involved . the typical radiographic features include eccentric nodular soft tissue masses ( particularly suggestive of tophus when they are dense ) , close erosions ( also suggestive when located at a distance from the joint and when associated with an elevated bony margin ) , preservation of the joint space , exuberant bony proliferation , and lack of periarticular osteoporosis . when the clinical , biological , and radiographic findings are uncertain , an ultrasound may be particularly helpful , as this technique may demonstrate more tophi [ figure 10 ] and erosions than plain films , more synovitis than a clinical examination , suggestive hyperechoic aggregates in the synovium or in the joint fluid , which may be aspirated , and the double contour sign ( an irregular hyperechoic band over the superficial margin of the articular cartilage , related to crystal deposition).[4244 ] calcium pyrophosphate dihydrate deposition ( cpdd ) disease is characterized by articular and periarticular tissue deposits ( hyaline cartilage , fibrocartilage , and other soft - tissue structures ) , even in the spine [ figure 11 ] . unlike gout , in which the increase in serum urates leads to supersaturation and deposits in the joints , in cpdd , the calcium deposits usually appear in the cartilage in the absence of any serum abnormality . even though such deposits may be seen in asymptomatic patients , they may be associated with the development of severe arthropathies or with acute pain related to the migration of the crystals in the synovial fluid , mimicking septic arthritis . gout of the first mtp joint in conventional radiography : excentric dense nodular soft tissue mass ( arrows ) , large erosions ( arrowhead ) , lack of periarticular osteoporosis , and exuberant bony proliferation ( curved arrow ) . gout involvement of the mtp ( a ) shows the tophus ( arrow ) with hyperechogenic microcrystal deposits ( arrowheads ) and a thickened synovium ( curved arrows ) . another patient with tophus deposition in the dorsal midfoot ( b ) demonstrates microcrystal deposits ( arrowhead ) with acoustic shadows ( thick arrows ) . calcium pyrophosphate dihydrate deposition disease demonstrated by deposits of microcrystals in the menisci ( arrow ) , articular cartilage ( arrowhead ) , and periarticular soft tissue ( curved arrow ) . elderly patients are more prone to infection because of an increased incidence of predisposing disorders ( diabetes mellitus , peripheral vascular disease , poor dentition , ) , immunosuppression and surgical procedures in this population ( dental extractions , open heart surgery , prosthetic joint replacement , ) . the infectious agents affecting elderly patients are similar to those found in the younger population ; although in the elderly , evidence shows an increased incidence of infection by nosocomial organisms ( due to institutionalization and hospitalization ) . these infections can have a relatively benign course , as in the case of cellulitis , or serious consequences , as with necrotizing fasciitis . in patients over 80 years of age , another peculiarity of the elderly , uncommon in adults , is acute hematogenous osteomyelitis that mainly affects the spine . attention should , moreover , be paid to the diagnosis of tuberculosis , as early manifestations are subtle and advanced disease mimics other infections , granulomatous diseases , and malignancy . tuberculosis may affect the spine ( about 50% of cases ) , with potential neurological consequences , or any joint , causing deformity [ figure 12 ] . it should be noted that elderly men may present with bone lesions , as a manifestation of reactivation of the disease . as there are no pathognomonic imaging signs of musculoskeletal tuberculosis , the most conclusive means of reaching a diagnosis is by biopsy and culture , as chest radiographs and skin tests may not be positive in the elderly . as regards imaging techniques , magnetic resonance imaging is the modality of choice for musculoskeletal infection , as it best delineates the extension of soft tissue infections . computed tomography , ultrasound , radiography , and nuclear medicine studies are considered to be ancillary . in some circumstances ultrasound can be very useful , as it allows the aspiration of fluid collected in the joints or soft tissue , the evaluation of structures adjacent to orthopedic hardware , or the assessment of small peripheral joints . besides , in the case of debilitated elderly patients , a mobile ultrasound device can be easily moved close to their beds . ( a ) on conventional radiography there is mild osteoporosis , extensive soft tissue swelling , and large marginal erosion ( arrowhead ) , ( b ) on doppler ultrasound the synovitis ( arrows ) and bone erosion ( arrowhead ) are well - depicted . paget 's disease is a bone disorder characterized by an increase in osteoclast - mediated bone resorption accompanied by osteoblast - mediated formation of new bone of inferior quality . the frequency of this condition is decreasing , but it still exists in 1 5% of persons over 50 years of age , varying according to geographic areas.[5356 ] the radiographic signs include an advancing wedge of bone resorption , an accentuation and coarsening of the bone trabeculae along lines of stress , cortical thickening , and enlargement of the bone [ figure 13 ] . as a result , secondary osteoarthritis , insufficiency fractures , bowing of the bones , and even spinal cord or nerve root compression can be observed . in a minority of cases , sarcomatous degeneration may develop , mainly in the humerus , pelvis , and proximal femur , with a frequency believed to range from 0.9 to 2% . paget 's disease affecting the iliac bone demonstrated with coarsening of the bone trabeculae and cortical thickening ( arrows ) . tumors : tumors can be revealed by bone pain , pathological fractures , or disability , or be discovered as an incidental finding . primary bone tumors in elderly patients are not common , but , due to the risk of metastases , any newly discovered bone lesion or one developing within a known pre - existing lesion must be assumed to be malignant until proven otherwise . metastases : bone metastases are a common complication of malignant disease , especially of the breast , prostate , and lung . although they are often multiple , a solitary metastasis is still more common than a primary neoplasm . plain films are relatively insensitive for the detection of bone metastases , especially subtle lesions . mr imaging is superior to ct in the detection of malignant marrow infiltration , and it has a better contrast resolution for visualizing soft tissue and spinal cord lesions . even with the advances in mri , especially for bone marrow screening , bone scintigraphy continues to be used as an effective method for initial evaluation of the whole body for bone metastases . sarcomas : in the elderly population , many sarcomas are secondary to a pre - existing disorder of the bone , such as paget 's disease , or lesions treated by radiation . chondrosarcoma must also be kept in mind , especially when isolated osteolytic lesions of the pelvis are seen , as this is the most common primary sarcoma of bone in adults . the cartilaginous nature of the lesion may be suggested by the presence of calcifications or by a lobulated hyperintense lesion in an mri [ figure 14 ] . chondrosarcoma of the pelvis in ( a ) conventional radiography appears as an isolated osteolytic lesion ( arrows ) , ( b ) coronal t2-weighted image demonstrates a lobulated hyperintense lesion ( arrowheads ) . myeloma : myeloma is a characteristic disease in the elderly population , with a peak incidence in the eighth decade that results from an unregulated , progressive proliferation of neoplastic monoclonal plasma cells that accumulate in the bone marrow , leading to anemia and marrow failure . osteolytic lesions may be present on plain films , but differentiating osteoporotic vertebral compression fractures from those associated with myeloma is a common diagnostic dilemma , as bone loss may be the first sign revealing the latter disease . indeed , widespread osteoporosis , due to cytokine - mediated osteoclast activation , is common in patients with myeloma . this possible association must be kept in mind , particularly if other features are present , including unexplained back or bone pain . special attention is required in this population , as an early diagnosis can avoid delay in treatment , which is associated with increased morbidity and mortality . besides , a better understanding of musculoskeletal diseases can lead to the implementation of effective preventive measures , thus reducing public health expenditure , and improving the quality of life in the elderly . migraine is the leading neurological cause for seeking medical care , and is associated with significant disability in the sufferer . the greatest impact is on migraineurs with headaches on more days than not , a condition defined as chronic migraine ( cm ) . cm is defined as at least 15 days of headache per month in which at least eight of the days fulfill migraine criteria and/or are treated with specific migraine medications , in the absence of a diagnosis of medication overuse headache . patients with cm often had a history of episodic migraine that began in adolescence or early adulthood , reporting a process of transformation marked by headaches that become more frequent over years . among migraineurs , defining risk factors for cm , or for the progression of episodic migraine to cm , identified risk factors include medication overuse , obesity , sleep problems , and psychiatric comorbidity [ 712 ] . studies in both community and tertiary settings have demonstrated an association between migraine and several psychiatric conditions [ 8 , 9 ] . however , the frequency of psychiatric disorders in both setting has not been compared before in a single study . furthermore , differences in methods of studies based in community or tertiary centers prevent appropriate comparison . population studies fail to conduct face - to - face assessments , while clinic - based studies carry the potential for selection bias . studies focusing on best methods to address this gap are of interest , and one strategy is to compare data obtained from the community with those from specialty care , where methods of collection have been virtually identical , and that was the scope of this study . we compared demographic data and psychiatric comorbidity in a sample of individuals with cm from the community with another from a tertiary care clinic . in light of the fact that patients suffering from migraine and comorbid psychiatric disorders are greater health - care service users , we hypothesized that the frequency of psychiatric disorders , notably depression , is higher in patients followed in tertiary care . community data were gathered in capela nova , a city from the state of minas gerais , brazil . according to the 2000 brazilian census , its population was 2,066 inhabitants ( 1,631 over the age of 10 years ) . the present study is part of an observational , cross - sectional and population - based study conducted in two phases [ 14 , 15 ] . initially , community health workers from the family health program directly interviewed all inhabitants aged 10 years or older for headache symptoms in the previous year . the family health program works through family health - care teams which are composed of one physician , one nurse , one auxiliary nurse , and four to six community health workers , and are assigned to specific geographical areas with defined populations of 6001,000 families . activities provided by family health - care teams take place at primary care facilities , in patients homes , and in the community . in the first phase of our study ( screening phase ) , trained community health workers screened for the occurrence of headaches using the following question : have you had any headache episode over the last 12 months ? those who screened positive were asked about headache frequency in the past month , and those reporting 15 days or more of headache were offered in - person assessment by neurologists with expertise in headache medicine . three neurologists independently examined participants , and headaches were diagnosed according to the second edition of the international classification of headache disorders . subsequently , all individuals aged 18 years or older with cm were assessed for psychiatric comorbidities . psychiatric assessment was performed by an experienced psychiatrist using the mini international neuropsychiatric interview ( mini ) . the same investigators involved in the community - based assessment used the very same procedures to diagnose consecutive patients attended at a university - based headache center in the first half of 2006 . this center is located in belo horizonte and is the only headache clinic in the state of minas gerais , brazil . the study followed the guidance of the regulatory norms of the brazilian national health council ( resolution 196/1996 ) which is in accordance with the helsinki declaration . the protocol and all forms were reviewed and approved by the local ethics research committee . data were transferred to epi - info 2000 by a study coordinator and analyzed using spss 12.0 . the relative frequencies of psychiatric comorbidities were stratified by headache type , and confidence intervals were calculated . discrete data were compared between groups using the chi - square test or the fisher test ( when anticipated values were small ) . for continuous non - parametric variables , the mann community data were gathered in capela nova , a city from the state of minas gerais , brazil . according to the 2000 brazilian census , its population was 2,066 inhabitants ( 1,631 over the age of 10 years ) . the present study is part of an observational , cross - sectional and population - based study conducted in two phases [ 14 , 15 ] . initially , community health workers from the family health program directly interviewed all inhabitants aged 10 years or older for headache symptoms in the previous year . the family health program works through family health - care teams which are composed of one physician , one nurse , one auxiliary nurse , and four to six community health workers , and are assigned to specific geographical areas with defined populations of 6001,000 families . activities provided by family health - care teams take place at primary care facilities , in patients homes , and in the community . in the first phase of our study ( screening phase ) , trained community health workers screened for the occurrence of headaches using the following question : have you had any headache episode over the last 12 months ? those who screened positive were asked about headache frequency in the past month , and those reporting 15 days or more of headache were offered in - person assessment by neurologists with expertise in headache medicine . three neurologists independently examined participants , and headaches were diagnosed according to the second edition of the international classification of headache disorders . subsequently , all individuals aged 18 years or older with cm were assessed for psychiatric comorbidities . psychiatric assessment was performed by an experienced psychiatrist using the mini international neuropsychiatric interview ( mini ) . the same investigators involved in the community - based assessment used the very same procedures to diagnose consecutive patients attended at a university - based headache center in the first half of 2006 . this center is located in belo horizonte and is the only headache clinic in the state of minas gerais , brazil . the study followed the guidance of the regulatory norms of the brazilian national health council ( resolution 196/1996 ) which is in accordance with the helsinki declaration . the protocol and all forms were reviewed and approved by the local ethics research committee . data were transferred to epi - info 2000 by a study coordinator and analyzed using spss 12.0 . the relative frequencies of psychiatric comorbidities were stratified by headache type , and confidence intervals were calculated . discrete data were compared between groups using the chi - square test or the fisher test ( when anticipated values were small ) . for continuous non - parametric variables , the mann in the community of 1,605 interviewed inhabitants , 57 ( 3.6 % ) had headaches on at least 15 days for at least three consecutive months . 43 ( 75.4 % ) had cm and 41 consented to being assessed by the psychiatrist ( 95.3 % participation rate ) . in the headache center , 43 patients had cm ( n = 453 , 9.5 % ) and all consented in participating in the psychiatric assessment . sociodemographic profiles were similar between groups with the exception of the mean number of years of formal education , lower in the community relative to the headache center ( table 1).table 1demographic characteristics in individuals with chronic migraine from the community and a clinic - based samplecommunity ( n = 41)headache center ( n = 43)p valuegender men7 ( 17.1 % ) 2 ( 4.7 % ) 0.09 * * women34 ( 82.9 % ) 41 ( 95.3 % ) education ( years of study ) 826 ( 78.8 % ) 5 ( 11.6 % ) < 0.001 * 9115 ( 15.2 % ) 15 ( 34.9 % ) 122 ( 6.1 % ) 23 ( 53.5 % ) age mean ( sd)41.2 ( 17.2)35.7 ( 12.6)0.19 * * * range13731863 * chi - square , * * fisher , * * * mann whitney demographic characteristics in individuals with chronic migraine from the community and a clinic - based sample * chi - square , * * fisher , * * * mann whitney among individuals from the community , 65.9 % of cases were diagnosed with any current psychiatric disorder , relative to 83.7 % in those from the headache center ( p = 0.06 ) . the relative frequencies of some specific diagnoses were remarkably high in both groups , despite not being statistically different . in the headache center , the most prevalent disorders were simple phobia ( 41.9 % ) , generalized anxiety disorder ( 34.9 % ) and major depression ( 32.6 % ) . in the community , the same disorders were also the most common ones : generalized anxiety disorder ( 39.0 % ) , phobias ( 29.3 % ) , and major depression ( 29.3 % ) . bipolar disorder was not seen in the community and was diagnosed in two cases from the headache center . the frequency of antidepressants use was similar in the headache center ( 51.2 % ) and in the community ( 44.4 % ) ( p = 0.51 ) . table 2 summarizes these data.table 2current psychiatric comorbidities in individuals with chronic migraine from the community and a clinic - based sampleheadache center ( n = 43)community ( n = 41)p valueany diagnosis36 ( 83.7 % ) 27 ( 65.9 % ) 0.06*one or two psychiatric diagnoses21 ( 48.8 % ) 16 ( 39.0 % ) 0.36three or more psychiatric diagnoses15 ( 34.9 % ) 11 ( 26.9 % ) 0.42major depression14 ( 32.6 % ) 12 ( 29.3 % ) 0.74*dysthymia9 ( 20.9 % ) 9 ( 22.0 % ) 0.99**bipolar disorder2 ( 4.7 % ) 0 ( 0 % ) 0.23**generalized anxiety disorder15 ( 34.9 % ) 16 ( 39.0 % ) 0.69*specific phobia18 ( 41.9 % ) 12 ( 29.3 % ) 0.23*obsessive compulsive disorder9 ( 20.9 % ) 10 ( 24.4 % ) 0.70*somatization7 ( 16.3 % ) 3 ( 7.3 % ) 0.31**eating disorders2 ( 4.7 % ) 1 ( 2.5 % ) 0.99*alcohol abuse0 ( 0.0 % ) 2 ( 4.9 % ) 0.23 * * * chi - square , * * fisher current psychiatric comorbidities in individuals with chronic migraine from the community and a clinic - based sample * chi - square , * * fisher to the best of our knowledge this is the first study to compare the frequency of psychiatric comorbidity of cm in community and tertiary care clinic samples . the frequency of psychiatric disorders in cm was elevated in both settings , tending to be higher in the tertiary care sample . while psychiatric comorbidity in episodic migraine has been well established in the literature , psychiatric disorders have been less studied in cm . only a few studies have addressed psychiatric comorbidities of cm in population - based samples , finding increased levels of depression and anxiety disorders even in comparison with episodic migraine patients [ 11 , 20 ] . one limitation of these studies was the use of self - report questionnaires rather than clinical interview in ascertaining psychiatric diagnosis . in the present study we found that up to a third of our patients in each setting had depression . a similar rate was described in the american migraine prevalence and prevention ( ampp ) study , a population - based survey based on mailed questionnaires . in that study , depression was assessed by self - report of a physician diagnosis and by the patient health questionnaire ( phq-9)depression module . one interesting result from the ampp study was that cm patients were twice as likely to have depression as assessed by phq-9 in comparison with episodic migraine patients [ 30.2 and 17.2 % respectively ; or ( 95 % ci ) = 2.0 ( 1.67 to 2.40 ) , p < 0.001 ] . cm patients were also approximately twice as likely to report anxiety [ 30.2 vs. 18.8 % respectively ; or ( 95 % ci ) = 1.8 ( 1.51 to 2.15 ) , p < 0.001 ] . the international burden of migraine study ( ibms ) also found higher levels of anxiety and depression in cm when compared with episodic migraine . regarding anxiety syndromes , generalized anxiety disorder and phobias seem to be comorbid with the migraine spectrum [ 2024 ] . interestingly the frequency of obsessive compulsive disorder was significantly high ( between 20 and 25 % ) in cm patients in comparison with its prevalence in the general population ( 2 % ) . only few previous studies have pointed out this association between obsessive compulsive disorder and migraine that may be associated with underlying serotonin system dysfunction . bipolar disorder is also comorbid with migraine ; migraineurs without aura are 2.4 times more likely to have bipolar disorder type 1 , and the ratio increases to 7.3 when the diagnosis is migraine with aura [ 13 , 22 ] . for bipolar disorder type 2 , we failed to detect this association , likely because of small size of the samples . drug abuse has not been traditionally associated with migraine [ 22 , 24 ] , but a recent study reported that illicit drug abuse may be more frequent in migraine patients with depression or post - traumatic stress disorder . demographic profiles were similar in both groups , and the vast majority of individuals with cm were women . it must be highlighted that the assessed community was from a small city , while the patients at the headache center mainly came from a large urban center . nonetheless , studies of migraine that have enrolled subjects with different demographic features also found striking similarities regarding the risk of psychiatric comorbidities , once more pointing to shared biological factors as a plausible mechanism for the comorbidity [ 22 , 24 ] . specific genotypes coding d2 dopaminergic receptors , dysfunction in tyramine conjugation , changes in the metabolism of serotonin and catecholamines and in estrogen levels have been considered to explain the comorbidities [ 2730 ] . it is worth mentioning , however , that we have comprehensively and systematically assessed almost all patients with cm from an entire population of a small city . we did not assess the differential disability associated with headache and psychiatric disorders in the individuals . finally , our findings were not adjusted for other confounding factors , such as parameters of headache severity ( intensity , duration associated symptoms ) , obesity , sleep disorders , use of psychotropic medication and household income . we partly justify these latter limitations by arguing that the demands on patients and resources in conducting these missing assessments could jeopardize the community assessment , since most interviews were conducted in participant households . in conclusion , the present study suggests that psychiatric comorbidity in cm is elevated in the community and clinical settings , tending to be more common in cm patients from a headache center .

musculoskeletal disorders are among the most common problems affecting the elderly . the resulting loss of mobility and physical independence can be particularly devastating in this population . the aim of this article is to present some of the most frequent musculoskeletal disorders of the elderly , such as fractures , osteoporosis , osteoarthritis , microcrystal disorders , infections , and tumors . although the association between episodic migraine and psychiatric comorbidities is well documented , few studies have focused on the comorbidity with chronic migraine ( cm ) and discrepancies exist between population - based and clinic - based data . the objective of this study is to compare demographic and psychiatric comorbidity correlates between cm samples drawn from the community and tertiary care . all inhabitants from a city borough were interviewed for the presence of headaches occurring 15 or more days per month . cm was diagnosed after subjects had been interviewed and examined by a headache doctor . participants were also assessed with a structured interview by a psychiatrist , who assigned diagnoses based on the dsm - iv . the same investigators assessed all patients consecutively seen in a university - based outpatient headache center over a 4-month period . the samples consist of 41 individuals from the community and 43 from the headache center . sociodemographic profiles were similar between groups with the exception of the mean number of years of formal education . among individuals from the community , psychiatric diagnoses were present in 65.9 % of cases , relative to 83.7 % in those from the headache center ( p = 0.06 ) . phobias ( 41.9 vs. 29.3 % ) and depression ( 32.6 vs. 29.3 % ) were more frequent in patients from the headache center , but this difference did not reach statistical significance . thus the frequency of psychiatric disorders in patients with cm was elevated in both settings , being higher in the specialty care clinic .

m1dg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-10(3h)-one ) is one of many endogenous exocyclic lesions derived from the reaction of dna with bifunctional electrophiles generated by lipid , protein , or dna peroxidation . being an endogenous constituent of human and rodent genomes , m1dg is mutagenic in bacterial and mammalian cells and leads to mispairing when replicated in vitro by multiple different dna polymerases . we have previously developed a monoclonal antibody against m1dg and incorporated the antibody into analytical schemes using immunoaffinity purification of m1dg followed by mass spectrometric detection . these studies indicated that adult humans excrete m1dg at a rate of 12 fmol / kgd in urine . subsequent investigations into the metabolism and elimination of m1dg revealed that administration of m1dg to rodents at doses from 8 mg / kg to 6 pg / kg leads to its rapid disappearance from plasma due to oxidation and formation of the single , stable metabolite , 6-oxo - m1dg ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-6,10(3h,5h)-dione ) . in both humans and rats , xanthine oxidase appears to be responsible for this oxidation , while aldehyde oxidase also plays a role in m1dg metabolism in humans . because 6-oxo - m1dg is the sole metabolite of m1dg in rats , it appears to be an ideal surrogate for m1dg as an in vivo biomarker for oxidative stress . all studies of m1dg metabolism in vivo have been conducted with exogenously administered deoxynucleoside . thus , it is not known if 6-oxo - m1dg is actually present in unadulterated intact animals . in order to address this question , we developed a highly specific monoclonal antibody ( mab ) against 6-oxo - m1dg and covalently linked the antibody to sepharose beads . the resultant antibody - sepharose matrix ( the gel ) was used for the immunoaffinity purification of 6-oxo - m1dg from urine and feces followed by lc - ms / ms quantification against the stable isotope - labeled internal standard , [ n5]-6-oxo - m1dg . utilizing this method , we report for the first time that 6-oxo - m1dg is endogenously produced in rodents and is excreted in urine and feces . na2co3 , nahco3 , nacl , water , acetonitrile , mariculture keyhole limpet hemocyanin ( mcklh ) , hcl , acetic acid , and methanol were obtained from thermo fisher scientific ( waltham , ma , usa ) . abts ( diammonium salt ) , thimerosal , kh2po4 , na2hpo4 , kcl , 30% h2o2 , tween 20 , bovine serum albumin ( bsa ) , dimethylformamide ( dmf ) , dimethylsulfoxide ( dmso ) , k2co3 , sodium periodate , sodium methoxide , nabh4 , hypoxanthine , xanthosine , xanthine , ethyl cis-3-iodoacrylate , ethyl cis-3-bromoacrylate aminopterin , ethanolamine , cyanogen bromide - activated sepharose beads , diethylene glycol , 2-deoxyguanosine , sodium hydroxide , formic acid , sodium azide , glycine , and citric acid were obtained from sigma - aldrich ( st . louis , mo , usa ) . horseradish peroxidase - conjugated goat antimouse igg ( h + l ) [ 0.8 mg / ml ] and igg ( fc ) were purchased from jackson immunoresearch ( west grove , pa , usa ) . phosphate buffered saline ( pbs ) was purchased from invitrogen ( carlsbad , ca , usa ) . the synthesis of 6-oxo - m1guo was based on a previously described synthetic scheme with some modifications . guanosine was dissolved in a minimum amount of a dmf / dmso mixture ( 1:1 , v / v ) . k2co3 ( 1.5 equiv ) was added to the solution and the reaction mixture was held at 6065 c for 18 h. ethyl cis-3-iodoacrylate ( 0.25 equiv per h for 5 h ) was added to the reaction mixture . the residue was dissolved into a minimum amount of methanol , and sodium methoxide ( 1.5 equiv ) was added ( dropwise in a 0.5 m solution ) . 6-oxo - m1guo was purified on biotage sp1 flash chromatography system ( biotage , uppsala , sweden ) using the following gradient ; 1% to 5% acetonitrile in 30 column volumes followed by a 5% to 15% gradient in 10 column volumes . separation was achieved on a 12 + m c18 column , and the purified compound showed a single sharp peak by reverse - phase hplc . h nmr ( 600 mhz , dmso - d6 ) : 8.65 ( d , 1h , h8 , j = 7.8 hz ) , 8.29 ( s , 1h , h2 ) , 6.24 ( d , 1h , h7 , j = 8.4 hz ) , 5.82 ( d , 1h , h1 ) , 5.48 ( d , 1h , h2oh ) , 5.20 ( d , 1h , h3oh ) , 5.03 ( d , 1h , h5oh ) , 4.49 ( d , 1h , h2 ) , 4.15 ( d , 1h , h3 ) , 3.91 ( d , 1h , h4 ) , 3.633.55 ( m , 2h , h5 ) . the h nmr may be seen in figure a of the supporting information . 6-oxo - m1dg and its stable isotope - labeled analogue , [ n5]-6-oxo - m1dg , were synthesized as previously described . briefly , anhydrous dg ( 1.6 g , 6 mmol ) and anhydrous k2co3 ( 0.93 g , 6.75 mmol ) were dissolved in anhydrous dmf ( 18 ml ) . one milliliter of a 0.55 m solution of ethyl cis-3-bromoacrylate in anhydrous dmf was added to the reaction every 15 min for 4 h. the reaction was stirred for an additional 2 h then cooled and filtered . the filtrate was evaporated under high vacuum and the residue was dissolved in water ( 4 ml ) and 5% acetic acid ( 4 ml ) . the product was purified by reversed - phase ( c18 ) medium pressure liquid chromatography with a biotage sp1 apparatus ( biotage ) using a prepacked 25 75 mm biotage flash 25 + s cartridge ( kp - c18-hs ) and a flash 25 c18 samplet for sample loading . a pure final product , 6-oxo - m1dg ( 0.92 g , 50.6% ) , was obtained . [ n5]-6-oxo - m1dg was synthesized by substituting [ n5]-dg for dg ( cambridge isotope laboratories , andover , ma , usa ) and employing the scheme described above . the h nmr of 6-oxo - m1dg and its stably labeled analogue matched the spectrum previously reported for this compound . mass spectrometric analysis of 6-oxo - m1dg showed an [ m + h ] peak at m / z 320 , while analysis of [ n5]-6-oxo - m1dg showed an [ m + h ] peak at m / z 325 . the m / z = 325 corresponds to the 6-oxo - m1dg value plus 5 additional mass units from the 5 n atoms incorporated into the [ n5]-dg starting material . a m / z 320 peak was not observed for [ n5]-6-oxo - m1dg , indicating that no unlabeled 6-oxo - m1dg was present in the internal standard . 6-oxo - m1guo ( 12 mg ) was dissolved in 750 l of 100 mm aqueous sodium periodate . protein ( 20 mg of bsa or mcklh ) was reconstituted with 700 l of pbs preadjusted to a ph of 9.5 with 5% k2co3 . the 6-oxo - m1guo solution and the protein after 1 h , 45 l of a 1 m diethylene glycol solution was added to the mixture ( to quench excess oxidizing agent ) followed by 700 l of 0.45 m nabh4 ( aqueous ) . after another 12 h , the ph of the mixture was adjusted to 7.0 with 1.0 m formic acid . the mixture was kept at this ph for 1 h. then , the ph was increased to 8.5 by the careful addition of 1 m aqueous ammonium hydroxide solution . this mixture was dialyzed against pbs buffer twice for 24 h. the sample was lyophilized and stored at 4 c . scheme 1 shows the conjugation reaction of 6-oxo - m1guo with a lysine residue of the carrier protein . figure b of the supporting information depicts the scheme for preparation of the conjugated protein . four balb / cj mice and four a / j mice ( the jackson laboratory , bar harbor , me , usa ) were injected subcutaneously with 50 g of 6-oxo - m1guo - klh and freund s complete adjuvant ( primary boost ) . four wk after the initial immunization , the mice were boosted subcutaneously ( first boost ) with the same dose of conjugate , but with the substitution of incomplete adjuvant ( which was also used in subsequent boosts ) . two wk after the first boost , the mice were tail bled and antibody titers were assessed by direct and competitive elisa as described below . a second boost was administered 4 wk subsequent to the first , and after 2 wk , sera were again extracted and subjected to elisa evaluation . a third boost was administered 25 wk after boost two , and the sera were collected and screened approximately 2 wk later . on the basis of the cumulative elisa data , a single balb / cj mouse ( balb / cj r ) showing the most selective and concentrated anti-6-oxo - m1dg titer was chosen for a fourth and final boost given intraperitoneally and lacking adjuvant to prepare it for splenocyte extraction . four days after the final boost , the mouse was sacrificed by cervical dislocation and the spleen harvested . splenocytes were isolated and subjected to polyethylene glycol - mediated fusion with both sp/20 and ns1 murine myeloma cells ( obtained from the university of virginia and the university of nebraska medical univeristy school of dentistry , respectively ) and allowed to recover for 24 h in liquid culture . the products of the fusion were evenly distributed into 24 96-well plates , with 12 plates corresponding to fusions of sp/20 cells and splenocytes and 12 plates corresponding to fusions of ns1 cells and splenocytes . cells were grown in iscove s modified dulbecco s medium with 20% fetal bovine serum ( invitrogen ) . hybridomas were selected by growing in the presence of aminopterin ( 5 10 m , sigma ) and ht supplement ( 1:100 dilution , invitrogen ) for 14 days with media and aminopterin replenishment every 3 days . supernatant was removed and screened for antigen - specific antibodies by elisa , with 6-oxo - m1dguo - bsa conjugate used as the antigen ( described below ) . positive hybridoma supernatants were rescreened in the presence of xanthine and 6-oxo - m1dg . xanthine was employed as a competitor to identify antibodies not specific to 6-oxo - m1dg . clones exhibiting anti-6-oxo - m1dg activity and specificity in addition to positive growth and productivity were selected and plated on 24-well plates . of these , five clones were chosen based on activity , specificity , growth , and productivity as determined by direct and competitive elisa analysis ( described below ) and then subsequently subjected to two rounds of subcloning . the clones were subsequently cryopreserved . under vanderbilt institutional care and use committee protocol # m-07 - 109 , the vanderbilt antibody and protein resource core is permitted to use freund s complete adjuvant in the manner described above . the following solutions were prepared as follows and their use is described below : ( a ) carbonate - bicarbonate coating buffer ( ph 9.6 ) was prepared from na2co3 ( 1.59 g / l ) , and thimerosal ( 0.10 g / l ) ; ( b ) pbs - tween ( ph 7.4 ) was prepared from nacl ( 8.00 g / l ) , tween 20 ( 1.00 ml / l ) , and thimerosal ( 0.10 g / l ) ; ( c ) bsa ( 5.0 g ) mixed in pbs - tween ( 500 ml ) ; ( d ) 1 nm abts solution in 70 mm citrate - phosphate buffer ( ph 4.2 ) was prepared from citric acid ( 5.64 elisa quality microtiter plates ( thermo fisher scientific ; 96-well immulon 2hb flat bottom microtiter plates or 384-well 4hb flat bottom plates ) were coated with 50 , 75 , or 100 l of a 10 g / ml solution of 6-oxo - m1guo - bsa antigen in carbonate - bicarbonate coating buffer ( a ) and incubated at 4 c overnight . plates were washed three times with 100 l of pbs - tween ( b ) using a bio - tek elx405 automatic microplate washer ( winooski , vt , usa ) , then incubated with 100 l of pbs - tween ( b ) for 30 min at 37 c . aliquots of murine serum dilutions , hybridoma supernatants , or purified antibody ( depending on the stage of the antibody development process ) were then incubated in the coated wells with or without the addition of varying concentrations of 6-oxo - m1dg and structural analogues ( figure 1 ) in a final volume of 100 l pbs for 60 min at 37 c . for wells that included 6-oxo - m1dg or structural analogues , the serum dilution used was 1:5000 in pbs - tween ( b ) , and the compounds depicted in figure 1 were preincubated in the dilute murine serum for 45 min prior to the serum addition step . this allowed the nonspecific antibodies to bind the structural analogues , theoretically leaving behind only 6-oxo - m1dg - specific antibodies . initial analysis revealed that xanthine was recognized nearly as well as 6-oxo - m1dg , which led to the use of this structural analogue in further competition studies . 6-oxo - m1dg , m1dg , and structural analogues that were used as competitive antigens during elisa analysis of murine sera , hybridomal supernatant , and purified antibodies . the plates were then washed three times with pbs - tween ( b ) . aliquots of diluted horseradish peroxidase - conjugated goat antimouse igg fc region - specific or igg ( h + l ) secondary antibody ( diluted 1:5000 in pbs - tween / bsa ) were added , and the plates were incubated at 37 c for 60 min . after 60 min , the plates were washed three times and 100 l of a freshly prepared abts solution ( 1.8 l of h2o2 per 1 ml of abts ) was immediately added to each well . to determine peroxidase activity , the absorbance at 414 nm was measured after 15 and 30 min for each well using a bio - tek powerwave ht 340 plate reader with gen5 software . selected clones were scaled up , inoculated into one - liter bioreactors ( wilson wolf manufacturing , new brighton , mn , usa ) and grown for 34 wk . purification of mab from the bioreactor supernatants was achieved by affinity chromatography on protein - g sepharose ( ge healthcare , piscataway , nj , usa ) followed by a final desalting step into pbs . purified mab were isotyped and subsequently quantified by sds - page electrophoresis followed by infrared coomassie staining . purified anti-6-oxo - m1dg mab was loaded onto pd-10 columns ( ge healthcare ) equilibrated with 25 ml coupling buffer ( 0.1 m nahco3 , 0.5 m nacl , ph 8.0 in water ) . cyanogen bromide - activated sepharose beads ( 3.75 g ) were prepared for conjugation by rapid swelling in 1 mm hcl followed by filtering , rinsing with 400 ml of 1 mm hcl and then 10 ml of coupling buffer . the sepharose beads were transferred to the antibody solution and gently rotated at 4 c for about 20 h. the mixture was then filtered and washed with 500 ml of coupling buffer , and the residue was divided into four parts . each quarter was placed in 14 ml of ethanolamine solution ( 1.0 m , ph 8.0 ) and gently rotated overnight at 4 c . the sepharose beads were then filtered and washed with a low ph acetate buffer ( 0.1 m acetic acid , 0.1 m naoh , and 0.5 m nacl ) followed by a 0.1 m tris hcl buffer ( 0.5 m nacl , ph 8.0 ) . each wash used 75 ml of buffer , and the wash cycle was repeated two more times . the resulting residue was suspended in 20 ml of 0.1 m tris buffer with 0.5 m nacl and 0.2% nan3 ( ph 8.0 ) , and the final product ( the gel ) was stored at 4 c . the gel was used to recover 6-oxo - m1dg and [ n5]- 6-oxo - m1dg from various fluids by the following general procedure . an aliquot of gel was prepared for use by rinsing with the following solutions : 50 ml of 0.1 m glycine ( ph 2.7 ) , 20 ml h20 , 50 ml methanol , and 50 ml pbs . this cleaning procedure was necessary to remove any 6-oxo - m1dg that had bound to the mab during preparation . the ph of the fluid to be analyzed was adjusted to 7.08.0 with acetic acid or ammonium hydroxide . an aliquot of the washed and reconditioned gel was added to the sample , and the suspension was mixed gently for 14 h. the sample / gel suspension was filtered through a precleaned empty polypropylene spe cartridge fitted with a polypropylene frit ( sigma - aldrich ) . the filtered gel was washed with 10 ml of pbs , and the analytes were eluted with 2 ml of methanol . the eluant was dried under n2 gas , capped , and stored at 20 c . the sample was reconstituted in 9:1 water / methanol ( v / v ) immediately prior to lc - ms / ms analysis . urine and feces were collected at 24 h intervals and stored at 20 c following collection . urine samples were analyzed by thawing , adding 200500 fmol of [ n5]-6-oxo - m1dg , and centrifuging the samples at 4000 g for 25 min at 4 c . an aliquot of supernatant was diluted 1015-fold with pbs , and the ph of the final solution was adjusted to 7.5 0.5 with either acetic acid or sodium hydroxide . the gel ( prepared as described above ) was introduced after ph adjustment , and the suspension was rotated gently end - over - end for 14 h. the suspension was filtered and the analyte eluted from the gel as described above . the weighed sample was transferred to a clean vessel with 40 ml of pbs , and 200500 fmol of [ n5]-6-oxo - m1dg were added . dispersion of the sample was accomplished by rotating end - over - end for 23 h followed by sonication for 35 min . the suspension was then filtered through a mesh screen , with the collected solid material rinsed with 10 ml pbs . the supernatant was transferred to a clean vessel , diluted roughly 5 times with pbs , and the antibody gel ( prepared as described above ) was added . the suspension was rotated gently end - over - end for 14 h , filtered , and the analyte eluted from the gel as described above . final lc - ms / ms analysis of gel - purified samples was accomplished on a thermo surveyor autosampler and ms pump in - line with a thermo quantum triple quadrupole mass spectrometer ( thermo fisher scientific ) . the mass spectrometer was equipped with an electrospray source and operated in positive ion mode . 6-oxo - m1dg and its internal standard , [ n5]-6-oxo - m1dg , were detected via selected reaction monitoring ( srm ) with the following transitions , respectively ; m / z 320 204 and 325 209 . in both cases , the transition corresponds to cleavage of the glycosidic bond and loss of the deoxyribose moiety , with the positive charge remaining on the base . the analytes were chromatographed on the following reverse - phase gradient system ; 2% b to 15% b in 5 min , 15% b to 80% b in 1.5 min , followed by a 1 min hold at 80% b. component a was water and component b was methanol / acetonitrile ( 3:1 ) , and each component contained 0.1% acetic acid ( v / v ) . the column used was a phenomenex c18 ( phenomenex , torrence , ca , usa ; 10.0 0.2 cm , 3 m ) held at 3540 c . the column was equilibrated at the initial conditions for 3 min prior to each injection . 6-oxo - m1dg was quantitated by stable isotope dilution against [ n5]-6-oxo - m1dg . lc - ms / ms data was acquired and processed by xcalibur software ( thermo fisher scientific ) . the overall scheme of the antibody generation and isolation process is depicted in figure 2 . eight mice ( four balb / cj and four a / j ) were injected with 6-oxo - m1 g conjugated to klh as described in the materials and methods section . at 6 , 10 , and 35 wk after the initial innoculation , the mice were tail bled , and direct and competitive elisa analyses of the sera were performed ( figure 2 , step 1 ) . all three bleeds from all 8 mice showed robust responses against the 6-oxo - m1g - bsa antigen , indicating that the immune systems of the mice were producing significant amounts of antibodies against the 6-oxo - m1 g portion of the innoculant . competitive elisa screenings were executed to assess antibody specificity , with 6-oxo - m1dg and a series of structural analogues ( figure 1 ) serving as competing antigens . eight mice were inoculated with adduct / protein conjugate , followed by tail bleeding and testing for the presence of 6-oxo - m1dg antibodies using direct elisa analysis ( step 1 ) . splenocytes from a single mouse , balb / cj r , were fused with myeloma cells , plated on twenty - four 96-well plates , and screened using direct and competitive elisa analyses ( step 2 ) . from this screen , five parental cell lines were chosen for subcloning and further screening ( step 4 ) . on the basis of these final screenings , the cell line 6c9ba4c12 was chosen for scale - up and antibody purification ( step 5 ) . figure 3 depicts a competitive elisa analysis of the 35-week serum from mouse balb / cj r. this analysis employed 6-oxo - m1dg as the competing antigen and a robust concentration dependent decrease in optical density is observed , indicating the presence of anti-6-oxo - m1dg antibodies in the sera from this mouse . the results of similar analyses of the sera of all 8 inoculated mice can be seen in supporting information figure c. supporting information figure d displays the results from competitive elisa analyses of the 35-week serum from all 8 inoculated mice where m1dg is employed as the competing antigen . increasing amounts of m1dg do not result in a decrease in optical density , indicating that m1dg does not bind to the anti-6-oxo - m1dg antibodie(s ) present in the sera . 6-oxo - m1dg antibodies are present in the serum of mouse balb / cj r. the serum of mouse balb / cj r was subjected to competitive elisa analysis with 6-oxo - m1dg used as the competitor . the displayed results represent the average of spectroscopic readings at 15 and 30 min postaddition of abts substrate . on the basis of the elisa analysis of murine sera , a single mouse ( balb / cj r ) was chosen for hybridoma production . splenocytes from balb / cj r were fused with both sp/20 and ns1 myeloma cells ( figure 2 , step 2 ) , and 1670 hybridomas were formed from 2304 fusions wells . elisa analysis revealed that of the 1670 hybridomas , 180 showed production of anti-6-oxo - m1dg antibodies . these were grown in 24-well plates ( figure 2 , step 3 ) , and the supernatants were subjected to further elisa screenings . on the basis of the screening data , five hybridomas were selected for subcloning and further screenings ( figure 2 , step 4 ) . figure 4 shows a representative elisa analysis of the supernatants from two subclones ( b and e2 ) of parental hybridoma cell line 6c9 in which xanthine and 6-oxo - m1dg are employed as competing antigens . the presence of unbound 6-oxo - m1dg decreases the optical density of both b and e2 supernatants , indicating the presence of anti-6-oxo - m1dg antibodies . for subclone e2 , xanthine has a similar effect , demonstrating that e2 s mab are not specific to 6-oxo - m1dg . however , xanthine has no effect on the optical density of subclone b supernatant , suggesting that mab from subclone b are specific to 6-oxo - m1dg . on the basis of these data , subclone b was subjected to further subcloning but e2 was not . supporting information figure e illustrates that cell line 6c9ba4 , a subclone of hybridoma 6c9b , retained activity against 6-oxo - m1dg but did not react to xanthine . the specificity of antibodies produced by two hybridoma cell lines ( b and e2 ) subcloned from the parental hybridoma cell line 6c9 were assessed using competitive elisa analysis . the displayed results represent the average of spectroscopic readings of subclones e2 and b at 15 and 30 min postaddition of abts substrate . on the basis of a review of the cumulative elisa data , a final daughter cell line was selected from each parental hybridoma line , and these daughter cell lines were cryopreserved . the elisa data indicated that antibodies from the cell line 6c9ba4c12 displayed the most promising expression and specificity . thus , this cell line was subjected to large - scale expression and antibody purification . figure 5 shows the elisa responses of the mab purified from 6c9ba4c12 in the presence of 6-oxo - m1dg and structural analogues . only 6-oxo - m1dg attenuated the optical density , indicating a specificity of the purified mab for 6-oxo - m1dg over close structural analogues . purified antibodies from the hybridomal subclone ( 6c9ba4c12 ) were screened for specificity for 6-oxo - m1dg in the presence of structural analogues using competitive elisa analyses . the purified anti-6-oxo - m1dg mab was covalently linked to sepharose beads as described above . the purification and analysis protocol outlined in materials and methods was used to quantify aliquots of 10 mm potassium phosphate ( ph 8.0 ) spiked with varying amounts of 6-oxo - m1dg ( 0.25 , 2.5 , and 25 pmol ) and 1.0 pmol of the internal standard , [ n5]-6-oxo - m1dg ( n = 3 for each concentration of 6-oxo - m1dg ) . as shown in table 1 , the experimentally determined amount of 6-oxo - m1dg was within 12% of the known amount at each level . the average percent recovery of 6-oxo - m1dg ranged from 94% to 74% , while the internal standard was recovered at an average rate of 79% . thus , the gel was able to bind 6-oxo - m1dg from an aqueous solution and released the analyte in the presence of methanol . the amount detected , % accuracy , and % recovery values are shown as the mean s.d . the limit of detection ( lod ) of the described lc - ms / ms system was established at approximately 10 fmol 6-oxo - m1dg on - column ( supporting information figure f , top ) by analyzing a series of increasingly dilute 6-oxo - m1dg solutions . given this lod and the percent recovery observed from urine and fecal samples ( approximately 30% ) , the limit of quantification of the assay was estimated to be 50 fmol per sample . additionally , the lc - ms / ms system provided a linear response to 6-oxo - m1dg solutions over a 5000-fold concentration range and a single [ n5]-6-oxo - m1dg concentration ( supporting information f , bottom ) . these results indicate that the gel provided a sufficient level of recovery of the analyte and its internal standard as well as sufficient capacity for the expected levels of 6-oxo - m1dg present in rat urine and feces . they also demonstrate that the lc - ms / ms analysis of the purified sample provided sensitive and accurate quantification . urine and feces collected from male sprague dawley rats over 3 days were analyzed as described in the materials and methods section . 6-oxo - m1dg was detected in urine from only one subject , animal 3 , at a rate of 188 fmol / kgd . however , 6-oxo - m1dg was eliminated in feces in all the animals at a rate of 3501893 fmol / kgd . thus , it appears fecal elimination is the main route by which that 6-oxo - m1dg is excreted from the body . values are given as the mean amount observed in fmol / kgd s.d . ( n = 35 for feces and 2 for urine ) . while the amounts of 6-oxo - m1dg are low , the assay provided a sufficient signal - to - noise ratio to permit accurate quantification . the upper trace ( m / z 320 204 ) shows the analyte , while the bottom trace ( m / z 325 209 ) represents the [ n5]-6-oxo - m1dg internal standard . the inset of figure 6 is the internal standard alone after recovery from pbs . there is no peak in the 6-oxo - m1dg trace of the inset , which is illustrative of the fact that there was no isotopic impurity in the internal standard that could contribute to the 6-oxo - m1dg signal . a representative lc - ms / ms chromatogram of 6-oxo - m1dg and the internal standard [ n5]-6-oxo - m1dg isolated from rat feces is displayed . m1dg is an endogenous dna adduct detectable in the genomic dna of humans and rodents . its presence is associated with peroxidative damage to cellular constituents , particularly polyunsaturated fatty acids , and dna . although many studies have shown that the lipid peroxidation product , malondialdehyde , can react in vitro with deoxyguanosine to form m1dg , recent studies in e. coli , with defined polyunsaturated fatty acid content in membrane phospholipids , indicate that this is not a major source of m1dg . rather , the dna peroxidation products , base propenals , appear to account for nearly all of the m1dg generated when bacteria are exposed to peroxynitrite ( onoo ) . thus , although m1dg appears to be a reliable marker of oxidative damage to cells , it primarily reflects oxidative damage to dna . m1dg is repaired by nucleotide excision repair and is excreted in urine at rather low levels in humans ( 12 3.8 fmol / kgd ) . a major contributor to the low levels of m1dg is its oxidative metabolism to 6-oxo - m1dg . this suggests 6-oxo - m1dg may be superior to m1dg as a noninvasive marker of oxidative damage . in the present manuscript , we describe the generation of a monoclonal antibody that exhibits sensitive and specific binding to 6-oxo - m1dg and its use in a quantitative assay suitable for analysis of urine and feces . in the assay , the sepharose - bound mab is used to isolate 6-oxo - m1dg from urine and feces . the purified sample is subjected to lc - ms / ms analysis where 6-oxo - m1dg is quantified via stable isotope dilution against the synthetic internal standard , [ n5]-6-oxo - m1dg , which is introduced to the sample prior to antibody purification . the antibody gel shows excellent recovery of 6-oxo - m1dg from buffer solutions . the analyte is recovered at 74% or greater from 10 mm potassium phosphate over a 100-fold range of 6-oxo - m1dg , 0.25 pmol to 25.0 pmol . the internal standard was recovered at 79% ( 1.0 pmol ) from these same solutions , and spiked buffer solutions were quantified accurately to 12% . these data demonstrate that 100 l of the antibody gel is able to bind at least 26 pmol of 6-oxo - m1dg from solution , and lc - ms / ms detection of the purified sample is quantitative . the recovery of [ n5]-6-oxo - m1dg from saline solutions was greater than 78% but its recovery from urine and feces was only 29% . this suggests a matrix effect where other nucleosides , deoxynucleosides , and/or endogenous congeners compete with 6-oxo - m1dg for antibody binding sites . although there was minimal cross - reactivity of the antibody with individual members of a panel of exocyclic adducts and purine oxidation products ( figures 1 and 5 ) , it is possible that high levels of low - avidity nucleosides in urine and feces compete with the low levels of endogenous 6-oxo - m1dg for binding sites on the mab . 6-oxo - m1dg was detected in the feces of each of three different sprague dawley rats but was detected in the urine of only one of the rats ( table 2 ) . the fecal levels of 6-oxo - m1dg ( 3501893 fmol / kgday ) were significantly higher than the urinary levels ( 188 fmol / kgday ) . this is consistent with previous findings that demonstrate m1dg is excreted mainly in urine , whereas 6-oxo - m1dg is excreted mainly in feces . because 6-oxo - m1dg is present endogenously in rats , it suggests that its metabolic precursor , m1dg , is also produced endogenously in rats and metabolized to 6-oxo - m1dg . this idea is consistent with the finding that m1dg is present in rodent and human genomic dna and with studies from this laboratory demonstrating that 6-oxo - m1dg is the sole metabolite of m1dg . it may be argued that endogenous 6-oxo - m1dg is generated in the nucleoside pool rather than arising from direct damage to dna . however , this seems unlikely if one considers that base propenals are the principal precursors to m1dg . base propenal formation is triggered by abstraction of the hydrogen from the c-4 position of the deoxyribosyl unit of double - stranded dna . the base propenal produced then reacts with a deoxyguanosine residue in duplex dna to form m1dg . in comparison , the yields of base propenals are much lower when deoxynucleosides are oxidized instead of duplex dna . furthermore , m1dg formation as a reaction of base propenals and free deoxyguanosine is a bimolecular process . if base propenals are formed in duplex dna , they are generated in close proximity to deoxyguanosine residues in the dna duplex , which should maximize the probability of their reaction with a deoxyguanosine residue . one or both of these compounds may have been ingested , then excreted or metabolized . it was not possible to control for this possibility under the reported experimental regime , so future studies with animals fed nucleic acid - free diets will be required to test this hypothesis . the ability to measure excreted 6-oxo - m1dg has the potential for clinical relevance in assessing the levels of oxidative dna damage in humans . since the basal levels of both deoxynucleoside adducts are low , they should provide high sensitivity to increases triggered by oxidative damage . given that the analytical method described herein is applicable to feces and urine , the possibility exists for preclinical or clinical studies conducted in a noninvasive fashion . a 60-year - old woman ( height , 162 cm ; weight , 61 kg ) visited pain clinic with tactile allodynia and electric shock - like pain in the left dorsal scapular area around the t3 dermatome , which had been diagnosed as phn about 1 month previously and attack of the herpes zoster was 1 year ago . the 100-mm visual analogue scale ( vas ) of allodynia and electric shock - like pain was rated between 70 and 80 mm on a scale from 0 ( no pain ) to 100 ( worst pain imaginable ) . the interlaminar epidural block was performed at the t3 - 4 space by the paramedian approach with 5 ml of 0.2% ropivacaine and 20 mg of triamcinolone acetate . pregabalin and morphine at doses of 150 mg and 10 mg , respectively , twice a day , amitriptyline at a dose of 10 mg before sleep and topical lidocaine patches were prescribed . dosages of all drugs were adjusted depending on the side effects during the follow - up period . epidural blocks were repeated twice with a 1-week interval and the continuous intravenous infusion of ketamine ( 60 mg ) was performed over a period of 1 hour twice a week under careful monitoring . after 1 month elapsed , electric shock - like pain was reduced to a vas score of 30/100 , but allodynia was not diminished ( vas score of 70/100 ) . after 4 months elapsed , we decided to administer magnesium sulfate via the intravenous route . and it was done with continuous intravenous infusion of 1,000 mg of magnesium sulfate in 50 ml of normal saline for 1 hour . before and after infusion , the serum magnesium levels were checked . after magnesium therapy , she felt very good about her pain and the vas of allodynia was reduced to 40 - 50 . at 1-week follow - up , she was very satisfied with the treatment and reported the reduction of allodynia on the dorsal scapular area of up to 50% ( vas 25 - 30/100 ) . however , the serum magnesium level had increased above normal range ( 2.3 meq / l to 2.9 although it was below the serum level reveals of the adverse effect , we decided to stop intravenous infusion of magnesium sulfate . for more accurate and safe delivery of magnesium to the target site , we applied magnesium using the transforaminal epidural injection technique . with the patient 's informed consent , left t3 tfemi identification of the t3 nerve root sheath and epidural space was performed using contrast media ( fig . 1 ) . then , 100 mg of magnesium sulphate and 1 ml of 0.2% ropivacaine ( total volume , 2 ml ) was carefully injected . tfemi was repeated twice with a 1-week interval ( total of three times ) and the degree of pain decreased gradually during the follow - up period . one week after the last procedure , the vas score of allodynia decreased to 15/100 and all medications except pregabalin were discontinued . the vas was 10/100 throughout 1-month follow - up , and pregabalin had also been tapered . to our knowledge , no previous report has described about the magnesium administration by the transforaminal epidural route in patients with neuropathic pain . here , in our report of phn patient , this treatment resulted in effective pain relief . previous studies have demonstrated the anti - allodynic effects of nmda receptor antagonists in neuropathic pain disorders . among the currently available nmda receptor antagonists , ketamine is the most widely used one for the treatment of neuropathic pain . however , ketamine is not always effective and psychomimetic side effects are frequent . magnesium can antagonize nmda receptor channels by blocking calcium influx in a voltage - gated manner . intravenous administration of magnesium is efficacious in the management of various conditions associated with neuropathic pain , including phn . demirkaya and colleagues revealed 1 g i.v . mg sulfate is effective in the treatment of migraine attacks and collins and colleagues reported that 70 mg / kg magnesium sulphate infusions in 4 hours for 5 days reduced pain in patients with complex regional pain syndrome . whether intravenous administration of magnesium can achieve a sufficient concentration in the cerebrospinal fluid to block nmda receptors is unclear and studies have reported on the limited efficacy of magnesium when administered via the intravenous route . furthermore , even if the dose of intravenously administered magnesium is not sufficient to present toxicity , patients are still at risk of magnesium overdose . neuraxial administration of magnesium is an " off - label " use , and the safety of this technique in human subjects is still undetermined . however , animal studies showed that intrathecally administered magnesium was free of neurotoxicity , and recent studies have demonstrated the safety of magnesium administration via the epidural or intrathecal route in humans . in fact , the exact site of action of epidurally administered magnesium ( i.e. , spinal or supraspinal ) remains unclear . however , comparison with previous reports regarding intravenous magnesium administration suggested that the low dose epidural magnesium used in our patient was unlikely to result in systemic effects . in conclusion , tfemi showed a favourable result in the treatment of intractable allodynia associated with phn . this study was performed in only a single case , and further investigations are required to determine the efficacy of tfemi in the management of allodynia in patients with phn .

oxidative stress triggers dna and lipid peroxidation , leading to the formation of electrophiles that react with dna to form adducts . a product of this pathway , ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-10(3h)-one ) , or m1dg , is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway . in vivo , m1dg is oxidized to a primary metabolite , ( 3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-6,10(3h,5h)-dione , or 6-oxo - m1dg , which is excreted in urine , bile , and feces . we have developed a specific monoclonal antibody against 6-oxo - m1dg and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo - m1dg from biological matrices . the purified analyte is quantified by lc - ms / ms using a stable isotope - labeled analogue ( [ 15n5]-6-oxo - m1dg ) as an internal standard . healthy male sprague dawley rats excreted 6-oxo - m1dg at a rate of 3501893 fmol / kgd in feces . this is the first report of the presence of the major metabolite of m1dg in rodents without exogenous introduction of m1dg . although postherpetic neuralgia ( phn ) is a common chronic pain syndrome , the pathophysiology of this disorder is not well known and management is often very difficult . n - methyl - d - aspartate ( nmda ) receptor antagonists are known to be effective in phn , and magnesium , a physiological blocker of nmda receptors , is widely used to treat various chronic pain disorders . here , we present a case of the phn refractory to conventional treatment , which was treated successfully with transforaminal epidural injection of magnesium sulphate at the affected dermatome .

central retinal vein occlusion ( crvo ) is a common sight - threatening vascular disease with prevalence rates ranging from 0.1%1 to 0.5%2 in the older adult population . crvo is characterized by occlusion of the central retinal vein and consecutive damming of the venous blood flow . the occlusion may occur at the level of or posterior to the lamina cribrosa.3 despite its prevalence , the pathogenesis of crvo is not completely understood . a combination of vascular , anatomic , and inflammatory factors may contribute to its pathophysiology.4 current treatment options include intravitreal injections of steroids5 or anti - vascular endothelial growth factor ( anti - vegf ) agents.68 fluorescein angiographic ( fa ) assessment is the most important tool for an adequate evaluation of the disease severity and for proper classification between the two types of crvo , ischemic and nonischemic . ischemic crvo is associated with a significant upregulation of vegf , a poor prognosis for visual acuity , and has a higher incidence of secondary neovascular glaucoma ( nvg ) ranging from 20% to 60% compared to nonischemic crvo with no risk of developing nvg.9 given these significant differences with various outcomes and risk profiles , it is crucial to differentiate between ischemic and nonischemic crvo . conventional fa images the retinal circulation within the posterior pole . however , it is not known if conventional fa devices are able to capture areas of interest regarding the intraocular metabolism of vegf , such as the peripheral retina . earlier findings suggested an association between peripheral retinal ischemia and an increased production of vegf.10,11 currently , two ultrawide - field systems are commercially available to perform fa and image the peripheral retina . one method uses a wide - field contact lens system ( ocular staurenghi 230 slo retina lens ; ocular instruments , inc , bellevue , wa , usa ) with the heidelberg retina angiograph ( hra ; heidelberg engineering gmbh , dossenheim , germany).12,13 the second available system uses the optos scanning laser ophthalmoscope ( optos plc , dunfermline , uk ) , which is a special non - contact - lens - based system that provides visualization up to the peripheral retina.1416 it has been shown that ultrawide - field fa ( uwfa ) is useful for the assessment of several retinal pathologies17 including retinal vein occlusions,18 diabetes,19,20 uveitis,21 retinal vasculitis,22 choroidal masses,23,24 retinal detachment,25 and retinopathy of prematurity.26 the aim of this study was to evaluate a possible association between peripheral retinal nonperfusion obtained with uwfa and the number of intravitreal ranibizumab injections needed in patients with crvo . this prospective interventional study was performed between june 1 , 2012 , and february 1 , 2014 . the institutional review board of ludwig - maximilians university in munich approved the study design , and patients care adhered to the tenets of the world medical association declaration of helsinki . all patients gave written informed consent for both participation in the study and for fa . inclusion criteria were diagnosis of crvo ( as revealed by retinal hemorrhages and dilated retinal veins in all four quadrants of the fundus ) with active center - involving macular edema and central subfield thickness > 250 m detected by spectral - domain optical coherence tomography ( sd - oct , heidelberg engineering , heidelberg , germany ) . patients without macular edema , with previous focal or panretinal photocoagulation , and with degenerative disorders of the posterior pole and/or retinal periphery were excluded . additional intravitreal injections were administered in the presence of active center - involving macular edema and central subfield thickness > 250 m , as determined by sd - oct . all patients underwent comprehensive ophthalmologic examination that included best - corrected visual acuity ( bcva ) measurement , slit - lamp biomicroscopy , applanation tonometry , indirect ophthalmoscopy , and sd - oct before injections and at each follow - up visit . uwfa using the optos 200t imaging system was obtained in each case before the first injection . all included patients received three intravitreal injections of 0.50 mg ranibizumab ( lucentis , genentech , inc . , south san francisco , ca , usa and novartis pharma ag , basel , switzerland ) every four weeks and then were examined monthly with follow - up oct scans of the macula . they received additional intravitreal injections if macular edema was detected by sd - oct scans at follow - up visits . sd - oct volume scans ( 2015 with 19 horizontal sections , art 9 , sd - oct , heidelberg engineering , heidelberg , germany ) of the macula were obtained for each study eye to measure the central subfield thickness ( cst ) in m by using heidelberg sd - oct software ; significant macular ischemia was ruled out by uwfa . fa images were acquired approximately one minute ( arteriovenous phase ) and 45 minutes ( late venous phase ) after intravenous injection using the optos 200t scanning laser ophthalmoscope ( optos plc ) after standard intravenous infusion of 5 ml of sodium fluorescein ( 10% ) by one experienced technician for all included cases . fa images were compressed into high - quality jpeg files ( figures 1 and 2 ) and analyzed for retinal nonperfusion by two experienced ophthalmologists ( kaa and fs ) . the far peripheral retina was defined as the area between the ampullae of the vortex veins and the ora serrata . the wide - field color images and wide - field fa images were used to identify the vortex vein ampullae , which were located near the ocular equator.27 based on another published work , we defined retinal nonperfusion as at least five disc areas of hypofluorescence28 ( representing retinal nonperfusion or capillary dropout ) or areas of microvascular pathology ( multiple microaneurysms and significant perivascular leakage ) on uwfa . five disc areas was also the mean size of peripheral retinal nonperfusion of all our patients with crvo . because of the effect of upper and lower eyelid and eyelashes , the far periphery in the horizontal plane alone ( the nasal and temporal retina ) was analyzed . collected parameters included demographic information , previous ocular history , number and dates of intravitreal injections , central subfield thickness , peripheral retinal ischemia , central intraretinal fluid , visual acuity , and intraocular pressure ( throughout the study period ) as well as the occurrence of any complications . data were collected and analyzed using spss software ( version 20.0 , ibm corporation , armonk , ny , usa ) . spearman rho test was used for correlation analysis . a p - value of < 0.05 was considered statistically significant . this prospective interventional study was performed between june 1 , 2012 , and february 1 , 2014 . the institutional review board of ludwig - maximilians university in munich approved the study design , and patients care adhered to the tenets of the world medical association declaration of helsinki . all patients gave written informed consent for both participation in the study and for fa . inclusion criteria were diagnosis of crvo ( as revealed by retinal hemorrhages and dilated retinal veins in all four quadrants of the fundus ) with active center - involving macular edema and central subfield thickness > 250 m detected by spectral - domain optical coherence tomography ( sd - oct , heidelberg engineering , heidelberg , germany ) . patients without macular edema , with previous focal or panretinal photocoagulation , and with degenerative disorders of the posterior pole and/or retinal periphery were excluded . additional intravitreal injections were administered in the presence of active center - involving macular edema and central subfield thickness > 250 m , as determined by sd - oct . all patients underwent comprehensive ophthalmologic examination that included best - corrected visual acuity ( bcva ) measurement , slit - lamp biomicroscopy , applanation tonometry , indirect ophthalmoscopy , and sd - oct before injections and at each follow - up visit . uwfa using the optos 200t imaging system was obtained in each case before the first injection . all included patients received three intravitreal injections of 0.50 mg ranibizumab ( lucentis , genentech , inc . , south san francisco , ca , usa and novartis pharma ag , basel , switzerland ) every four weeks and then were examined monthly with follow - up oct scans of the macula . they received additional intravitreal injections if macular edema was detected by sd - oct scans at follow - up visits . sd - oct volume scans ( 2015 with 19 horizontal sections , art 9 , sd - oct , heidelberg engineering , heidelberg , germany ) of the macula were obtained for each study eye to measure the central subfield thickness ( cst ) in m by using heidelberg sd - oct software ; significant macular ischemia was ruled out by uwfa . fa images were acquired approximately one minute ( arteriovenous phase ) and 45 minutes ( late venous phase ) after intravenous injection using the optos 200t scanning laser ophthalmoscope ( optos plc ) after standard intravenous infusion of 5 ml of sodium fluorescein ( 10% ) by one experienced technician for all included cases . fa images were compressed into high - quality jpeg files ( figures 1 and 2 ) and analyzed for retinal nonperfusion by two experienced ophthalmologists ( kaa and fs ) . the far peripheral retina was defined as the area between the ampullae of the vortex veins and the ora serrata . the wide - field color images and wide - field fa images were used to identify the vortex vein ampullae , which were located near the ocular equator.27 based on another published work , we defined retinal nonperfusion as at least five disc areas of hypofluorescence28 ( representing retinal nonperfusion or capillary dropout ) or areas of microvascular pathology ( multiple microaneurysms and significant perivascular leakage ) on uwfa . five disc areas was also the mean size of peripheral retinal nonperfusion of all our patients with crvo . because of the effect of upper and lower eyelid and eyelashes , the far periphery in the horizontal plane alone ( the nasal and temporal retina ) was analyzed . collected parameters included demographic information , previous ocular history , number and dates of intravitreal injections , central subfield thickness , peripheral retinal ischemia , central intraretinal fluid , visual acuity , and intraocular pressure ( throughout the study period ) as well as the occurrence of any complications . data were collected and analyzed using spss software ( version 20.0 , ibm corporation , armonk , ny , usa ) . each graded variable was tested for normal distribution . spearman rho test was used for correlation analysis . a p - value of < 0.05 was considered statistically significant . fifty - four eyes of fifty - four consecutive patients with treatment - nave crvo were enrolled in this study . twenty - eight patients ( 52% ) were male , and twenty - three eyes ( 42.6% ) were located on the right side . seven patients ( 12.9% ) had known glaucoma , 36 patients ( 66.6% ) had systemic hypertension , and 22 patients ( 40.7% ) were pseudophakic . all patients had center - involving macular edema confirmed by macular leakage seen in fa and retinal thickening in sd - oct images . tables 1 and 2 show the baseline and final bcva and cst . as shown in the tables , there were significant improvements in the visual acuity within and between groups , whereas changes in cst were not significant . twenty - four eyes ( 44% ) showing less than five disc areas of retinal nonperfusion ( group 1 ) that received a mean number of 4.122.73 intravitreal ranibizumab injections . thirty eyes ( 56% ) showed more than five disc areas ( group 2 ) of retinal nonperfusion in uwfa and received a mean number of 9.323.84 intravitreal ranibizumab injections ( p<0.001 , mann whitney u - test ) . there was a significant positive correlation between the size of peripheral retinal nonperfusion measured in terms of disc areas and the number of injections in each group . there was also a significant correlation between the size of retinal nonperfusion and final bcva in each group . there was no significant correlation between the size of retinal nonperfusion and final cst in each group . we did not observe any eye with neovascularization of the disc or neovascularization elsewhere during the observation period ; therefore , panretinal photocoagulation was not performed during this study . furthermore , no cases of injection - related adverse events like retinal detachment or endophthalmitis were encountered . venous occlusive disease of the retina is the second most common retinal vascular disorder after diabetic retinopathy.29 it typically affects patients between 40 and 80 years of age.30 usually , a decrease of visual acuity is the result of macular edema , which can lead to permanent visual loss and even legal blindness in severe cases . applying uwfa , we found a significant correlation between the total number of intravitreal ranibizumab injections and the size of nonperfusion in the peripheral retina : crvo patients with peripheral retinal nonperfusion received more intravitreal injections for treatment of their macular edema than patients without peripheral retinal nonperfusion . we hypothesized that nonperfused peripheral retina could be a source of increased intraocular vegf levels with consecutive macular edema . this observation could explain the requirement for more frequent intravitreal anti - vegf injections of in crvo patients with macular edema and peripheral retinal nonperfusion . this finding is important because an evaluation of the peripheral retina using uwfa may be a prognostic factor and allows the ophthalmologists to estimate the requirement for more or fewer intravitreal injections based on the peripheral retinal perfusion status . looking at the perfusion of the peripheral retina in those patients , a more precise evaluation of the estimated treatment costs within a defined health care system may be feasible , including a better evaluation of injection - related complications such as endophthalmitis or other complications . interestingly , the lack of observed neovascularizations in any of the patients even with extended areas of peripheral retinal nonperfusion might be attributed to good patient compliance , strict follow - up visits every 4 weeks , and immediate treatment in case of detected macular edema . the results of our study differ from those published by spaide31 and singer et al.32 spaide evaluated 22 patients treated with ranibizumab for crvo and found that the area of peripheral nonperfusion was not correlated with the number of injections . the follow - up time of his study was longer than ours , but the sample size was smaller . singer et al32 evaluated 32 patients with retinal vein occlusion and refractory macular edema using sd - oct and uwfa . those patients were retreated with intravitreal injections with anti - vegf and with dexamethasone intravitreal implant . there was no significant difference in the number of anti - vegf ( 1.8 vs 1.6 , p=0.438 ) or dexamethasone implant ( 1.4 vs 1.6 , p=0.364 ) treatments given between the > 10% and 10% nonperfusion groups , nor was there any difference regarding the time to recurrence ( 3.4 vs 4.3 months , p=0.440 ) . in singer et al s series , there were 13 crvo patients with previously failed contiguous therapy with intravitreal ranibizumab and dexamethasone implant . in contrast , we enrolled previously untreated ( treatment - nave ) patients ; furthermore , the number of our cases was higher than in the abovementioned studies . we previously reported that peripheral retinal nonperfusion correlates significantly with intravitreal ranibizumab injections in patients with brvo and macular edema.33 rehak et al34 evaluated 22 crvo patients in a randomized clinical trial and suggested the selective laser photocoagulation of peripheral areas of nonperfusion may further improve the visual outcome and decrease the number of needed ranibizumab reinjections in crvo patients . there are only few published articles regarding the size of peripheral retinal nonperfusion using uwfa . we calculated the mean size of peripheral retinal nonperfusion for all enrolled patients , which was five disc areas , and then the patients were divided into two groups according to that cut - off point . however , the authors totally agree that this approach is not an established step due to lack of evidence . moreover , there are several problems in the interpretation of fluorescein angiograms . the grading of the ultrawide - field angiograms is not uniform because there are eyelash artifacts that influence the entire amount of interpretable fundus , and the graders can also change the clarity of the images that may cause intergrader variability . another drawback of our study design was that we performed uwfa for each patient at only one single time point . thus , we were not able to demonstrate potential dynamic angiographic changes in nonperfusion areas over the follow - up time . in conclusion , uwfa is a precise tool for the detection and quantification of retinal nonperfusion which correlated with the number of needed intravitreal ranibizumab injections in our patients with crvo . retinal nonperfusion could be considered as a prognostic factor for a more precise patient management . small bowel obstruction ( sbo ) is one of the most feared complications after a gastric bypass . it can be chronic or acute and can range from a nuisance to the patient to a life - threatening emergency sbos related to adhesions occur after laparoscopic roux - en - y gastric bypass ( lgb ) , but ih formation , secondary to the reconstruction of the small intestine , is also of concern to bariatric surgeons . multiple defects in the mesentery of the bowel can occur and may lead to ih and , in an antecolic bypass , a defect is associated with the roux limb passing over the transverse colon . the reported incidence of sbo after lgb varies widely , perhaps because the technique used in the operation also varies widely , not only in placement of the roux limb , but regarding closure or nonclosure of the mesenteric defects . the disparity in reports leads to confusion regarding the true incidence and even the causes of sbo after lgb . in addition , the closure of mesenteric defects is questioned in a substantial body of literature . the objective of this study was to examine the incidence and characteristics of sbo after antecolic antegastric bypass with nonclosure of the mesenteric defect of the jejunojejunal ( jj ) anastomosis . following health insurance portability and accountability act guidelines , the author performed a retrospective chart review of a series of consecutive lgbs performed over a 3-year period and recorded in a prospectively maintained database . the follow up of the patients included office visits at 1 week , 1 month , 3 months , 6 months , and then , yearly . all procedures were performed by the same surgeon using the antecolic antegastric technique . in no case was the mesenteric defect closed . any patients presenting with signs and symptoms of an sbo were emergently taken to the operating room . the policy of our bariatric program has always been to explore aggressively any patient with suspicion of an sbo , whether based on physical examination , radiographic studies , or history . patients with an sbo were usually taken to the operating room from the emergency department . those who underwent revisional bariatric surgery or conversion to open operation during the primary surgery were excluded . revisional procedures were not included because adhesions may have formed from the previous bariatric surgery that could have confounded the results . all patients had given informed consent prior to surgery for their deidentified data to be used for study purposes . the gastric pouch was sized to 20 ml with an orogastric balloon with 4 to 5 staple loads with seamguard ( w. l. gore , flagstaff , arizona ) staple - line reinforcement ( slr ) and the echelon 60-mm stapler ( ethicon endo - surgery , cincinnati , ohio ) . the small bowel was divided 40 cm from the ligament of treitz , and the mesentery had minimal division . the only division of the mesentery was from the same staple load that divided the small bowel . the roux limb was measured to 100 to 120 cm , depending on the body mass index ( bmi ) of the patient . the gastrojejunal anastomosis was hand sewn in 2 layers with absorbable sutures over a 34 french bougie . the jejunojejunostomy was formed with a single firing of the same stapler , and the enteroenterostomy was closed with the stapler . in all cases , an intraoperative endoscopy was used to check the gastrojejunal anastomosis , and drains were used in all male patients because of higher technical difficulty and higher rates of mortality and morbidity ; in patients with a bmi over 50 , for the same reasons ; and in selected cases . there was one conversion to an open operation for an incomplete malrotation ; there was no mortality . the gastric pouch was sized to 20 ml with an orogastric balloon with 4 to 5 staple loads with seamguard ( w. l. gore , flagstaff , arizona ) staple - line reinforcement ( slr ) and the echelon 60-mm stapler ( ethicon endo - surgery , cincinnati , ohio ) . the small bowel was divided 40 cm from the ligament of treitz , and the mesentery had minimal division . the only division of the mesentery was from the same staple load that divided the small bowel . the roux limb was measured to 100 to 120 cm , depending on the body mass index ( bmi ) of the patient . the gastrojejunal anastomosis was hand sewn in 2 layers with absorbable sutures over a 34 french bougie . the jejunojejunostomy was formed with a single firing of the same stapler , and the enteroenterostomy was closed with the stapler . in all cases , an intraoperative endoscopy was used to check the gastrojejunal anastomosis , and drains were used in all male patients because of higher technical difficulty and higher rates of mortality and morbidity ; in patients with a bmi over 50 , for the same reasons ; and in selected cases . there was one conversion to an open operation for an incomplete malrotation ; there was no mortality . there were 249 primary lgbs performed in the study period ( january 1 , 2011 , through december 31 , 2013 ) . sbo in 4 cases was caused by ih ( incidence of 1.6% ) and by adhesions in 11 ( 73% ) ( figure 1 ) . in the cases with ih , the most common locations were from the jj to the roux limb ( n = 5 ) , followed by those forming from the jj to the abdominal wall ( n = 3 ) , and 1 each from the jj to the colon and from the jj to the common channel ( figure 2 ) . in all cases , the adhesions were treated with laparoscopic lysis . the average time to sbo was 8.1 months ( range , 121 ) from the initial surgery . sbo is common after lgb and can be easily managed or can just as easily lead to disaster if not recognized and treated promptly . the surgical literature has many articles that discuss ih formation , and an area of contention has been closure versus nonclosure of the mesenteric defects . most authors seem to agree that if a retrocolic gastric bypass is performed , the mesocolic defect must be closed , which can lead to a 0% rate of ih formation , although that number can climb as high as 15% . elms et al showed that , in almost 2 400 patients who underwent antecolic antegastric bypass , ih formed in only 1.1% , primarily at the mesenteric defect . in those cases the defects had all been closed . cho et al had a 0.2% ih formation rate in 1 400 patients who underwent antecolic antegastric bypasses with no mesenteric closure and no division of the mesentery . rodriguez et al also reported that minimal division of the mesentery could lead to a decrease in ih formation . they showed that , with closure of the defect but with wide opening of the mesentery , there was a 14.4% ih rate ; if the defect was closed and the mesentery was not widely opened , the ih rate dropped to 1.1% . abasbassi et al showed a lower ih formation rate with no division of the mesentery , but also no closure of the mesentery . this cumulative experience could indicate that a way to decrease ih formation is to perform a minimal division of the mesentery . obeid et al used mixed techniques in both retro- and antecolic bypasses , some with closure of defects , some without . in 679 patients with an antecolic roux limb , the majority of all the defects closed . in the retrocolic group , almost 4% of these had closure of the mesenteric defect , 8.4% had nonclosure ; 3.8% were in antecolic and 8.5% in retrocolic bypasses . this wide range of techniques makes an overall analysis of the literature difficult . in 2010 in jsls , hope and colleagues examined the incidence of ih formation after documented closure of mesenteric defects . permanent sutures were used to close these defects , but 15 of 18 patients in the series presented with open defects , causing ih . these outcomes indicate that closure of the defects may not be as permanent as surgeons would like to believe , and indeed this very situation was the catalyst for this paper . in our lgbs , we were closing all defects , but our ih formation rate seemed to be climbing . we noticed most of the mesenteric defects were open at reoperation , and so we switched to nonclosure . as demonstrated here , the published papers on the subject have reported a wide range of ih incidence , from 0% to 15.5% . another complicating factor may be that many of the papers published in this area are from surgical programs training fellows , and the association of postsurgical complications with performance of operations by fellows is not known . in this study , the author specifically examined the incidence and cause of sbo in antecolic antegastric bypasses . in no case was either the mesenteric or antecolic defect closed . the interesting finding was that , of an overall incidence of sbo of 6% , only 1.6% of the total was caused by ih formation , all at the jj mesenteric defect . this outcome compares favorably with the ih formation rate reported by other authors who did or did not close the defects . also , the incidence of sbo caused by adhesions is higher in the current study than in some others . elms et al reported that 47.6% of the sbos in their series were secondary to adhesions , mostly at the jj , compared with our rate of 73% . the opposing end of the adhesion had multiple locations , but the staple line of the jj was always at one end , perhaps because of the intense inflammatory reaction that it generates . a hand - sewn technique could be used to close the common enterotomy and might reduce the incidence of sbo slightly , but the cut end of the biliopancreatic limb still has an exposed staple line . the strength of this study is in the homogeneity of the technique used , as this was a single - surgeon series . however , its strength is also its chief weakness , as the results may not be reproducible by others . the number of sbos is also small enough to raise the possibility of a type ii error . the author lives in a geographically isolated part of the country , with no other surgeons who are willing to care for bariatric patients , and so it was more likely that all the sbos would be treated by the same bariatric surgeon . another limitation of the study is that it was a small series with a short follow - up . although only the years of 2011 through 2013 were examined , and the average time to sbo was 8.1 months , it is possible that the sbo rate will climb in these same patients as time passes , and some of the patients are likely to be lost to follow - up . another confounding factor is that an slr was used early in the series at the jj , but not in later operations . there may be an association of slr with adhesion formation , but the connection is not clear . the most common location of adhesions was at the jj , were slr was used , but about half of the sbos caused solely by adhesions to the jj occurred after the use of slr was abandoned , so there are probably not enough cases to tell if the use of slr increases sbo . the usual weaknesses of a retrospective study apply to this paper as they do to all other studies about ih formation . surgeons should remember that the mainstay of treatment of sbo after lgb is an operation . an isolated roux limb obstruction can be decompressed by vomiting , and the placement of an ngt involves risk of a perforation . the surgeon should move the patient quickly to the operating room once an sbo is diagnosed or even suspected , as bowel ischemia can lead to extensive bowel resection and nutritional debility or even death . these operations can often be performed laparoscopically , but laparotomy is also safe and effective . the incidence of sbo caused by ih with nonclosure of the mesenteric defect is similar to that in other series where the defect was closed . regardless of the cause of the sbo , operation remains the definitive treatment and should not be delayed in the gastric bypass patient .

purposeto evaluate the association between the size of peripheral retinal nonperfusion and the number of intravitreal ranibizumab injections in patients with treatment - nave central retinal vein occlusion ( crvo).methodsfifty - four patients with treatment - nave crvo and macular edema were included . each patient underwent a full ophthalmologic examination including optical coherence tomography imaging and ultrawide - field fluorescein angiography . monthly intravitreal ranibizumab injections were applied according to the recommendations of the german ophthalmologic society . two ophthalmologists quantified the areas of peripheral retinal nonperfusion ( group 1= less than five disc areas , group 2= more than five disc areas ) . correlation analyses between the size of nonperfusion with best - corrected visual acuity , central subfield thickness , and the number of intravitreal injections were performed.resultsbest-corrected visual acuity improved significantly after intravitreal injections ( p<0.001 , both groups ) . final central subfield thickness after treatment did not significantly differ between both groups ( p=0.92 , p=0.96 , respectively ) . mean number of injections in group 1 and group 2 was 4.122.73 and 9.323.84 , respectively ( p<0.001 ) . there was a significant positive correlation between areas of nonperfusion and the number of injections in each group . ( r=0.97 , p<0.001 ; r=0.94 , p<0.001 , respectively).conclusionperipheral retinal nonperfusion in patients with crvo correlates significantly with the number of needed intravitreal ranibizumab injections . ultrawide - field fluorescein angiography is a useful tool for detection of peripheral retinal ischemia , which may have direct implications in the diagnosis , follow - up , and treatment of these patients . background and objectives : there is a wide variation of reported incidence of small bowel obstruction ( sbo ) after laparoscopic roux - en - y gastric bypass ( lgb ) . there is also wide variation in technique , not only in placement of the roux limb , but also regarding closure or nonclosure of the mesenteric defects . the objective of this study was to examine the incidence and characteristics of sbo after antecolic antegastric bypass with nonclosure of the mesenteric defect of the jejunojejunal anastomosis.methods:this is a retrospective review of a series of consecutive lgbs over a 3-year period . all procedures were performed by the same surgeon using the same technique . in no case was the mesenteric defect closed . a prospectively maintained database was used for data collection . patients who returned with an sbo were the study group , and those who underwent revisional bariatric surgery or conversion to open operation were excluded.results:there were 249 primary lgbs performed during the study period ; 15 of the operations were followed by sbo , for an incidence of 6.0% . four cases were caused by an internal hernia ( ih ) , for an incidence of 1.6% , and 11 were caused by adhesions , which accounted for 73% of the sbos.conclusions:sbo after lgb is a relatively common complication . the incidence of sbo from ih with nonclosure of the mesenteric defect is similar to that in other series where the defect is closed . regardless of the cause of the sbo , operative treatment of the patient who has a gastric bypass remains the definitive standard and should not be delayed .

the following section describes the treatment procedure for patients with deep infection following tka using modified static spacers . the original prosthesis was removed , followed by intensive irrigation and wide debridement of the infected soft tissue . a 36 fr - diameter straight thoracic catheter ( mallinckrodt medical , athlone , ireland ) and a steinmann pin measuring 3.0 mm in diameter and 22 cm in length vancomycin ( 2 g ) was then added to the gentamicin bone cement ( depuy , warsaw , in , usa ) . at the late liquid stage of the cement , the steinmann pin was inserted into the tube , and the prepared cement was delivered through the tube . during this procedure , the tube was removed from the cement rod using a surgical knife . finally , a cement rod measuring 9 mm in diameter and 22 cm in length was formed ( fig . an entry hole was created at the center of the distal femur and proximal tibia for the insertion of the cement rod . the rod was then inserted into the femur and tibia through this hole . during insertion , it was important to place the center of the cement rod on an imaginary joint line . the proximal medullary canal of the tibia was filled with antibiotic - impregnated cement up to the surface of the proximal tibia , while the surgical assistant maintained proper anatomic alignment and joint space . the space between the cement of the distal femur and the proximal tibia was filled with more antibiotic - impregnated cement . finally , the suprapatellar pouch and medial and lateral gutter space were filled with antibiotic - impregnated cement to reduce soft tissue adhesion ( fig . cylinder splint immobilization was required for three days after the operation , after which a fixed - angle knee brace was used and toe - touching ambulation was allowed until the reimplantation surgery . from april to november 2007 , the authors performed the static technique in four patients using the novel antibiotic - impregnated cement rod for the treatment of infected tka ( culture : staphylococcus in three cases and no bacteria in one case ) . follow - up laboratory studies , including erythrocyte sedimentation rate and c - reactive protein , culture study via knee aspiration and a frozen biopsy from the second - stage operation ( < 5 polymorpho - leukocytes / high power field ) were performed to confirm successful eradication of the infection . the second - stage reimplantation was performed when all the criteria for the validation of infection control were met ( fig . the second - stage reimplantation was performed using the rectus snipping approach , and more than 90 of flexion was obtained intra - operatively . in all four patients , no re - infection was evident after two and a half years of follow - up . the range of motion of the knee joints were respectively improved from 50 to 80 , 95 , and 100 , and the knee society scores were 70 , 86 , 65 , and 84 , respectively , in the last follow - up evaluation . fehring et al.8 ) emphasized the importance of resting the joint in septic joint conditions . others also reported that the static spacer technique provides more stability than the mobile spacer technique in patients with severe bone loss.5,8 ) the main advantage of this technique is the maintenance of a normally aligned lower limb during the interval period . this maintains knee stability in combination with early muscle strengthening exercises , including quadriceps setting exercise , and enables the patient to comfortably dress and manage him / herself during the intervening period . the cement rod and static spacer provide a stable gap between the femur and tibia , thereby minimizing soft tissue contracture and shortening of the lower limb . in addition , symmetric maintenance of the soft tissue of both medial and lateral gutters requires no additional soft tissue balancing at the second - stage reimplantation . the additional cost of a metal nail , however , and the difficulty in infection control due to biofilm formation around the metal nail can be troublesome . in addition , a metal nail can not be removed easily at the second stage reimplantation due to its hardness , and soft tissue adhesion of the femoral or tibial medullary canals . on the other hand , an antibiotic - impregnated cement rod enjoys advantages , such as inexpensive antibiotic delivery to the marrow spaces and easy removal by using a hercules cutter . in addition , a static spacer is anchored to the cement rod , which can prevent spacer migration and bone erosion . this feature is believed to generate less cement wear debris than with the conventional static spacer or mobile articulating spacer technique . during the conduct of the antibiotic - impregnated cement rod technique , antibiotic - impregnated cement was applied to the proximal tibia , distal femur , joint gap space , suprapatellar pouch , and both gutters in a stepwise manner . this technique enables easy removal of the cement and reduced soft tissue adhesion at second - stage reimplantation . mental fatigue is common and can be a disabling long - time condition following a stroke . it has been estimated that about 3070% of stroke survivors complain of fatigue [ 17 ] . even for those with an almost recovered stroke and without neurological and neuropsychological impairments , the person who suffers from mental fatigue is able to perform mental effort just for short periods , and , notably , it will take longer than normal to regain energy after being exhausted . accompanying symptoms , such as irritability , sensitivity to stress , concentration difficulties , and emotional instability may further impair social interactions [ 811 ] . however , attention has been paid to poststroke fatigue during the last 10 years , and fatigue is now generally held as a separate phenomenon [ 46 , 1214 ] . few investigations have been carried out to evaluate fatigue and cognitive functions following a stroke . leegard reported that fatigue is frequent following a stroke but did not find any related impaired cognitive functions . van zandvoort and coauthors investigated lacunar infarct and reported frequent difficulties relating to fatigue and a decreased cognitive performance under more demanding conditions . with the intention to increase knowledge about mental fatigue and cognitive difficulties related to stroke , we examined well - rehabilitated stroke participants , who had suffered from long - term mental fatigue for at least one year prior to examination . the subjects were examined for subjective self - reporting of mental fatigue , depression , and anxiety symptoms . neuropsychological tests were aimed at evaluating information processing speed , attention , and working memory . twenty - four participants , having recovered from neurological symptoms , but suffering from pathological mental fatigue for at least one year following a stroke and 24 healthy controls were included in the study . the study persons were recruited from an advertisement in a local , daily newspaper or from the neurological clinic at the local university hospital and were later included in intervention studies . the stroke subjects should have been healthy and at work before the stroke meaning that they had no known diseases but hypertonia was present among some participants . the type of stroke was obtained from medical records and self - reports ( see table 1 ) . the control participants were recruited from the local community , with no history of brain injury , stroke , psychiatric or neurological disorder , and no drug abuse , and they were fully able to work . they performed cognitive tests focused on information processing speed , attention , and working memory . the self - assessment for mental fatigue is a multidimensional questionnaire containing 15 questions and is adapted from rdholm et al . . the self - reported questionnaire covers the most common symptoms occurring after brain injury , stroke , or other neurological disorders affecting the brain [ 11 , 17 ] . the self - assessment scale for mental fatigue and related items has been evaluated , and the 14 questions had adequate internal consistency with a cronbach 's alpha of 0.94 . the questions concern fatigue in general , lack of initiative , mental fatigue , mental recovery , concentration difficulties , memory problems , slowness of thinking , sensitivity to stress , increased tendency to become emotional , irritability , sensitivity to light and noise , and decreased or increased duration of sleep as well as 24-hour variations . this cprs scale is used here for self - assessment of depression and anxiety . the neuropsychological tests included digit symbol - coding from the wais - iii , measuring information processing speed , digit span from the wais - iii , measuring attention and working memory ; verbal fluency test , fas ; trail making test ( tmt ) a and b , measuring visual scanning , divided attention and motor speed . in order to evaluate higher demands such as dual tasks , a series of two new trail making tests was constructed with three and four factors , respectively . months were added in part c and both months and days of the week in chronological order in part d. in the latter , the order of letters and digits was switched . a new computer test was constructed in our department including a single and a complex subtest . the single test included speed of mouse click in four squares , located in each corner of a bigger square ( 6 6 cm ) on the computer screen and was performed in a clockwise order . a mouse click outside the square was recorded as a miss , and a new click was necessary to do in order to be able to go on with the test . the complex sub - test also included the same mouse clicking procedure , but at the same time , the subject was asked to count how many instances of a specific digit between zero and nine , randomly chosen , he / she could see . after the 30 seconds , the subject was asked to report how many of the specific digit he / she had seen . it was possible to measure the difference in speed between a single and a complex task , variability over time , and errors made in counting digits . in the complex test , it was necessary to attend to both the square and the digits . a comparison between groups was done by t - test and analysis of covariance ( ancova ) . the mann - whitney u test was used when analyzing separate items included in the self - assessment scales . pearson 's correlation and linear regression were used for analysis of connections between variables . spss 16.0 for windows the control group had significantly more years of education than the stroke group ( t - test , p = 0.001 ) , and age was almost significantly different when comparing the two groups ( p = 0.055 , table 1 ) . accordingly , ancova , controlling for the variance for education and age , was conducted for all the variables analysed . the only significant gender difference found was that the control females were faster in the digit symbol - coding test , and an ancova , also controlling for sex , was conducted for this variable . a significant difference was found between the groups for the total sum of scores for mental fatigue . the mean value for the stroke group was 18.4 with a 95% confidence interval of between 16.420.5 . the mean value for the control group was 4.0 , with a 95% confidence interval of between 2.95.0 ( see figure 1 ) . the total score for the cprs scale , taken from the depression and anxiety subscale , was also rated significantly higher in the stroke group compared to controls ( see table 2 , figure 1 ) . the significant effect for mental fatigue remained after adjustment for depression ( p < 0.001 ) . all the separate items in the self - assessment scale for mental fatigue were rated significantly higher for the stroke group compared to controls . the cprs gave the following findings : without taking into account overlapping items , the items relating to sadness , emotional involvement , pessimistic thoughts , and zest for life rated significantly higher for the stroke group ( adjusted for multiple comparisons , as shown in figure 2 ) . among the stroke subjects , 74% reported a clear 24-hour variation with morning most frequently reported to be the best time of the day and afternoon and evening the worst . the participants from the stroke group were significantly slower on the test measuring information processing speed , primarily digit symbol - coding , and also reading speed and number of mouse clicks in the computer test . the stroke group was also significantly slower and made more errors in tmt d , the most demanding of the tmt tests . they were also significantly slower on tmt b , and produced fewer words on the verbal fluency test . the result for the simple mouse click sub - test was found to be significantly faster for the controls compared with the stroke subjects . the computer test which placed a simultaneous demand on speed , attention , and working memory , resulted in a fairly good speed for the stroke subjects , with no detected difference in speed compared to the controls . on the contrary , the stroke subjects made significantly more errors compared to the controls ( table 2 , figure 3 ) . the cognitive tests with significant results ( p < 0.05 , see table 2 ) were included in a linear regression model , using the enter method . digit symbol - coding ( p = 0.004 ) and the scores for number of errors in the computer test ( p = 0.018 ) were significant predictors for mental fatigue , while the other scores were not significant predictors for mental fatigue . digit symbol - coding and number of errors in the computer test also correlated significantly to the mental fatigue sum of scores ( r = 0.59 and r = 0.46 ) . the total scores for cprs , depression , and anxiety were on a significantly higher level compared to controls . the madrs , which provides a separate scale , has the same format as the cprs self - assessment scale depression , except that the items are graded in a different way . in the cprs scale , depression , the highest level is three for each separate cprs item , while the levels for the alternatives in the madrs scale are double for each separate item , with the highest level being 6 . according to madrs , a score between 12 and 20 is regarded as mild depression and 21 and above indicates a probable true depression . the mean level of eight for the stroke subjects corresponds to 16 on madrs , indicating an overall mild level of depression . there was a highly significant difference between the groups on their self - assessment of mental fatigue , with a mean value of 18 reported for the almost recovered stroke group and a mean value of four for the control group . the self - assessment scale has no cut - off value for fatigue , but our experience shows that a value of 15 and above indicates a clear problem with mental fatigue [ 9 , 23 ] . the clear difference in 24-hour variation also showed the specific exhaustion mental fatigue subjects experience during the active time of the day . it should be noticed that stroke victims were included due to the presence of mental fatigue during one year or more following the stroke . the data we have presented in our study do not include an indication of the frequency of mental fatigue after different types of stroke . however , on an individual basis , according to madrs , eight participants were not depressed , seven had an indicated mild depression , and nine had a probable , true depression . among the controls , two participants scored on a mild depression level , while the remainder scored below this level . the total sum of scores on the depression scales may be deceptive if a person is complaining of concentration difficulties and fatigue , but does not have a depressed mood , nor a lack of interest in and enjoyment of daily activities . in this study , three items were overlapping between mental fatigue and depression , and these items were rated on a higher level than the corresponding specific items on the depression sub - scale ( see figure 2 ) . depression and mental fatigue can occur on their own , but they sometimes occur simultaneously , as shown in this study . accordingly , we suggest mental fatigue and depression to be independent phenomena following a stroke . this also conforms with the findings from other studies [ 46 , 12 , 13 ] . the distressing exhaustion along with the bad memory , concentration difficulties , and not being able to perform simultaneous tasks are the phenomena many subjectively complain of following a stroke . it is important to include cognitive tests in the examinations in order to better understand the difficulties connected to mental fatigue and for the purpose of recommending treatment strategies . in this study , physically well - recovered stroke subjects , who had no medical problems except long - term mental fatigue , also showed decreased information processing speed and made more errors in demanding cognitive tasks compared to the control subjects . processing speed is also fundamental and important when considering cognitive functions of a higher order . few studies have been carried out which cover fatigue and cognitive performance following a stroke . however , studies have shown the connection between fatigue and decreased cognitive performance under more demanding conditions , while no cognitive impairments were detected , despite frequent fatigue . with more demanding and sensitive tests , including processing speed and complex attention , it may be possible to detect cognitive impairments accompanying mental fatigue . this is important , as mental fatigue and cognitive deficits will be an obstacle to almost recovered stroke subjects who are on their way to a return to work and previous activities , as every - day life today is complex , with high demands being placed on simultaneous and rapid decisions . in conclusion , mental fatigue is disabling for many people following an almost recovered stroke , and this is suggested to be related to cognitive impairments , primarily information processing speed , and attention . mental fatigue should also be treated as a separate phenomenon and should be differentiated from , and not confused with , depression . today , no specific guidelines exist for the treatment of mental fatigue , and research is urgently needed for this common yet distressing symptom .

the two - stage exchange arthroplasty ( one- or two - stage ) is believed to be the gold standard for the management of infections following total knee arthroplasty . we herein report a novel two - stage exchange arthroplasty technique using an antibiotic - impregnated cement intramedullary nail , which can be easily prepared during surgery using a straight thoracic tube and a steinmann pin , and may provide additional stability to the knee to maintain normal mechanical axis . in addition , there is less pain between the period of prosthesis removal and subsequent reimplantation . less soft tissue contracture , less scar adhesion , easy removal of the cement intramedullary nail , and successful infection control are the advantages of this technique . mental fatigue is for many a distressing and long - term problem after stroke . this mental fatigue will make it more difficult for the person to return to work and previous activities . the intention with this study is to investigate mental fatigue in relation to depression and cognitive functions . we examined 24 well - rehabilitated stroke subjects , who suffered from mental fatigue one year or more after a stroke , and 24 healthy controls . subjects were examined using self - assessment scales for mental fatigue , depression and anxiety , and cognitive tests . the results showed a highly increased rating for mental fatigue for the stroke group ( p < 0.001 ) . these participants also had a significantly higher rating on the depression ( p < 0.001 ) and anxiety ( p < 0.001 ) scales . furthermore , they had a slower information processing speed ( p < 0.001 ) and made more errors in a demanding attention and speed test ( p < 0.05 ) . among the cognitive tests , processing speed and errors made in an attention and speed test were significant predictors for mental fatigue . we suggest mental fatigue following a stroke to be related to cognitive impairments , primarily information processing speed . mental fatigue should also be treated as a separate phenomenon and should be differentiated from , and not confused with , depression , even if overlapping symptoms exist .

cigarette smoke ( cs ) is associated with the development of inflammation - related diseases such as chronic obstructive pulmonary disease and vascular diseases , including atherosclerosis and stroke . several studies have revealed that cs is a major contributor to vascular diseases because it accelerates the development of atherosclerotic plaques . the relationship between cs and the increased incidence of atherosclerosis has been reported [ 57 ] , which may be a consequence of direct endothelial damage , increased proliferation of smooth muscle in atherosclerotic lesions , and/or decreased vasodilation . endothelial damage has also been suggested as the initial cause of development of vascular diseases . in a previous study , it was shown that inhibition of oxidative stress exerts protection in human endothelial cells , which could be an effective strategy in the treatment of vascular diseases . a number of studies support that reactive oxygen species ( ros ) causing oxidative stress may play an essential role in mediating endothelial cell death . oxidative stress is a major factor in vascular diseases such as hypertension , stroke , and atherosclerosis . korean red ginseng ( krg ) is a popular traditional herbal medicine that has been widely used to treat several diseases such as cancer and vascular diseases . recent research shows that ginseng may have therapeutic potential in the treatment of alzheimer 's disease , diabetes , cancer , and cardiovascular diseases , through its antioxidant , antithrombotic , antihyperlipidemic , and anticancer effects [ 1215 ] . in endothelial cells , krg simulates no production in vivo and in vitro , suggesting that krg has antihypertensive effects . krg also promotes angiogenesis through the activation of the signaling pathway , indicating that krg can be implicated in potential angiogenic therapies for improving tissue repair , wound healing , and cardiovascular diseases . in addition , our previous study suggested that krg exerts a cytoprotective effect through the induction of heme oxygenase ( ho)-1 expression , suggesting a possible therapeutic mechanism of krg in cardiovascular diseases . it is well known that chronic inflammation contributes to the pathogenesis of many human diseases such as atherosclerosis . accumulating evidence suggests that krg is involved in the regulation of inflammatory responses , suggesting an anti - inflammatory effect of krg . cyclooxygenase ( cox ) catalyzes the conversion of arachidonic acid to prostaglandins that play vital roles in multiple physiological and pathophysiological processes , including inflammation . in particular , cox-2 is normally undetectable in most tissues and is induced in response to numerous stimuli . vascular diseases may , in part , be caused by cox-2 upregulation at sites of inflammation and vascular injury . cox-2 plays an important role in inflammation , therefore , inhibition of cox-2 expression may participate in the treatment of inflammation - related diseases such as vascular diseases . the objective of our study was to investigate the vascular protective effect of krg in acrolein - stimulated human umbilical vein endothelial cells ( huvecs ) . therefore , we examined the involvement of cox-2 expression via p38 mitogen - activated protein kinase ( mapk ) , intracellular ros , and apoptosis in acrolein - stimulated huvecs . krg powder was obtained from the korea ginseng corporation ( daejeon , korea ) . m199 medium and fetal bovine serum krg powder was soaked in water ( 1:25 , w / w ) for 3 h , and boiled for 40 min . following centrifugation at 1,900 g for 60 min , supernatants of ginseng extract were further centrifuged at 10,000 g for 30 min and lyophilized . the general composition of the product offered by the korea ginseng corporation is as follows : moisture 36% , solid volume 64% , ash 2.5% , total fat 0.05% , total crude saponin 70 mg / g , and total ginsenosides 20 mg / g . huvecs were maintained in m199 medium and supplemented with 10% fetal bovine serum , 1% penicillin and streptomycin , 10 ng / ml human fibroblast growth factor , and 18 mu / ml heparin . huvecs were grown to 80% confluence , maintained with fresh medium described above , and subcultured every 2 or 3 d. the cells were used within nine passages during these experiments . we applied 20 or 40 g of the whole cell lysate proteins to each lane and analyzed them with western blotting . western blotting was performed using primary antibodies as follows : anti - cox-2 , p38 mapk , phopho - p38 , cyclic amp - responsive element - binding protein ( creb ) , phospho - creb ( cell signaling , danvers , ma , usa ) and anti - glyceraldehyde 3-phosphate dehydrogenase ( abfrontier , seoul , korea ) . horseradish - peroxidase - conjugated anti - igg antibodies were used as the secondary antibody to detect the above - mentioned protein bands by enhanced chemiluminescence westsave - up ( abfrontier ) . the rna pellets were washed in 70% ethanol , dried completely , and dissolved in diethylpyrocarbonate to inhibit rnase . total rna was quantified using a nd-100 spectrometer ( nanodrop technologies , wilmington , de , usa ) . polymerase chain reaction ( pcr ) was performed using the synthesized cdna as a template and using specific primers for cox-2 or -actin as a loading control . the primer sequence for human cox-2 was 5-gacagtccaccaacttacaat-3 ( forward ) and 5-catctctccatcaattatctgat-3 ( reverse ) . the amplified products were resolved by 1% agarose gel electrophoresis , stained with ethidium bromide , and photographed under ultraviolet light . huvecs were cultured in a glass culture chamber slide ( falcon plastics , london ontario , canada ) and processed for immunofluorescence analysis . the amount of prostaglandin ( pg)e2 in the culture medium was measured using the pge2 eia kit according to the manufacturer 's protocol ( cayman chemical company , ann arbor , mi , usa ) . samples as well as standards were applied to a 96-well plate , precoated with goat anti - mouse igg , and incubated with pge2 acetylcholinesterase tracer and pge2 antiserum . all the wells were emptied , rinsed five times , and incubated with ellman 's reagent for 60 min in the dark with gentle rocking to produce 5-thio-2-nitrobenzoic acid , which has a strong absorbance at 405 nm ; the plate was read at 405 nm in an enzyme - linked immunosorbent assay reader ( el 800 ; bio - tek , winooski , vt , usa ) . we calculated the results using the standard curve , which were expressed as picograms per milliliter . intracellular ros in acrolein - stimulated huvecs is analyzed using a fluorescent dye , 2,7-dichlorofluorescein diacetate ( dcf / da ) . in the presence of oxidants , after 18 h incubation with 25 m acrolein in the presence or absence of krg , cells were stained with 10 m dcf / da , and fluorescence was analyzed by a facs vantage flow cytometer ( becton dickinson , san jose , ca , usa ) and fluorescence microscopy ( eclipse 50i ; nikon , japan ) . to clarify whether krg - mediated inhibition of acrolein - induced cox-2 expression plays a significant role in cytoprotection against oxidative stress , acrolein - stimulated cells were pretreated with krg ( 1 mg / ml ) or untreated , and cell death was measured by in situ terminal transferase dutp nick end labeling ( tunel ) assay . to measure dna fragmentation , the commercially available in situ death detection kit ( roche diagnostics , mannheim , germany ) huvecs were cultured in a glass culture chamber slide and fixed for 30 min in 10% neutral buffered formalin solution at room temperature . a tunel assay system was used , according to the manufacturer 's instructions , for examination under a fluorescence microscope , with excitation at 488 nm and emission at 525 nm . fluorescein isothiocyanate ( fitc ) ( bd pharmingen , san diego , ca , usa ) and propidium iodide ( pi ) staining of necrotic and apoptotic cells . cells were washed in pbs , resuspended in 100 l binding buffer containing 5 l annexin v fitc and 1 g / ml pi , and incubated for 10 min at room temperature in the dark . positioning of quadrants on the annexin v / pi dot plots was performed as previously described . data were expressed as mean standard deviation . statistical analysis was performed using one - way analysis of variance ( graphpad prism version 4 ; graphpad software , san diego , ca , usa ) followed by bonferroni 's multiple comparison test . a previous study found that acrolein in cs induces cox-2 expression in human endothelial cells . krg inhibited acrolein - induced cox-2 protein expression in a concentration - dependent manner ( fig . after pretreatment of acrolein - stimulated cells with krg , the cells were fixed , and cox-2 localization in huvecs was observed by immunofluorescence staining with an anti - cox-2 antibody followed by a fluorescence - tagged secondary antibody . immunofluorescence analysis showed that acrolein - induced cox-2 protein levels were inhibited in huvecs after treatment with krg ( fig . the induction of cox-2 expression is known to be responsible for pge2 release in the culture medium of cells stimulated with acrolein . acrolein increased pge2 secretion , which was dramatically reduced by krg ( fig . 2 ) . this result indicates that krg leads to the reduction of cox-2 protein expression and subsequently pge2 biosynthesis in acrolein - stimulated huvecs . thus , we examined the effect of krg on ros production in acrolein - stimulated cells . the shift to the right of the curve due to increased fluorescence indicates an increase in the intracellular levels of ros . the results indicate that ros generation in cells treated with acrolein increased compared to untreated cells , whereas krg inhibited acrolein - induced ros generation ( fig . these results indicate that krg may play a role in the inhibition of cox-2 expression via reduction of acrolein - generated ros in acrolein - stimulated huvecs . thus , to determine the upstream signaling pathway involved in krg - mediated cox-2 inhibition , we measured the activation of p38 and creb by detecting increased phospho - p38 and phospho - creb levels in acrolein - stimulated cells and found that phosphorylation of p38 and creb was strongly reduced by krg in acrolein - stimulated cells ( fig . 4 ) . these results demonstrate the role of p38 and creb signaling in the inhibition of acrolein - mediated cox-2 induction . fluorescence - activated cell sorting showed that while the number of apoptotic cells increased following treatment with acrolein , pretreatment with krg reduced the number of apoptotic cells ( fig . to confirm this result , we evaluated the presence of dead cells by tunel staining , which is widely used in detecting dna fragmentations in situ . the tunel assay indicates cell death , including apoptosis , by detection of the appearance of intensely stained nuclei , which indicates incorporation of labeled dutp into the 3-end of fragmented dna derived from apoptotic nuclei . as illustrated in fig . 5b , acrolein treatment significantly increased the proportion of tunel - positive cells , which was restored by krg pretreatment . these results revealed that the vascular protective effect of krg is mediated by the inhibition of cox-2 expression in acrolein - stimulated huvecs . in this study , we explored the inhibition of an inflammatory mediator , cox-2 , by krg water extract in huvecs . we found that krg inhibited both mrna and the protein level of cox-2 and its cytoprotective effect in acrolein - stimulated huvecs . there is increasing evidence that ,-unsaturated aldehydes in cs , including acrolein and crotonaldehyde play an important pathophysiological role in vascular diseases such as atherosclerosis and alzheimer 's disease . exposure to ,-unsaturated aldehydes is critical to the inflammatory response via activation of the proinflammatory signaling pathway and redox - sensitive transcription factors . furthermore , ,-unsaturated aldehydes increase oxidative stress , which plays a crucial role in the pathogenesis of vascular diseases via direct injury to the endothelium . cox-2 , a key enzyme for prostaglandin biosynthesis , is an inducible enzyme that is rapidly induced during inflammatory reactions . numerous studies have reported the involvement of cs in vascular diseases through cox-2 and endothelial no synthase activity . chronic inflammation plays an important role in vascular diseases , therefore , cox-2 may participate in the development of inflammation - related diseases , including vascular diseases . ginseng has been used as a general tonic for > 2000 years in east asia , and it has become a famous herbal medicine for treatment of various diseases , including vascular disorders . krg has been reported to have effective pharmacological activities , including antioxidant , anticarcinogenic , and ameliorative effects on blood circulation . recently , the diverse effects of several constituents of krg , including ginsenoside , on endothelial cells have been extensively studied . hien et al demonstrated the anti - inflammatory and antiatherosclerotic activities of ginsenoside rg3 in human endothelial cells , with a decrease of cell adhesion molecules and proinflammatory cytokines . moreover , the cytoprotective effect of ginsenoside rb1 in endothelial cell damage mediated by oxidized low - density lipoprotein has been reported . several constituents of red ginseng have been reported to regulate proliferation and migration and to protect oxidative stress - mediated damage in human endothelial cells . there is evidence demonstrating the presence of major ginsenosides including rb1 and rg1 in krg water extract . thus , these components could also contribute to the diverse retinue of protective actions of krg . a previous study showed that the induction of ho-1 expression may exert protective effects in krg - treated human endothelial cells . however , there have been no reports revealing the mechanism underlying krg - inhibited cox-2 expression in acrolein , ,-unsaturated aldehydes in cs , stimulated huvecs . we have established that the major signaling pathway of cox-2 ( i.e. , p38 mapk creb ) and intracellular ros generation are involved in this inhibition of cox-2 expression in acrolein - stimulated huvecs by krg . as mentioned above therefore , the inhibition of cox-2 expression following krg water extract treatment may be associated with its strong protective effect in acrolein - stimulated huvecs . in conclusion , we propose that the krg water extract may exert a cytoprotective effect through the inhibition of cox-2 induction and that this reduction of cox-2 in acrolein - stimulated huvecs is mediated by the p38 mapk 4cl4-coumarate : coa ligasecas9crispr - associated protein 9crisprclustered regularly interspaced short palindromic repeatsctcondensed tanningrnaguide rnammillionpamprotospacer adjacent motifpol iiirna polymerase iiirnairna interferencernaseribonucleasesnpsingle nucleotide polymorphisms / g ligninsyringyl - to - guaiacyl lignin ratiotrnatransfer rna 4-coumarate : coa ligase crispr - associated protein 9 clustered regularly interspaced short palindromic repeats protospacer adjacent motif single nucleotide polymorphism syringyl - to - guaiacyl lignin ratio since the first report of its programmable gene editing potential , the crispr / cas9 technology is revolutionizing all facets of biology from medicine to agriculture ( jinek et al . , 2012 ) . with its efficiency and simplicity , crispr / cas9 has quickly displaced its predecessors ( e.g. , zinc finger and transcription activator - like effector nucleases ) as the method of choice for genome editing ( carroll , 2014 ) . for agricultural applications that have traditionally depended on gene - silencing for rna modification , crispr / cas9 is a game changer . the previous methods , such as antisense or rna interference ( rnai ) , leave much to be desired as the degree , specificity and stability of gene silencing are not always predictable . this necessitates screening and characterization of a large number of transgenic lines for the desired trait . by contrast , crispr / cas9 is guided to disrupt the reading frame thereby protein function of the target gene by dna editing . 2012 ) , and both monoallelic and biallelic mutations are reported ( bortesi and fischer , 2015 ) . with biallelic editing , null mutations are readily obtained in primary transformants , though with varying efficiencies ( brooks et al . , 2014 ; zhang et al . , 2014 ; zhou et al . , 2014 , 2015 ) , interestingly , the most efficient and consistent crispr / cas9 editing was reported in hybrid populus tremula x alba clone 717 - 1b4 ( 717 ) , with biallelic mutations detected in all independent transgenic lines examined ( zhou et al . , 2015 ) . for woody perennials such as forest trees , fruit / nut trees and woody ornamentals with highly heterologous genomes and long generation times , crispr / cas9 affords a facile means to accelerate genetic improvement . the first application of crispr / cas9 genome editing in populus targeted the 4-coumarate : coa ligase ( 4cl ) gene family involved in phenylpropanoid metabolism ( zhou et al . , 2015 ) . one of the genes , 4cl1 ( potri.001g036900 ) , has been extensively characterized for its involvement in lignin biosynthesis . down - regulation of 4cl1 or its orthologs leads to reduced lignin content and altered lignin structure in a number of species ( boerjan et al . , thus , crispr / cas9 editing of 4cl1 served as a proof - of - concept study , allowing an assessment of its efficacy in perturbing lignin biosynthesis relative to previous methods . we generated 36 independent transgenic lines , and amplicon - sequencing of randomly selected events confirmed biallelic mutations in all cases ( zhou et al . , 2015 ) . as frequently reported for crispr / cas9 genome editing , small indels , especially 1-bp modifications , were the predominant patterns and predicted to disrupt the reading frame . consistent with this prediction , lignin content was reduced by 23% with a concomitant decrease of s / g lignin ratio by 30% in all transgenic plants ( zhou et al . , 2015 ) . wood discoloration is a known side effect of lignin modification frequently observed in transgenic plants with suppressed lignin gene expression . however , the coloration patterns reported in those cases lack the uniformity we observed for the crispr / cas9 mutants . for example , antisense downregulation of 4cl1 in populus resulted in patchy wood discoloration in only 5 of 14 transgenic lines ( voelker et al . , wood discoloration patterns of transgenic populus with down - regulated expression of cinnamoyl - coa reductase were highly variable among vegetatively propagated plants ( van acker et al . , 2014 ) , presumably due to the unstable nature of sense- or antisense - mediated post - transcriptional gene silencing . rnai - mediated gene suppression is considered a more effective alternative , with improved trait stability ( li et al . , 2008 ) . for instance , rnai - silencing of 4-coumaroyl - coa 3-hydroxylase in transgenic populus resulted in lignin reductions among 9 independent events that ranged from no change to 1560% ( coleman et al . , similarly , rnai silencing of 4cl in pinus led to wood discoloration in only 2 of 12 viable transgenic lines ( wagner et al . , 2009 ) . by contrast , the homogeneity of wood discoloration both within and across independent crispr / cas9-edited populus mutant lines is consistent with a null 4cl1 , and resembles that of naturally occurring brown midrib mutants of maize and sorghum ( sattler et al . , 2010 ) . the results serve as testament to the superiority of crispr / cas9 over previous gene silencing methods . ( 2015 ) with permission . a second gene , 4cl2 ( potri.019g049500 ) that is phylogenetically distinct from lignin - associated 4cls ( chen et al . , 2014 ) . has long been suggested to participate in flavonoid biosynthesis , based solely on its preferential expression in epidermis and roots ( harding et al . , 2002 ) . crispr / cas9 mutation of 4cl2 was therefore expected to yield functional evidence to substantiate this role . as with the 4cl1 case , biallelic mutations were observed in all independent transgenic lines surveyed by amplicon - sequencing ( zhou et al . , the metabolic consequence was examined in roots where flavonoid - derived condensed tannins ( cts ) are known to accumulate to high levels ( 20% dry weight ) in the experimental poplar genotype ( chen et al . , 2014 ) . crispr / cas9 mutations of 4cl2 resulted in significantly reduced levels of condensed tannins in roots , providing reverse genetics evidence in support of class ii 4cl function in flavonoid biosynthesis . after our initial publication , successful crispr / cas9 editing was reported for populus tomentosa ( fan et al . , 2015 ) . that study targeted a phytoene desaturase ( pds ) gene for mutagenesis by multiplexing 4 grnas , and observed a 50% frequency of albino phenotypes . the lower mutation rates compared to our study may be attributed to gene redundancy and sequence heterozygosity that were not accounted for during grna design ( see discussion below ) . regardless , selected albino transgenic lines were sequence - confirmed to harbor biallelic mutations ( fan et al . , 2015 ) , providing additional support for the superior phenotypic uniformity of crispr / cas9 mutants . a major concern in genome editing is the specificity of the dna modification , as off - target cleavage due to non - specific crispr / cas9 activity may cause unintended mutations . this is especially true for plant genomes that are characterized by recurring episodes of whole - genome , segmental and tandem duplications . we assessed crispr / cas9 specificity in populus by analyzing its on - target versus off - target activities among duplicated genes . the grna for 4cl1 was designed to discriminate against the paralogous 4cl5 ( potri.003g188500 ) , with 3 mismatches in the target sequence and one mismatch in the protospacer adjacent motif ( pam ) ( zhou et al . , 2015 ) . by designing consensus primers flanking the target site , we were able to sequence both 4cl1 and 4cl5 amplicons simultaneously to assess the specificity of crispr / cas9 . a custom program , ageseq ( analysis of genome editing by sequencing ) , was developed to facilitate variant detection ( xue and tsai , 2015 ) . no off - target cleavage in the 4cl5 locus was detected in 4cl1-edited lines , suggesting that crispr / cas9 editing in populus is highly specific ( zhou et al . , 2015 ) . negative experiment , where mismatches between a 4cl5-grna , designed based on the p. trichocarpa reference genome ( phytozome v3 ) , and the corresponding sequence of p. tremula x alba 717 used for transformation abolished cleavage in all transgenic lines examined ( zhou et al . , 2015 ) . in this case , the mismatches were in the form of 2 single nucleotide polymorphisms ( snps ) , one per allele , located near or in the pam . these results , together with the highly heterologous nature of woody perennials , suggest that off - target cleavage of crispr / cas9 is likely to be low in these species . the ability of crispr / cas9 to discriminate single - base differences makes it a powerful tool to investigate functional redundancy of highly homologous genes that are otherwise difficult to discern using gene silencing approaches . for instance , 4cl1 is the most abundantly expressed isoform in populus xylem , constituting > 85% of 4cl transcripts , with 4cl5 being a distant second , based on rna - seq data ( swamy et al . , 2015 ) . however , the crispr / cas9 knockout of 4cl1 reduced lignin content only by 23% ( zhou et al . , 2015 ) . because the 4cl5 locus was unmodified , it is likely involved in the residual lignin biosynthesis of the 4cl1-knockout mutants . in the populus study that targeted pds , 4 grnas were designed based on one gene model ( potri.014g148700 ) without considering the genome duplicate ( potri.002g235200 ) that shares a high degree ( 93% ) of coding sequence identity . while the genome sequence of p. tomentosa is not yet available , analysis of the p. trichocarpa and p. tremula x alba orthologs revealed different degrees of sequence variation between the 2 duplicates in the grna target regions . these differences ( plus unknown allelic heterozygosity ) likely contributed to the variation in the reported editing efficiencies of different grnas ( fan et al . , 2015 ) . examples include designing multigene - targeting grnas based on conserved sequences of homologous genes ( brooks et al . , 2014 ; jacobs et al . , 2015 ) , or multiplexing individual grna cassettes ( brooks et al . , 2014 ; fan et al . , 2015 ; zhang et al . , 2014 a recent study hijacked the trna processing machinery for efficient processing of up to 8 , and potentially more , grnas from one single construct ( xie et al . , the grna is positioned downstream of trna as a polycistronic gene with repeating trna - grna units for multiplexing ( xie et al . , 2015 ) . because mature grnas are released following cleavage of the polycistronic transcript by the trna processing machinery , there is no sequence constraint at the 5 end of grnas ( xie et al . , 2015 ) . this greatly simplifies grna design , as any sequences upstream of pam ( ngg ) can be used ( e.g. , n20-ngg or other truncated lengths ( fu et al . , 2014 ) ) , unlike with the standard design where grna is transcribed directly from the pol iii u6 or u3 promoter using a specific transcription initiation sequence ( e.g. , gn19-ngg or an19-ngg , respectively ) ( wang et al . , 2008 ) . in some cases , grna stacking , either by re - transformation or via controlled crosses of primary transformants may be necessary in order to discern individual gene functions . as discussed above , numerous studies have exploited sequence polymorphisms for developing gene- or allele - specific grnas to achieve precision editing by crispr / cas9 . however , few studies have addressed the added complexity of sequence polymorphisms on crispr / cas9 genome editing , probably because the common plant models , such as arabidopsis , rice , soybean and tomato , are selfing species with homozygous genomes . we argue that sequence polymorphisms are of concern in genome editing of most flowering plants , especially woody perennials that are outcrossing in nature . this is supported by the high frequency of genic snps reported for trees , one per 60 bp in populus tremula ( ingvarsson , 2005 ) or one per 16 - 33 bp in eucalyptus depending on the species ( klheim et al . , 2009 ) . a recent population genomics study of 544 populus trichocarpa individuals identified 17.9 million ( m ) snps by whole - genome resequencing ( evans et al . , 2014 ) . using a similar approach with both resequencing and rna - seq data , we uncovered over 10 m snps from a single individual , p. tremula x alba clone 717 that is commonly used in populus transformation ( xue et al . , 2015 ) . given the high levels of heterozygosity in tree species , the need to consider sequence polymorphisms in genome editing experiments can not be over - emphasized . in fact , most published genome sequences are , by default , consensus sequences of their ( pseudo)haploid genomes . thus , while several web - based grna design programs are available for plants , such as crispr - plant ( xie et al . , 2014 ) . and crispr - p ( lei et al . , 2014 ) , their utility for outcrossing species is limited because the preloaded genome sequences lack biallelic ( or multiploid ) coverage . furthermore , it is often the case as in populus that the species or genotype chosen for whole genome sequencing ( e.g. , , p. trichocarpa nisqually-1 ) is different from that routinely used for genetic transformation ( e.g. , p. tremula x alba 717 ) . to facilitate crispr / cas9 genome editing in 717 , we constructed a custom 717 genome by substituting > 10 m sequence variants into the p. trichocarpa reference genome ( xue et al . , 2015 ) . we then assessed the impact of sequence polymorphisms on grna specificity for 1000 randomly selected genes . grnas were designed based on the haploid genome of ( 1 ) p. trichocarpa or ( 2 ) 717 , and then cross - checked for snps or indels in the 717 target sequences . from the first analysis , we found that more than 57% of the grnas designed based on the p. trichocarpa genome correspond to regions that harbor snps or indels in 717 ( xue et al . the rates were slightly lower but nevertheless high ( 42 - 44% ) in the second analysis when the custom 717 genome was used , owing to allelic variations ( zhou et al . , 2015 ) . together , these data suggest that standard grna designs may suffer from a high probability of failure in crispr / cas9 genome editing , due to the frequent occurrence of snps / indels in outcrossers . the 717 sequences and associated blast and gene model query tools are available via aspendb ( http://aspendb.uga.edu/s717 ) . tool is particularly useful for screening custom grnas or pcr primers against known snps / indels in the p. trichocarpa or the custom 717 genome ( xue et al . , 2015 ) . a set of pre - selected grnas with no known sequence variants for 38,509 populus genes is provided ( zhou et al . , 2015 ) . the modified , variant - sensitive pipeline for grna design , called aspen crispr designer , is also available at aspendb and can be applied to other species . for outcrossing species lacking a genomic variant database , several approaches can be applied to identify snps / indels in the gene(s ) of interest prior to grna selection . rna - seq data from the transformation genotype , when available , is an excellent source for high - confidence snp discovery . provided that the gene of interest is reasonably expressed ( e.g. , , with a high read coverage ) , manual inspection of candidate grnas against rna - seq alignments using programs such as the popular integrative genomics viewer ( http://www.broadinstitute.org/igv ) is usually effective to confirm their specificity . in the absence of deep - sequencing data , pcr primers ( with or without degeneracy ) can be designed to amplify the target gene coding region based on a closely - related reference genome , or on conserved amino acid sequences of orthologs as in conventional rt - pcr cloning ( hu et al . , 1998 ) . as pam ( ngg)the only sequence constraint in grna design occurs frequently , it is not necessary to obtain full - length gene sequences . however , the use of multiple primer pairs is recommended to increase the likelihood of capturing both alleles . direct sequencing of pcr products to identify sequence variants is an established practice in molecular ecology research , and several programs have been developed to decode overlapping sanger chromatograms ( dmitriev and rakitov , 2008 ) . for researchers with convenient access to high - throughput sequencing , sequence reads can then be processed by ageseq to search for potential variants in the target gene sequences ( xue and tsai , 2015 ) . ageseq is available as a standalone program or a galaxy - compatible tool ( https://toolshed.g2.bx.psu.edu ) to support web - based data analysis . it should be noted that pcr amplification of noncoding sequences can be tricky due to the lower degree of sequence conservation ( xue et al . , 2015 ) . thus , for crispr / cas9 applications that target promoter or other noncoding sequences ( e.g. , transcriptional activation or chromatin modification ( sander and joung , 2014 ) ) , resequencing of the transformation clone for unbiased variant calling , as discussed above for populus 717 ( xue et al . , 2015 ) , is recommended . the populus study extended the ever growing list of plant species successfully genome - edited by crispr / cas9 ( bortesi and fischer , 2015 ; kumar and jain , 2015 ) to woody perennials . woody perennials suffer as experimental systems due to the lengthiness of genetic transformation and plant characterization . many forest and horticultural tree species and genotypes are notoriously recalcitrant to tissue culture regeneration with low transformation efficiencies ( litz and padilla , 2012 ; busov et al . , 2005 ) . limitations associated with other established technologies , such as incomplete silencing , position effects or epigenetic modifications of transgenes ( matzke and matzke , 1998 ) , further impede progress in transgenic tree research . even for species with a facile transformation system , such as populus tremula x alba 717 , the common practice of generating multiple independent events for preliminary screening prior to in - depth characterization of selected lines is resource - demanding and time - consuming ( fig . 2 ) . the ability of crispr / cas9 to generate null mutations in primary transformants , and the promise of phenotypic consistency among independent biallelic mutants as demonstrated in the populus study make a compelling argument that it may soon become acceptable to characterize only a few transgenic lines with sequence - verified mutations . high - throughput methods for mapping genome editing patterns ( xue and tsai , 2015 ) and transgene ( e.g. , t - dna harboring cas9 , grna and selectable marker ) insertion sites ( kanizay et al . , 2015 ) are expected to facilitate this effort . as depicted in figure 2 , primary transformants with confirmed editing can be used directly for in - depth characterization when multiple independent events are available as biological replicates ( zhou et al . , 2015 ) . even when only a few mutation events are obtained ( for difficult - to - transform species or from suboptimal transformation trials ) , the ability to bypass rna - level screening and to obtain stable mutations still confers advantages over the gene - silencing approaches . crispr / cas9 thus offers the enticing prospect of streamlined transgenic characterization and expedited large - scale functional characterization . figure 2.schematic comparisons of experimental approaches involving gene silencing vs. crispr / cas9 genome editing . ( a ) for gene silencing by antisense , sense or rnai approaches , knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation . expression knowledge is not necessary for crispr / cas9 editing at the dna level , and vectors with a pol iii promoter ( e.g. , u6 or u3 ) for grna expression and a cas9 under control of a constitutive ( e.g. , 35s ) promoter are widely applicable . ( b ) following regeneration of putative transgenic plants , conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired ( or maximum ) levels of gene suppression . it is common to screen a large number of events ( 10 ) to identify a minimum of 3 for subsequent analysis . this process can be greatly simplified with crispr / cas9 mutants , as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis . ( c ) for species with a robust transformation system , multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . in case of difficult - to - transform species for which few biallelic events may be obtained , micropropagation is necessary to obtain biological replicates before in - depth characterization , as is typically done using gene silencing . ( a ) for gene silencing by antisense , sense or rnai approaches , knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation . expression knowledge is not necessary for crispr / cas9 editing at the dna level , and vectors with a pol iii promoter ( e.g. , u6 or u3 ) for grna expression and a cas9 under control of a constitutive ( e.g. , 35s ) promoter are widely applicable . ( b ) following regeneration of putative transgenic plants , conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired ( or maximum ) levels of gene suppression . it is common to screen a large number of events ( 10 ) to identify a minimum of 3 for subsequent analysis . this process can be greatly simplified with crispr / cas9 mutants , as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis . ( c ) for species with a robust transformation system , multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . in case of difficult - to - transform species for which few biallelic events may be obtained , micropropagation is necessary to obtain biological replicates before in - depth characterization , as is typically done using gene silencing . biallelic mutations of the target gene(s ) in conjunction with the hemizygous nature of transgene ( e.g. , t - dna ) integration in primary transformants ( t0 ) means that dna - edited but transgene - free progenies can be readily obtained from t1 segregants , as demonstrated in annual species ( zhang et al . trait stacking with early flowering induction is thus highly desirable , for example by over - expression of flowering locus t ( ft ) as demonstrated in prunus ( plum ) , populus and eucalyptus ( hoenicka et al . , 2014 ; another concern for outcrossing species is that the progenies will be different from the parents , which necessitates additional screening and selection . we note , however , that introgressing transgenic / mutant events into elite genotypes is an established practice in crop and tree breeding programs , and hence not unique to crispr / cas9-edited mutants . a well - known tree example is the loblolly pine ( pinus taeda ) clone 756 that harbors a naturally occurring null allele of cinnamoyl alcohol dehydrogenase involved in lignin biosynthesis , and that is extensively used as a parent in several breeding programs in the us ( gill et al . , 2003 ) . another example is early - flowering ft - plum that serves as a parent in the fasttrack breeding program to expedite development of improved commercial cultivars ( callahan et al . , various molecular and genomics tools are available to facilitate progeny selection in transgenic breeding programs . recently , virus - based systems have been explored for transient expression of genome - engineering reagents ( ali et al . however , such systems still utilize t - dna to express the repurposed viral genome components , and the resulting plants are not transgene - free without crossing . these methods also depend on agroinfiltration to deliver the vector into plant cells , which unfortunately is not applicable to most woody species with an open minor vein structure in their leaves ( gamalei , 1989 ) . clearly , development of innovative methods will be necessary for facile removal of t - dna from the genome - edited plants without crossing . regardless , null segregants of crop cultivars derived from genome editing are indistinguishable from naturally occurring or chemically - induced mutants , and may therefore be exempt from gmo regulation in countries that adhere to product - based regulations ( voytas and gao , 2014 ) . this is expected to significantly reduce the timeline and financial burden associated with developing new crop varieties , thereby encouraging adoption of crispr / cas9-based genome editing for the forest products , fruit / tree nuts , and woody ornamental industries . the research in the tsai laboratory discussed in this article was supported by the department of energy , office of biological and environmental research ( grant no . de - sc0008470 ) , the department of agriculture , national institute of food and agriculture ( grant no . 2015 - 67013 - 22812 ) , and by the georgia research alliance - hank haynes forest biotechnology endowment .

cigarette smoke is considered a major risk factor for vascular diseases . there are many toxic compounds in cigarette smoke , including acrolein and other ,-unsaturated aldehydes , which are regarded as mediators of inflammation and vascular dysfunction . furthermore , recent studies have revealed that acrolein , an ,-unsaturated aldehyde in cigarette smoke , induces inflammatory mediator expression , which is known to be related to vascular diseases . in this study , we investigated whether korean red ginseng ( krg ) water extract suppressed acrolein - induced cyclooxygenase ( cox)-2 expression in human umbilical vein endothelial cells ( huvecs ) . acrolein - induced cox-2 expression was accompanied by increased levels of phosphorylated p38 in huvecs and krg inhibited cox-2 expression in huvecs . these results suggest that krg suppresses acrolein - induced cox-2 expression via inhibition of the p38 mitogen - activated protein kinase signaling pathway . in addition , krg exhibited an inhibitory effect on acrolein - induced apoptosis , as demonstrated by annexin v propidium iodide staining and terminal deoxynucleotidyl transferase - mediated dutp nick end - labeling assay . consistent with these results , krg may exert a vasculoprotective effect through inhibition of cox-2 expression in acrolein - stimulated human endothelial cells . abstractthe crispr / cas9 technology is a welcome breakthrough for genome editing , owing to its precision , efficiency , versatility and ease of adoption . we recently reported the first application of crispr / cas9 for biallelic mutations in stably transformed populus , extending the species range of this powerful technology to woody perennials . an underappreciated obstacle in genome editing of outcrossing species is the frequent occurrence of sequence polymorphisms that can render crispr / cas9 unproductive . we discuss experimental evidence as well as genome - wide computational analysis to demonstrate the sensitivity of crispr / cas9 to allelic heterozygosity , and highlight tools and strategies that can help deal with such sequence polymorphisms . with its specificity , crispr / cas9 offers a less equivocal means than previous approaches for discerning functional redundancy of paralogous genes that are prevalent in plant genomes . continuing improvements of the crispr / cas9 system for multiplex genome engineering should facilitate these efforts . the paradigm shift brought about by crispr / cas9 promises to accelerate not only basic research but also applied crop improvement progress .

like co@fe3o4 , which recently underwent extensive study to elucidate both the oxidative stability of the core and the phase of the shell , fept@fe3o4 core shell nanoparticles can be used as building blocks to form nanocomposites with enhanced magnetic properties with the potential for novel applications . these include magnetic data storage , catalysis , and targeted drug delivery . in terms of nanomagnetics specifically , our hope is that the presence of an inert oxide shell may function to inhibit agglomeration of the cores upon annealing ; this is a necessary step in creating an ordered l10 fept bimetallic structure with a higher magnetic coercivity . the determination of the core composition is important for tailoring their synthesis in order to ultimately achieve the desired 50:50 fept alloy composition . a solution of core shell particles was drop - cast onto a 3 mm holey carbon copper grid . edx data were acquired using an fei osiris tem equipped with a high brightness schottky x - feg gun and a super - x edx system comprising four silicon drift detectors , each approximately 30 mm in area and arranged symmetrically around an optic axis to achieve a collection solid angle of 0.9 sr . edx data were collected in the form of spectrum images , in which a focused electron probe was scanned in a raster across a region of interest in the scanning tem ( stem ) . at each point in the scan , structural information was obtained from the electron scattering incident on a high angle annular dark field ( haadf ) detector , and simultaneously , an edx spectrum was obtained by collecting x - rays emitted from the local volume probed by the electron beam . the resulting edx spectrum image was a three - dimensional data set whose ( x , y ) axes correspond to the position of the probe and whose z axis corresponds to the energy of the detected x - ray . spectrum images were acquired with a probe current of approximately 0.7 na , an acceleration voltage of 200 kv , a spatial sampling of between 0.5 and 1 nm / pixel and 50100 ms / pixel dwell times . x - ray intensities were obtained by fitting a model of the edx spectra to the experimental data using weighted least - squares and atomic fractions were quantified from intensities using the cliff lorimer quantification . the edx model and the quantification were implemented in hyperspy and will be available in future releases of the software . figure 1a displays a haadf stem image obtained during the acquisition of a spectrum image enclosing a cluster of 13 co@fe3o4 nanoparticles . although the particle morphologies are seen to vary slightly from one particle to another , the majority of particles have a round core approximately 20 nm in diameter surrounded by a thin shell approximately 5 nm in thickness . the edx elemental maps for cobalt , iron , and oxygen ( figure 1b d ) , obtained by integration of the element s background - subtracted k - line x - ray peak , show that individual particles are comprised of a cobalt core surrounded by a shell composed of iron and oxygen , as expected . the largest particle in the lower left region of the map appears to have more iron in the core compared to the smaller particles . these conventional edx element maps show the location of the various elements , but the composition of the particle core , for example , can not be determined by elemental mapping due to the presence of the shell above and below the core in projection . elemental maps of ( b ) cobalt , ( c ) iron , and ( d ) oxygen display the location of the various elements with respect to the particle morphology ( scale bar = 50 nm , greyscale = x - ray counts ) . the edx spectrum image data of the co@fe3o4 nanoparticle cluster shown in figure 1a was subsequently processed using bss methods in hyperspy . first , the spectral dimension in the data set was binned by four from 5 ev / channel to 20 ev / channel in order to increase the number of counts per channel . next , we note that the binning step is necessary in order to optimize the accuracy of the variance stabilization channel . the first three principal components , pc#0 , pc#1 , and pc#2 , exhibited significantly greater variance than the remaining components ( figure 2a ) , which suggests that there are only three phases present in the sample . that being the case , those three pca components should be a linear combination of the spectra and distribution maps of those phases , but the mixing matrix is unknown . next , we compute numerically the first derivative of the pca spectral components in order to diminish the correlation caused by the edx background , and we use fastica to estimate the mixing matrix and compute the independent components ( ics ) ic#0 , ic#1 , and ic#2 ( figure 2b ) and their distribution maps ( figure 2c e ) from the pca results . component independence is a much more stringent property than uncorrelatedness imposed by pca . if we disregard the small copper peaks contained in all the independent components , likely originating from the copper support mesh , we see that ic#0 contains cobalt x - ray peaks , ic#1 iron and oxygen peaks , and ic#2 a carbon peak . the three ics appear to belong to the three phases present in the originally scanned area : the core , shell , and supporting film . this hypothesis is explored further in the next section . at this point , however , it is important to note that , unlike in the conventional edx mapping shown in figure 1 , no elements were selected prior to performing ica , and thus , the analysis is free of external bias , except for the choice of the number of components . result of bss by pca and ica of an edx si of a co@fe3o4 core ( b ) corresponding independent component spectra contain the expected x - ray lines for the elements present . independent component maps ( c e ) show that ( c ) ic#0 is concentrated in the nanoparticle cores , ( d ) ic#1 in the shells , and ( e ) ic#2 everywhere on the carbon supporting film ( scale = 50 nm , greyscale = normalized weighting ) . we now move on to the analysis of a second cluster of particles comprised of a bimetallic iron / platinum core surrounded by an iron oxide shell . a crystalline core surrounded by a polycrystalline oxide shell is observed in the representative high - resolution stem haadf image shown in figure 3 . the particle morphologies were found to have a mean core diameter of approximately 3.3 nm and mean shell thickness of approximately 1.7 nm ( figure 4a ) . also visible were pure iron oxide particles ( in the lower right - hand corner of figure 4a ) . ica of a cluster of bimetallic platinum / iron nanoparticle seeds coated by fe3o4 shells . ( b ) scree plot of the first 50 principal components showing the first three components lying above the noise . ( c e ) element maps of ( c ) platinum , ( d ) iron , and ( e ) oxygen . ( f h ) . the ic maps ( f ) ic#0 , ( g ) ic#1 , and ( h ) ic#2 and ( i ) the corresponding ic spectra . from the selected element maps ( figure 4c e ) , it is clear that , with the exception of the two particles in the lower right - hand corner , the particles are comprised of a platinum rich core surrounded by an iron oxide shell . however , it is not clear from the maps alone whether iron is present in the core . by conventional elemental mapping , one can not tell whether the particles contain a pure platinum core surrounded by an iron oxide shell or whether iron is alloyed with platinum to form a bimetallic core . we now address these questions by performing bss on the same edx spectrum image using the same procedure detailed before . once again , by inspecting the scree plot ( figure 4b ) , we find that the sample consists of three phases . the spatial distribution of ic#0 is concentrated in the particle cores , ic#1 in a shell around the cores , and ic#2 is approximately uniformly distributed over the map ( figure 4f h ) . if we again disregard the spurious copper peak , ic#0 contains iron and platinum x - ray peaks , ic#1 iron and oxygen peaks , and ic#2 a single carbon peak ( figure 4i ) . from this analysis it appears that the ica components represent the different phases present in the edx spectrum image ; ic#0 , ic#1 , and ic#2 genuinely represent the bimetallic fept cores , iron oxide shells and the carbon support , respectively . in order to verify the accuracy of the ica results , we evaluated whether ic#0 represented the true composition of the core by analyzing bare fept seed particle clusters obtained from the same chemical synthesis but extracted prior to the shell addition step . being made from the same synthesis , the composition of the bimetallic seed particles are expected to match to the composition of the bimetallic cores in the core shell particles analyzed in figure 4 . a total of 12 edx sis were acquired in order to capture and analyze multiple fept bimetallic seed clusters . the segmentation of one of the edx sis is shown in figure 5a , b . to accurately extract the intensity of the fe k and pt l peaks , a model composed of one gaussian per x - ray line and a background based on kramers and small expressions as developed elsewhere was used , as shown in green in figure 5c . the only free parameters of the model were the area of the gaussian and the height of the background , which was negligible . the mean reduced over the fit of all particles was 1.01 , indicating that the discrepancies between the model and the data are in accordance with the poisson noise variance . the intensities of fe k and pt l peaks in the fitted model were quantified using the cliff lorimer method . the obtained compositions are plotted for each particle in figure 5d along with the fitting error estimated from poisson statistics . the raw data were decomposed using pca on all fept seed data and the first three components were retained for noise reduction . the 103 individual fept bimetallic seeds analyzed were found to have a mean composition of 82.0 at . % pt , which lies well within one standard deviation of the average bimetallic seed composition . the data points in figure 5d are displayed in order of ascending particle size . as such , the calculated compositions on the right - hand side tend to have a smaller error bar on account of the signal noise ratio being higher for larger particles . the compositions of the larger seed particles are also closer to the mean composition and to the composition of ic#0 , confirming the homogeneity of the composition and the validity of the bss analysis . summary of the composition of 103 bare fept bimetallic nanoparticles extracted from a synthesis prior to the shell addition step . ( b ) segmentation of the edx spectrum image prior to quantification of each particle . ( c ) fitting of the edx spectrum from a single seed ( circled in ( b ) ) to a model spectrum to determine the fe k and pt l peak intensities . ( d ) particle seed compositions obtained by quantifying the fitted intensities from 103 different particles ( error bars = 1 standard deviation ) . a comparison of raw edx spectra extracted from fept bimetallic seed particles and from pure fe3o4 particles with ic#0 and ic#1 , respectively , is provided in figure 6 . in both cases , the ics were scaled by a constant to obtain a best fit to the raw spectra . despite the strong overall agreement in each case , the carbon peak difference in both cases is caused by the separation of carbon into a different component ( ic#2 ) . the difference in the shell cu peaks are not due to a compositional difference as the cu signal is spurious in origin . the iron oxide particle spectrum also contains small silicon and sulfur peaks which likely originate from residue on the carbon film . the difference in the pt m core peak may be due to the attenuation of pt m x - rays in the shell of the core shell nanoparticles and in nearby particles along the trajectory to the detector . the strong overall similarity between the raw and ic spectra provide direct evidence showing that the spectral components extracted by ica from the core shell spectrum image data are strongly representative of the buried core , surrounding shell , and carbon support compositions . comparison between raw edx spectra extracted from a fept bimetallic seed particle ( top ) and from an iron oxide particle ( bottom ) with ic#0 and ic#1 , respectively . the fept seed and fe3o4 x when analyzing beam - sensitive materials , the main limitation to the accuracy of the bss analysis method that we propose is the intensity of the edx signal achievable without inducing significant sample damage . in our case , despite the use of a high efficiency edx system , the number of collected x - rays is low . in order to avoid the artifacts that arise when using the variance - stabilizing transformation out of its domain of application , we have binned the data by four in the spectral dimension from 5 ev / channel to 20 ev / channel . given that the resolution of our edx detector is approximately 130 ev at mn k and that there were no overlapping x - ray lines , in our case , the increase in the number of counts per channel ( and hence in the accuracy of the analysis ) comes without significant resolution loss and , therefore , should not have any adverse effect in the analysis . a blind source separation method based on pca and ica has been applied to the analysis of edx spectrum images of core the analysis has accurately determined the number of phases in the analyzed volume ( core , shell , and supporting film ) as well as their spectra and distribution maps . we have confirmed the accuracy of the analysis by comparing the calculated spectra from the platinum iron core and the iron oxide shell to those obtained from these structures in isolation , and the excellent agreement suggests that bss , therefore , can be used to accurately analyze edx data . the use of ica on edx spectrum image data promises to be a powerful technique for extracting buried compositions at the nanoscale in a variety of materials , and further testing on the method s applicability to different systems is now being initiated . ionic calcium ( ca ) controls multiple cellular signaling processes in all eukaryotic cells , including proliferation , gene expression , and neurotransmitter release . ca - binding proteins such as calmodulin play a pivotal role in ca signal transmission and amplification . increases in the concentration of intracellular ca activate specific protein targets , among which the ca / calmodulin - dependent protein kinase ii ( camkii ) is a critical signal mediator . in response to an increase in intracellular ca , thr-286 of camkii the coupling of ca - calmodulin to one of the camkii subunits allows for the phosphorylation of an adjacent subunit at thr . calmodulin trapping , confers ca - calmodulin - independent kinase activity to the complex and thus prolongs the ca signal . thus , calmodulin trapping represents a molecular mechanism of memory , which is defined as the capacity to acquire , store ( consolidate ) , and retrieve ( evocate ) information . camkii is a major synaptic protein that is activated during the induction of long - term potentiation ( ltp ) by ca influx through n - methyl - d - aspartate ( nmda ) receptors . calmodulin trapping allows camkii to remain activated long after the initial ca signal has dissipated , suggesting that camkii is a memory molecule crucial for ltp . consistent with this notion , camkii - null mice present with impaired memory formation , and camkii is essential for genesis and maintenance of ltp in postsynaptic neurons . following presynaptic stimulation , camkii is activated in postsynaptic neurons , which creates a physiological imprint of the initial ca signal , and increases translocation of nmda receptors to the plasma membrane . because of its capacity to remain activated long after the initial pulse of ca signaling , camkii perpetuates ca effects and modulates gene expression and the epigenetic profile of postsynaptic neurons . camkii also participates in glucose - stimulated insulin secretion ( gsis ) , as multiple insulin secretagogues increase camkii activity . in the perfused rat pancreas , the dynamics of camkii activation correlate with the amplitude of gsis , and camkii activation is temporally associated with insulin secretion . camkii is essential for appropriate gsis and is involved in several steps of this process , including the synthesis of insulin granules , the modulation of cytoplasmic content of atp , and the activation of synapsin . importantly , camkii also regulates transcription factors central for - cell function such as creb and mafa . here , we investigated whether pancreatic - cells , like neurons , acquire and store the information contained in calcium pulses as a form of metabolic memory . indeed , we find that - cells retain memory of prior activation , and describe the molecular mechanism that contributes to this memory . min6 insulinoma cells or mouse or human pancreatic islets were distributed into three groups : control , pulse , and pulse + kn93 ( 10 m ) . initially , all groups were maintained in the same medium with 5.6 mm glucose for 24 h. after this acclimation period , the control group was exposed to 3-mm glucose for 24 h , while the pulse and pulse + kn93 ( 10 m ) groups were exposed to 30 mm of glucose for four 1-h periods intercalated with 7-hour periods of 3 mm glucose . kn93 ( a camkii inhibitor ) at 10 m was only present during the 30 mm glucose pulses . all groups were then maintained at 3 mm glucose for a 24-h consolidation period . insulin secretion of min6 cells and mouse pancreatic islets was conducted following standard procedures , and insulin was measured by ria . one hundred and fifty islets were handpicked under a light microscope and pre - incubated for 1 h in krbb ( krebs bicarbonate buffer ) containing 0.3 the medium was discarded and the islets incubated for an additional hour in 500-l krbb containing 2.8 or 16.8 mm glucose . subsequently , the supernatant was collected to evaluate insulin secretion by ria . for perifusion assays , 150 treated human islets were placed into a perifusion chamber ( millipore , billerica , ma , usa ) . a computer - controlled fast - performance hplc system allowed programmable rates of flow and concentrations of the appropriate solutions held in a 37 c water bath . islets were perfused for 80 min with krbb in the absence of glucose , followed by krbb with increasing concentrations of glucose ( glucose was ramped from 0 to 30 mm at 0.75 mm / min ) . at the end of each experiment , islets were tested for maximal insulin secretion by adding 30 mm kcl to the perifusate . samples were collected at 2 ml / min and insulin content was determined by using ria . statistical analyses were performed using student 's t - test or a two - way anova with a bonferroni 's posttest , as required . statistical significance was set at p < 0.05 . in order to investigate metabolic memory of insulin - producing - cells , we first had to develop a paradigm in which such memory was evident . as detailed below , we established that exposure of cultured min6 - cells to four 1-h pulses of high glucose , interspersed with 7-h intervals of low glucose and thus mimicking postprandial glucose spikes in vivo , were able to elicit robust metabolic memory . the detailed experimental paradigm is outlined in figure 1a . as shown in figure 1b , min6 - cells exposed to the glucose pulse regimen showed higher insulin secretion both when exposed to low ( 2.8 mm ) or high ( 16.8 mm ) glucose than - cells , which had been cultured continuously at low glucose . this increase in insulin secretion was accompanied by a significant rise in the levels of phosphorylated camkii , consistent with the notion that camkii might be a molecular mediator of this metabolic memory ( figure 1c and d ) . importantly , both the increases in basal and stimulated insulin secretion in the glucose pulse group were abolished by kn93 ( figure 1b ) , a specific camkii inhibitor . next , we evaluated the kinetics of the - cell response to acute glucose exposure , and observed that the pulse group secreted more insulin at basal ( time zero ) and stimulatory concentrations of glucose ( 16.7 mm ) between 5 and 60 min following stimulation ( figure 1e ) . this effect persisted even after glucose concentrations were returned to 2.8 mm . in parallel , the phosphorylation of camkii was higher in the glucose pulse group at both glucose concentrations , and remained elevated even after the glucose concentration was reduced to 2.8 mm ( figure 1f ) . thus , min6 - cells had acquired metabolic memory of prior exposure to glucose pulses , which they retained through a 24-h consolidation period . it was important to determine if metabolic memory is confined to min6 insulinoma cells , or if it is also a property of primary human - cells . therefore , we cultured human islets from multiple non - diabetic deceased organ donors ( see table 1 for donor information ) and exposed them to pulses of 30 mm glucose in the same paradigm as described above . pulse treatment increased insulin secretion at basal ( 2.8 mm ) and stimulatory ( 16.8 mm ) glucose concentrations ( figure 2a ) . as was the case in min6 - cells , the pulse group showed increased camkii phosphorylation at both basal and stimulatory glucose concentrations ( figure 2b ) . next , we determined the human islet response to a glucose challenge in the islet perifusion assay . islets from the glucose pulse group showed both increased first and second phase insulin secretion compared with islets from the control group ( figure 2c and d ) . as before , these effects were abolished by the camkii inhibitor kn93 ( figure 2a d ) . we also tested metabolic memory of - cells using isolated mouse pancreatic islets , with similar outcome ( extended data figure 1 ) . we also found , using live cell calcium imaging of mouse islets , that the increases in cytoplasmic calcium levels following acute glucose stimulation , representing the penultimate step in insulin secretion , are significantly higher in the pulse group compared with controls ( extended data figure 1c ) . to investigate the molecular mechanisms by which the glucose pulse paradigm induces memory in - cells , we evaluated the expression of essential mediators of the insulin secretory response . the glucose pulse paradigm induced expression of glucokinase ( gck ) , the main glucose sensor of - cells ( figure 3a ) , the voltage - gated ca channel ( cav1.2 ) ( figure 3b ) , snap25 , an essential component of the exocytotic machinery ( figure 3c ) , and mafa , a key transcription factor of the insulin gene and the mature - cell phenotype in general ( figure 3d ) . all the effects of the pulsed glucose paradigm on protein levels were reduced or abolished by treatment with kn93 during the high glucose exposure only . finally , we asked if metabolic memory of - cells is dependent on glucose metabolism itself , or if this memory can be produced by membrane depolarization alone . to this end , we treated human islets with pulses of 30 mm kcl , which causes membrane depolarization , opening of voltage - gated ca channels , and insulin secretion without a prior increase in intracellular atp levels . as shown in figure 3e , depolarization by potassium chloride during the acquisition period produced similar , if somewhat lower , increases in pcamkii and psynapsin levels 24 h after the last kcl pulse . thus , increases in intracellular calcium elicited by membrane depolarization are sufficient to activate camkii and produce metabolic memory . total camkii protein levels were not affected by any of the treatments used ( data not shown ) . glucose - stimulated insulin secretion is a complex process that translates glycolytic flux and elevated atp production to increased cytoplasmic ca levels and finally fusion of insulin granules with the plasma membrane . this process is accompanied by increased phosphorylation of camkii ( extended data figure 2 ) , a process now shown to be part of the establishment of metabolic memory in - cells . cerasi and colleagues had previously reported time - dependent potentiation of insulin secretion , in that exposure of rat islets to high glucose ( 27.7 mm ) up to 60 min prior to the test stimulation with glucose increased subsequent insulin secretion . however , these studies focused on analyzing acute effects of high glucose pre - treatment , and did not yet investigate the molecular mechanism underlying the phenomenon . here , we demonstrate that human and mouse pancreatic - cells are able to acquire , consolidate , and retrieve information , induced by high glucose exposure , and determined that this ability is dependent on camkii . in conclusion , we have shown here that , like neurons , human and mouse - cells are able to acquire , store , and retrieve information in a process similar to neuronal ltp . this process , presented schematically in figure 4 , is dependent on the activation of camkii , as is the case for neuronal memory . these findings provide further evidence that the similarity of neurons and - cells on the transcriptome and epigenome level is not accidental , but an important aspect of the biology of these embryologically distinct cell types . metabolic memory of - cells likely represents a useful evolutionary adaptation to variation in food availability . as periods of food abundance in nature can vary dramatically in length , metabolic memory of repeated carbohydrate loads in - cells ensures higher insulin secretion and thus more efficient uptake of excess glucose into skeletal muscle and adipose tissue for long - term storage , contributing to the adaptation to periods of starvation . given the pivotal role of altered insulin secretion in metabolic disorders , future work will need to establish to what extent metabolic memory of pancreatic - cells contributes to the pathophysiology of these diseases .

the chemical composition of core shell nanoparticle clusters have been determined through principal component analysis ( pca ) and independent component analysis ( ica ) of an energy - dispersive x - ray ( edx ) spectrum image ( si ) acquired in a scanning transmission electron microscope ( stem ) . the method blindly decomposes the si into three components , which are found to accurately represent the isolated and unmixed x - ray signals originating from the supporting carbon film , the shell , and the bimetallic core . the composition of the latter is verified by and is in excellent agreement with the separate quantification of bare bimetallic seed nanoparticles . ca2+/calmodulin - dependent protein kinase ii ( camkii ) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators . therefore , we hypothesized that pancreatic - cells acquire and store the information contained in calcium pulses as a form of metabolic memory , just as neurons store cognitive information . to test this hypothesis , we developed a novel paradigm of pulsed exposure of - cells to intervals of high glucose , followed by a 24-h consolidation period to eliminate any acute metabolic effects . strikingly , - cells exposed to this high - glucose pulse paradigm exhibited significantly stronger insulin secretion . this metabolic memory was entirely dependent on camkii . metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and ca2 + -dependent vesicle secretion , and by elevated levels of the key - cell transcription factor mafa . in summary , like neurons , human and mouse - cells are able to acquire and retrieve information .

health is not only related to the absence of the disease , therefore we need to conceptualize and operationalize what health is . increasingly , we have come to understand that information about functional status is needed in order to appreciate the full picture regarding the health of an individual or a population . an individual 's health fundamentally includes their capacity to carry out the full range of actions , activities and tasks required to fully engage in all areas of human life . the health state of a person can be described in terms of capacity to carry out a set of tasks or actions . in addition , the health state also includes changes in body functions and/or structures arising from a health condition . the impact of the health state on a person 's life can be understood by measuring performance of tasks and actions in the person 's real - life or actual environment . the full picture of the health experience can further be appreciated by taking into cognizance the value that people place on levels of functioning in given domains in association with a health condition . plainly , the concept of functional status is integral to health and its achievement . two individuals with identical diagnoses may have utterly different levels of functioning that determine their actual health status . without fsi , our picture of the health of an individual , or a population , is flawed and incomplete . fsi has , of course , long been collected in various ways and used clinically , especially in rehabilitative medicine ; physical , occupational and speech and language therapy ; and in nursing home and home care settings . fsi is essential for needs assessment as well as the development and monitoring of rehabilitative interventions to restore or maintain functions . it is also essential in this area of health care because the aim of therapy is to assist patients in maximizing their capacities to perform activities needed for their lives . although no one doubts that restoring functioning is restoring health ( the ultimate purpose of all forms of health care ) some clinicians , focusing exclusively on acute - care needs , do not see the need to collect or utilize fsi . in most countries with a sophisticated health administrative data collection and utilization infrastructure , a wide variety of information what is often missing is information that would link diagnosis and treatment with health outcomes that are fully meaningful to the patient 's life , namely information about the presence of decrements in capacity to carry out tasks and actions in areas of life as well as how these decrements play out in the person 's actual , real - life environment ( deyo and patrick , 1989 ; lubetkin et al . , 2003 ) . there is growing recognition that there is a gap in health administrative records : the failure to collect or disseminate fsi across all health care settings . unless fsi becomes an essential part of administrative records , the potential value of these data will be lost , not merely to clinicians , but to health administrators concerned about management and quality of care issues , health researchers , and public health agencies . this insight is clearly expressed in a report by the national committee on vital and health statistics ( ncvhs ) ( 2001 ) : without functional status information , the researchers , policymakers , and others who are already using administrative data have at best a rough idea of how people , individually and collectively , are doing and at worst they are making erroneous assumptions and decisions . the report outlines in some detail the benefits of routinely collecting fsi across the entire health care delivery system and throughout all care settings . fsi can serve management needs of all the stakeholders in the health care system clinicians , providers , payers , patients , and government regulatory bodies . this is true especially with respect to evaluating outcomes , comparing treatment modalities , and predicting and managing costs . this links directly to debates of modes of service provision , single or multiple payer , managed care , fee - for - service , or some hybrid mixture . the policy and research applications of fsi are evident for local health management and quality control , and in the broader arena of public health . policy decisions about priorities must be made at the level of individual clinics or hospitals , local or regional health care agencies , or at the level of government planning and budgeting . given the importance of getting the complete picture of health outcomes , fsi is an essential input into evidence - based policy decisionmaking . researchers in all areas of health and social policy , at all levels , need valid and reliable data about functional status in order to make informed decisions . for example , it is a matter of debate whether , as the world 's population lives longer and ages , they will be unhealthy and pose a greater burden on health systems . there is some evidence suggesting that elderly persons today are functioning at higher levels than before . without reliable information on levels of functioning , this debate would be unresolvable because it would not be possible to detect functional status , since the disease morbidity may not have changed very much . compression of morbidity occurs when disability or decrement in functioning is postponed more than longevity is extended , as for example with the effects of exercise or better eating habits . the direct test of compression ( or extension ) of morbidity depends on the effects of reduced health risks on cumulative lifetime disability ( fries , 1980 , 2001 ; vita et al . , , fsi is a crucial element for the description of health states and quantification of overall health status in individuals that can be aggregated to a summary measure of population health . at the who , the use of fsi is in this area , in particular , because this data ( collected by the world health survey now in the field in more than 70 countries ) feeds into ongoing endeavors to determine levels and distributions of health this survey would be inconceivable without information on health outcomes that describe health on multiple dimensions in terms of levels of functioning in a parsimonious set of domains . it is commonly known that the demographic trends toward an older population , at least in developed counties , will create unprecedented burdens on all age - sensitive social policies , such as social security and other pensions , retirement , unemployment , and long - term care . aging , according to a recent organization for economic cooperation and development ( oecd ) ( 2001 ) report , is the principal factor currently driving pension spending costs . since age - sensitive social programming constitutes between 40 and 60 percent of total public spending , the impact of aging is considerable . to comprehend the nature and magnitude of its social impact , those responsible for policies from transportation and housing to employment and taxation , will need reliable data on functional status and how it plays out in the lives of the aging population . for fsi to be available for this wide variety of uses , however , it must be routinely and consistently collected across the entire health care delivery system , preferably in some electronic format . nonetheless , before contemplating the systemwide changes required to collect fsi , a classification that provides a common language and framework to describe the universe of functioning and disability is required . in order to complement the classification scheme , a comprehensive coding system that creates consistent and comparable data across all settings of care and a method of routinely capturing and disseminating these data ( in a mode and manner consistent with social interests in preserving privacy ) linked to measurement tools for clinical and related encounters the foundation of a new structure for collecting fsi is , therefore , a standard classification and coding system that will make it feasible for fsi to be included in administrative data . as the ncvhs report stated : while the international classification of diseases ( icd ) has served us well for more than a century in characterizing diagnoses , it is now time to complement it with a parallel system for characterizing functional status . although the committee argued that more research , analysis , testing , and demonstration projects are required before final recommendations can be made , it concluded that : the concepts and conceptual framework of the icf have promise as a code set for reporting functional status information in administrative records and computerized medical records . in the committee 's view , the icf is the only existing classification system that could be used to code functional status across the age span . in this article , we want to briefly describe the extensive international developmental process that lead to the revision of the original international classification of impairments , disabilities and handicaps ( icidh ) ( world health organization , 1980 ) and produced the icf . we also want to describe the basic principles and structure of the icf , in particular , to show its value in the context of collecting fsi for administrative records . the primary mandate of who is the production and dissemination of reliable and timely information about the health of populations . who 's 1947 constitution requires that : each member shall provide statistical and epidemiological reports in a manner to be determined by the health assembly . countries have long reported causes of death or mortality statistics based on who 's ( 1992 ) international statistical classification of diseases and related health problems ( icd-10 ) . though useful for calculating life expectancy for different countries , however , who recognized that these data did not capture the overall health status of living populations . missing was information about non - fatal health outcomes , i.e. , functioning and disability across all areas of life . to meet this need , who ( 1980 ) issued a tool for the classification of the consequences of disease , namely the icidh . a considerable academic literature built up around clinical and other uses of the icidh , but much of this literature was critical of the underlying model of disability . responding to these critiques and an international call for an updated version , who launched a revision process in 1993 to address what many viewed as an urgent international need for a framework for measuring and reporting the health as functional status at both individual and population levels . over the next 10 years , who 's international collaborating centers and governmental and non - governmental organizations , including groups representing persons with disabilities , engaged in the systematic revision of the icidh . from an exhaustive literature search of existing classifications and assessment tools , the who revision team developed a 3,000-plus item pool of potential classification domain names for areas of human functioning at the body , person , and social levels . all efforts were made to ensure that the icidh-2 , as it was initially named , would be a suitable classification for all domains of functioning associated with both physical and mental health conditions . adopting the strategy of computer software development , alpha and beta drafts were prepared from 1996 forward . the original 1980 icidh had only been approved for field - trial purposes . in light of that , the who team felt for icidh-2 to have the necessary credibility and legitimacy to serve as the international standard language of health and functioning , that the revision process should include several years of field trials and other tests . the first phase of field trials concentrated on the cross - cultural and linguistic applicability of the model and classificatory structure and language of the icidh-2 . the intent of this phase of field trials was to establish the conceptual and functional equivalence of the items contained within the classification . stn et al.(1999a , b ; 2000 ) provide the rationale for the methodologies and presentation and analysis of the 15-country field trials . these results fed into further international collaboration in which the who team relied on a global network of who collaborating centers , non - governmental organizations , disability groups , and individual experts and key informants . the next revision phase began in 1999 when a series of expert drafting teams were assembled in geneva to produce the beta 2 draft . this draft was used for the second round of international field trials , these focusing on questions of reliability , utility , and feasibility of use . once the results of these tests were collected and analyzed , a pre - final draft was produced in early fall 2000 as a result of an intensive editing process grounded in the expert input being received from around the world . the icidh-2 , unlike its predecessor , was from the outset developed in multiple languages , primarily to identify and respond to cross - cultural and linguistic differences that might affect the usefulness of the classification . the collaborating centers and others provided constant input at this stage as the language and classification structures were redrafted and refined in multiple iterations . the draft was put on the internet for comment from a wide range of individuals , including both providers and consumers . after presentation before the executive board in december 2000 , the classification was put on the agenda of the fifty - fourth world health assembly and renamed the icf . the new title reflected the philosophy of moving beyond the consequence of disease approach and highlighted functioning as a component of health . in may 2001 , it was unanimously endorsed , member states were urged to use the icf in their research , surveillance and reporting as appropriate . with its approval , the icf became a member of the who family of international classifications . whereas icd-10 provides the codes for mortality and morbidity , icf provides the codes to describe the complete range of functional states that capture the complete experience of health . the icd-10 and icf are , therefore , complementary and who encourages users to utilize both together , wherever applicable . this will ensure a more meaningful and complete picture of the health of people or populations . soon after its official release , who 's director general , gro harlem bruntland , announced that the icf is who 's framework for measuring health and disability at both the individual and population levels . who has already implemented icf as the basis for its extensive world health survey program , demonstrating its use as a global and universal tool . to improve health , tools are needed to measure health , and in particular to measure the changes in health brought about by interventions . icf is the ruler with which we will take precise measurements of health and disability . ( brundtland , 2002 . ) from the public health perspective , the usefulness of icf goes beyond that of the measuring of population health and the effectiveness of internationally coordinated interventions funded by initiatives , such as the global fund to fight aids , tuberculosis and malaria . in addition , with the icf as their framework , countries will be able to identify social factors such as education , transportation , or housing , both as determinants of health , and social factors influenced by improvements in health . making these links will further support the relationship between health and economic development . in short , we have in the shape of a little red book , an extraordinarily versatile tool a swiss army knife for health ministries , researchers and decision - makers . ( brundtland , 2002 . ) undoubtedly the primary reason that icf can plausibly claim to be a universal tool for classifying states of functioning and disability is that the underlying model of the icf reflects our best understanding of the complex phenomena of functioning and disability in a manner that is , to the greatest extent possible , theory - neutral and therefore compatible with whichever theoretical account of how disability arises , at the individual and population levels , that evidence may confirm . it is the conceptual basis for the definition , measurement , and policy formulations for all aspects of disability . a paradigmatic shift in the thinking with regard to disability that is captured in the icf is the stress placed on health and levels of functioning . heretofore , disability has been construed as an all or none phenomenon : a distinct category to which an individual either belonged or not . the icf , on the other hand , presents disability as a continuum , relevant to the lives of all people to different degrees and at different times in their lives . disability is not something that happens only to a minority of humanity , it is a common ( indeed natural ) feature of the human condition . the icf is for all people , not just people traditionally referred to as disabled and isolated as a separate group . icf thus mainstreams the experience of disability and recognizes it as a universal human experience . by shifting the focus from cause to the full range of lived experiences , it places all health conditions on an equal footing , allowing them to be compared using a common metric the ruler of health and disability . from emphasizing people 's disabilities , and labeling people as disabled , we now focus on the level of health and functional capacity of all people . decrements in functioning may be the result of decrements in intrinsic capacity or problems with body functions or structures ; or they can result from features of the person 's physical , human - built or social environment that lead to problems in performance over and above decrements in capacity . very likely , decrements in functioning are the result of both processes . yet , the extent to which intrinsic decrements in capacity or environmental factors are the cause is not a matter that can be determined a priori . moreover , icf is grounded in the principle of universality , namely that functioning and disability are applicable to all people , irrespective of health condition , and in particular that disability or decrement in functioning at one or more levels is not the mark of a specific minority class of people , but is a feature of the human condition , which is , epidemiologically speaking , over the lifespan , a universal phenomena . in addition , icf is committed to the principle of parity , which states that the functional status is not determined by background etiology , and in particular by whether one has a physical rather than mental health condition . much time , effort , and international collaboration has gone into the development of the icf . it is no longer plausible to insist that the icf is a medical classification of people with disability , that it reduces all issues of functional status to underlying medical conditions , that it ignores the often salient role of the physical and social environment in the creation of restrictions of participation experienced by persons with functional problems . the revision process has produced a classification that has already stood up to rigorous tests of validity , reliability , and cross - cultural applicability . it is , as the ncvhs has concluded , the only existing classification system that could be used to code functional status across the age span . we now turn to the structure of icf as a classification system , in part to show why the committee has correctly assessed the value of the icf as a coding system for functional status , suitable for use in administrative records . the model that informs icf , portrays functioning and decrements in functioning , or disability , as a dynamic interaction between health conditions ( diseases , disorders , and injuries ) and contextual factors . contextual factors include environmental factors , that is , all aspects of the physical , human - built , social , and attitudinal environment that create the lived experience of functioning and disability . although not classified in icf , contextual factors also include personal factors such as sex , age , coping styles , social background , education , and overall behavior patterns that may influence how disability is experienced by the individual . the terms functioning and disability in the icf are the general or umbrella terms for , respectively , the positive and negatives aspects of the interaction between an individual ( with a health condition ) and that individual 's contextual factors ( environmental and personal factors ) . in the icf , health condition is the umbrella term for disease ( acute or chronic ) , disorder , injury or trauma . a health condition may also include other circumstances such as pregnancy , aging , stress , congenital anomaly , or genetic predisposition . the icf interactive model identifies three levels of human functioning : functioning at the level of body or body part , the whole person , and the whole person in their complete environment . these levels in turn define three aspects of functioning : body functions and structures , activities , and participation . disability similarly denotes a decrement in functioning at one or more of these levels that is , an impairment , activity limitation or participation restrictions . table 2 shows the complete list of all of the chapters found in the three classifications included in icf . under each of these chapters are second , third , and in some instances , fourth levels of categories , arranged in a hierarchical , tree - branch - stem - leaf , arrangement . this structure makes it possible for icf to be used as a classification tool for systematically describing situations of human functioning and problems with functioning . this complex information is organized by icf by means of a hierarchical coding system , thereby creating a common international language for functioning and disability . icf organizes information by means of several classifications distributed into two parts : ( 1 ) a component of functioning and disability that includes the component of the body with the body function and body structure classifications , and the component of activities and participation that includes all domains denoting aspects of functioning from an individual and social perspective organized into a single classification , and ( 2 ) a component of contextual factors that has a list of environmental factors organized from the individual 's most immediate to the wider environment . the classifications in the first part identify all of the domains of functioning from basic physiological functions and body structures , to simple and complex actions , tasks , social performances and relationships . the environmental factors list provides a tool for identifying those features of a person 's physical , human - built , social and attitudinal environment that , in interaction with the domains of functioning , constitute the complete lived experience of human functioning and disability . within the contextual factors part , besides the environmental factors , the icf recognizes the existence of personal factors as another component , but provides no classification of these . domains are a practical , meaningful set of related physiological functions , anatomical structures , actions , tasks , or areas of life . domains make up the different chapters and blocks within each component ( world health organization , 2001 ) . in order for these domains to capture descriptive information about functioning and disability in particular cases , they must be used in conjunction with qualifiers that record the presence and severity of a problem or decrement in functioning at the body , person , and social levels . for the classifications of body function and structure , the primary qualifier indicates the presence of an impairment and , on a five - point scale , the degree of the impairment of function or structure ( no impairment , mild , moderate , severe , and complete ) . in the case of the activity and participation list of domains , two essential qualifiers are provided to capture the full range of relevant information about disability . the performance qualifier is used to describe what an individual does in their current or actual environment , including whatever assistive devices or other accommodations the person may use to perform actions or tasks and whatever barriers and hindrances exist in the person 's actual environment . because the current environment always incorporates the overall social context , performance might be understood as involvement in the lived experience of disability . the capacity qualifier describes an individual 's inherent ability to execute a task or an action . operationally , this qualifier identifies the highest probable level of functioning of a person in a given functional domain at a given moment without any specific assistance . for measurement purposes , this level of capacity presumes a standardized assessment environment , namely one that reveals the inherent capacity of a person in a specific functional domain without any particular enhancements . the environmental factors list can be used to describe such a standard assessment environment in order to ensure that results across different studies can be compared by holding this environment constant . intuitively , the performance qualifier captures what people actually do in their lives , whereas the capacity qualifier identifies the person 's inherent capacity without explicit environmental facilitation ( or hindrance ) . who is developing a standard application guide that will operationalize the constructs of capacity and performance with respect to individual items that form the classification . table 3 shows how data can be organized to reflect the role of these two qualifiers used for the domains of the activity and participation classification . as a general matter of describing functioning and disability phenomena fully and accurately , the performance / capacity having access to both performance and capacity data enables icf users to determine the gap between capacity and performance . if capacity is less than performance , then the person 's actual or current environment has enabled him or her to perform better than what data about their capacity would predict : the environment has facilitated performance . on the other hand , if capacity is greater than performance , then some aspect of the environment is acting as a barrier to a level of performance that is feasible in a more suitable environment . icf thus makes it possible to measure the effect of a person 's environment on their decrement in functioning , given their health condition . the environmental factors classification can be used to identify specific features of the person 's actual environment that are barriers or facilitators in general for the person or with specific regard to each item of the person 's body functions , body structures or activities and participation that have been described . it can also be used , as previously stated , to describe specific testing environments where capacity has been measured . for its use as a classification of functional status relevant for health administrative records , icf provides a complete classification of both body and person level domains of functioning . given that it has been designed for a multiplicity of uses and users , there is far more in icf than could ever be plausibly integrated into a viable coding system for health records , although it remains the ultimate lexicon to which any coder , for clinical or research purposes , could turn . clearly , for implementation purposes in this area , a simplified checklist of items is needed . such a checklist was produced and used during the beta 1 and 2 field - testing phase in the revision process ( world health organization , 2001 ) . this checklist , which takes less than 30 minutes to complete , is currently being extensively tested in clinical studies in different disorders in order to study its feasibility , reliability , and concurrent validity with existing assessment instruments as part of a larger project to define core sets of items that may be used in rehabilitation settings for specific conditions and across several disorders ( stucki et al . , 2002 ) . the core sets of items with their corresponding scales could also be then converted into even shorter assessment instruments . the challenge for incorporating the icf into clinical and administrative records beyond a lexicon and framework lies in identifying this parsimonious set of domains or items that captures decrements in functioning across different health conditions and a smaller subset of domains or items that uniquely describe the decrements of functioning that typify a given health condition . in addition , the mapping of instruments ( that measure functioning and disability that are already in use ) onto icf categories will allow a ready crosswalk between measurements already being made at points of encounter to a common framework ( cieza et al . , 2002 ) . the use of the icf in larger population based surveys will also provide data on norms and distributions of health , functioning and disability that will enable the setting of appropriate thresholds for a multitude of purposes . table 4 maps the domains of the icf that have been included in different waves of the world health survey that ought to be included as a minimum or ideal set for information systems . these domains are also included on the icf checklist , which is designed to be a clinical tool . primary data collection strategies with regard to functional status , in a manner that is truly comparable , are in their infancy especially for international use and for use across population groups . further tools need to be developed , and standards and procedures established , so that these data become meaningful and usable . as a final issue , it must be mentioned that the icf has been conceived as a dynamic classification that will not only serve multiple users requiring different levels of detail , but also will continue to evolve with advancements in science . the classification is flexible in its structure such that it can be expanded in the level of detail ( for example , the fourth level ) for specific uses , or new codes added where gaps have been left in the numbering system . a set of operational rules will specify the procedure for this evidence - based expansion , adaptation , or revision of the classification . a common language for describing fsi is the key to ensuring comparability of data from a myriad of sources as well as in providing users with a tool for precise and accurate communication with each other . the recognition that a description of health and health - related outcomes must go beyond a narrow view of health restricted to the absence of disease , as well as that the definition of disability must move beyond the narrow impairment - based view that has been traditionally adopted to define a minority population , will go a long way in bridging the gap between health and disability data . it will also fill the void in existing health outcomes data while measuring the impact of interventions and monitoring them over time . health records must include functioning information in order to ensure a complete description of health states . the icf is the common language and framework that users will employ from now on . in the same way that all languages grow , evolve , and flourish over time and are adapted and modified to express new ideas , the icf will have a multitude of applications where it will be creatively used such that it continues to be a living classification . as with all new languages , it will be important to develop tools to learn this new language . toward this end , who is developing standardized application manuals and web - based learning courses that will use state - of - the art pedagogic methodology to assist end users . its usefulness in describing functional health status information will be one of the measures of its success . the centers for disease control and prevention ( cdc ) and united states preventive services task force ( uspstf ) recommend universal hiv screening for all pregnant women entering prenatal care [ 1 , 2 ] . this screening enables hiv - infected women and their infants to benefit from appropriate and timely interventions such as antiretroviral medications . when the recommended antiretroviral and obstetric interventions are used , a woman who knows of her hiv infection early in pregnancy now has a less than 2% chance of delivering an hiv - infected infant . without intervention , this risk is approximately 25% in the united states [ 36 ] . testing for hiv began in 1985 with the introduction of the enzyme immunoassay ( eia ) . in order to account for false positive results using screening tests in a low - prevalence population , confirmatory testing has been implemented using a western blot or immunofluorescence assay . in a low - prevalence population , the false positive rate using the eia is increased compared to a high - prevalence population , and the positive predictive value of any test will always depend on the prevalence of the condition in the population tested . in testing performed by the cdc , the eia positive predictive value ranges from 71 to 83% in populations with hiv prevalence from 0.5 to 1% . some investigators believe that the presence of alloantibodies account for the increased false positive rate associated with pregnancy , transfusions , transplantation , and autoimmune diseases . however , risk factors specific to pregnancy that account for this are poorly understood . conversely , a recent large retrospective study found that the false positive hiv eia rate was lower in pregnant women compared to nonpregnant individuals ( 0.14% versus 0.21% ) . our objective was to determine if any maternal characteristics correlated with false positive hiv eia testing at delivery . this was a review of all women who delivered at parkland memorial hospital in dallas , tex , from october 1 , 2005 through september 30 , 2008 . all women routinely received serum hiv testing at their initial prenatal visit and at time of presentation to labor and delivery for delivery via the opt - out approach with the abbott commander hiv ab hiv-1/hiv-2 ( rdna ) eia ( abbott laboratories , abbott park , ill ) . hiv test results performed at the time of delivery were analyzed in this study . a woman was considered hiv negative if eia testing was negative . positive test results were confirmed with the fluorognost immunofluorescent assay ( ifa ) hiv-1 ( sanochemia corp , stamford , conn , usa ) . women were considered to be falsely positive if eia results were positive and the ifa was negative . women delivered in this time period were identified through the obstetric operations database and linked to the pathology database for hiv , hepatitis b surface antigen ( hbsag ) , and rapid plasma reagin ( rpr ) results . maternal characteristics , including race , parity , age , singleton versus multiple gestation , and a diagnoses of diabetes or hypertension were obtained using the obstetrics operations database . laboratory results drawn 28 days prior to delivery through seven days after delivery were included . the study was approved by the institutional review board at the university of texas southwestern medical center . categorical data were reported as frequencies , and statistical significance was determined using analysis . statistical analyses were performed using sas , version 9.2 ( sas institute , cary , nc ) . a total of 47,794 women were identified who delivered during the study time frame . compared to hiv negative patients , false positive patients were more likely to be nulliparous ( 43% versus 31% , p < 0.001 ) and younger ( mean age 23.9 5.7 versus 26.2 5.9 , p < 0.001 ) . hiv positive patients were significantly older than false positive patients ( 27.4 versus 23.9 , p < 0.001 ) and hiv negative patients ( 27.4 versus 26.2 , p = 0.012 ) . of the 47,794 women , 47,391 ( 99% ) were hiv negative , 145 ( 0.3% ) had a false positive test , 172 ( 0.4% ) were true positives , and 86 ( 0.2% ) tested equivocal or were missing hiv results . the specificity of the hiv eia test was 99.7% with a positive predictive value of 54.3% . the sensitivity of the eia test was presumed to be near 100% , as the false negative rate using the eia has been previously demonstrated to be negligible in studies performed by the manufacturer . table 2 shows the prevalence of diabetes , hypertension , hepatitis b , and results of syphilis testing in the study population . hiv positive women were more likely to test positive for hepatitis b surface antigen ( 1% versus 0% , p = 0.002 ) and rpr ( 2% versus 0% , p = 0.02 ) . there was no significant difference in the prevalence of diabetes or hypertension between the three groups . there was also no significant difference in the rate of hbsag and rpr positivity between the hiv negative and false positive groups . when evaluating the interaction between nulliparity and age , there is a significant correlation between the two variables ; that is , parous patients are likely to be older and , therefore , nulliparity and age are not independent predictors of hiv false positivity . however , when the hiv testing groups ( positive , false positive , negative ) are stratified by parity , the comparison of age across the three groups remains significant only in nulliparous women ( table 3 , p = 0.0003 ) . the interaction between parity and age means that the difference in age between nulliparous and parous women is different depending on the hiv status . for example , in the hiv positive group the nulliparous and parous women are closer in age than in the false positive group . this was the first population - based study to evaluate risk factors for hiv false positivity in pregnant women presenting for delivery at a large urban institution . we found that younger and nulliparous women were more likely to have false positive testing using the hiv eia . the observed positive predictive value ( ppv ) of 54.3% was lower than the previously reported by the cdc ( ppv 7183% ) in a nonpregnant population , suggesting that pregnancy may be associated with a higher rate of false positivity . the low positive predictive value of the hiv eia in our study may have been impacted by the relatively low hiv prevalence ( 0.4% ) in our population . this information could be useful for counseling women who test positive for hiv at delivery and emphasizes the limitations of eia testing if used as a rapid test to determine the need for intrapartum and neonatal antiretroviral prophylaxis . the false positive rates of currently available rapid hiv tests have been reported to be much lower than has been found with eia testing in this study . evaluated the hiv false positive rate in over 900 pregnant women , most of whom were hispanic , using both the eia and a rapid point - of - care ( poct ) test , oraquick . they found that while there were no false positive tests with the oraquick , there were seven false positives using the eia ( ppv 100% versus 35.7% ) . in the mother - infant rapid intervention at delivery ( miriad ) study , jamieson et al . found that the ppv of the oraquick test was 90% while the ppv of the eia was 74% [ 12 , 13 ] . current recommendations by the cdc and the american college of obstetricians and gynecologists include rapid hiv screening for women presenting to labor and delivery with undocumented hiv status and for repeat hiv testing in the third trimester for women at high risk or who live in areas with high hiv prevalence [ 7 , 14 , 15 ] . a woman testing preliminarily positive for hiv in labor should be counseled that she may have hiv infection and that her neonate may be exposed , and immediate antiretroviral prophylaxis should be recommended without waiting for confirmatory test results . the results of our study may aid in counseling women if they test preliminarily positive for hiv using the eia , while awaiting confirmatory testing results . it remains to be determined whether the same risk factors for false positive hiv eia testing apply to the poct tests used in a real life setting . our study found that the positive predictive value of eia testing was only 54.3% and that younger nulliparous women were more likely to test falsely positive . importantly , our data may represent the real world performance of the eia testing in a large obstetric population - based setting . investigators also have noted a significant relationship between influenza vaccination and false positive screening for hiv antibodies [ 1618 ] . a potential reason for this cross reactivity is that there are similarities in homology between the transmembrane domains of the influenza envelope protein hemagglutinin and the hiv-1 envelope proteins . in our population , there were no significant differences in age or parity and influenza vaccination acceptance rates ( unpublished data ) . women in our study were from a single institution , with a predominantly hispanic background , and therefore the results may not be applicable to all populations . reasons for false positive hiv serology may vary depending on the geographical region . while we did find a significant association between young age , nulliparity , and hiv false positive testing , our study does not have the capability to identify a causal relationship or explain why this association may exist . in addition , our study can not address if these same risk factors for false positive testing apply to all other hiv tests . further studies are needed to confirm our findings , elucidate the biological mechanisms for increased hiv eia false positivity in young , nulliparous women , and compare this conventional testing method with contemporary rapid screening assays , including poct .

a common framework for describing functional status information ( fsi ) in health records is needed in order to make this information comparable and of value . the world health organization 's ( who 's ) international classification of functioning , disability and health ( icf ) , which has been approved by all its member states , provides this common language and framework . the biopsychosocial model of functioning and disability embodied in the icf goes beyond disease and conceptualizes functioning from the individual 's body , person , and lived experience vantage points , thereby allowing for planning interventions targeted at the individual 's body , the individual as a whole or toward the environment . this framework then permits the evaluation of both the effectiveness and cost effectiveness of these different interventions in devising programs at the personal or societal level . objective . to examine risk factors for false positive hiv enzyme immunoassay ( eia ) testing at delivery . study design . a review of pregnant women who delivered at parkland hospital between 2005 and 2008 was performed . patients routinely received serum hiv eia testing at delivery , with positive results confirmed through immunofluorescent testing . demographics , hiv , hepatitis b surface antigen ( hbsag ) , and rapid plasma reagin ( rpr ) results were obtained . statistical analyses included pearson 's chi - square and student 's t - test . results . of 47,794 patients , 47,391 ( 99% ) tested negative , 145 ( 0.3% ) falsely positive , 172 ( 0.4% ) positive , and 86 ( 0.2% ) equivocal or missing hiv results . the positive predictive value of eia was 54.3% . patients with false positive results were more likely nulliparous ( 43% versus 31% , p < 0.001 ) and younger ( 23.9 5.7 versus 26.2 5.9 years , p < 0.001 ) . hiv positive patients were older than false positive patients and more likely positive for hbsag and rpr . conclusion . false positive hiv testing at delivery using eia is associated with young maternal age and nulliparity in this population .

childhood obesity is a serious and growing public health problem that has arisen over the past three decades . the increasing occurrence of disorders such as type 2 diabetes during childhood is believed to be a consequence of this obesity epidemic . in addition to several behavioral and dietary risk factors , genetic predisposition is an important factor in the pathogenesis of childhood obesity . it is estimated that 4070% of adiposity variance can be explained by direct or indirect genetic factors . growth arrest - specific 6 ( gas6 ) , cloned in 1988 and characterized in 1993 , is a secreted vitamin k - dependent protein present in the human circulatory system [ 4 , 5 ] . initially , gas6 was shown to be upregulated in growth - arrested fibroblasts , suggesting that it plays a protective role in certain cellular stresses such as during apoptosis . gas6 expression is widespread in many tissues , including immune cells , endothelial cells , vascular smooth muscle cells , and adipocytes [ 79 ] . the protein is also a ligand for the tam ( tyro-3/axl / mer ) family tyrosine kinase receptor . the gas6/tam system has been implicated in cell survival and proliferation , cell adhesion and migration , hemostasis , and inflammatory cytokine release [ 4 , 10 ] . recently , the gas6/tam pathway was found to be involved in mediating adipocyte survival and proliferation in vitro [ 11 , 12 ] . experiments with mice fed a high - fat diet indicated that overexpression of gas6 might enhance body - fat accumulation , but blocking gas6 signaling using an axl antagonist could reduce body - fat mass and body weight . interestingly , transgenic animals that ectopically express the axl tyrosine kinase receptor also develop progressive obesity with elevated circulating proinflammatory cytokines and severe systemic insulin resistance . this protein - array study also revealed higher levels of axl mrna in subcutaneous adipose tissue of obese humans than their lean control counterparts had . this indicates that the axl receptor may be involved in the development of human obesity . in addition , some studies in transgenic mice indicate that gas6/axl signaling might recruit macrophages and other immune cells into the adipose tissue resulting in the production and secretion of proinflammatory mediators . this suggests that the gas6/axl signaling might play a role in the pathogenesis of obesity - associated systemic inflammation [ 8 , 16 , 17 ] . recent studies have indicated that systemic inflammation , a hallmark of childhood and adult obesity , is a pivotal mechanism linking obesity to insulin resistance and type 2 diabetes [ 1821 ] . although gas6 gene polymorphisms are reported to be associated with stroke , acute coronary syndrome , and type 2 diabetes [ 2224 ] , to our knowledge , both gas6 and axl gene polymorphisms associated with childhood obesity have not yet been identified . in order to address this issue , we conducted a community - based study to determine whether common variations in the gas6 and axl genes correlate with adiposity , systemic inflammation , insulin resistance among adolescents . the taipei children heart study - iii was an epidemiological survey that investigated obesity and cardiovascular disease risk factors among adolescents in taipei city during 2006 . the sampling method and results briefly , the survey included junior high school students in taipei city to collect a representative distribution of demographic , lifestyle , and biochemical characteristics to measure their risk for cardiovascular disease . those with autoimmune diseases , cancers , or active infection and those taking medications known to interfere with insulin or glucose metabolism were excluded . excluding any missing data , 727 adolescents ( 358 boys and 369 girls ) the institutional review board of the tri - service general hospital approved these studies and obtained informed consent from both parents and adolescents . all the participants completed a structured questionnaire detailing their gender , age , puberty development , and lifestyle characteristics ( including cigarette smoking , alcohol consumption , and physical activity ) . based on their responses , the subjects were divided into young adolescents with history of smoking , those without , and those who currently smoke . physical activity was divided into 5 levels based on amount of exercise per week : less than 1 h , 13 h , 35 h , 57 h , and over 7 h. survey questions concerning puberty onset included the development of the penis / testis and pubic hair for boys and development of breasts and pubic hair for girls . body weight was measured of barefoot students wearing light indoor clothing and was rounded to the nearest 0.1 kg . waist circumference ( wc ) was measured at the midway point between the inferior margin of the last rib and the crest of the ilium in a horizontal plane and was recorded to the nearest 0.1 cm . body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . to reduce extraneous variation between subjects , we collected blood samples from the students after 12 h fasting and who had consumed their usual diet for the previous 3 days . children who had recently attended a holiday or family celebration were contacted for a blood sample several weeks after the event . plasma glucose concentrations were determined by the glucose oxidase method by using the beckman glucose analyzer ii ( beckman instruments , fullerton , ca ) . the intra- and interassay coefficients of variation ( cvs ) for glucose were 0.6% and 1.5% , respectively . plasma insulin was measured using a commercial immunoradiometric kit ( biosource europe , nivelles , belgium ) . serum levels for high - sensitivity c - reactive protein ( hscrp ) were measured using the tina - quant ( latex ) high - sensitivity assay ( roche , mannheim , germany ) . serum il-6 concentrations were determined using a human high - sensitivity enzyme - linked immunosorbent assay ( elisa ) ( innotest , besancon , france ) . serum tnf- was measured with the biotrak high - sensitivity human elisa kit from amersham biosciences ( buckinghamshire , uk ) . insulin resistance was assessed using the homeostasis model assessment ( homa ) , in which the homa of insulin resistance ( homa - ir ) = fasting insulin ( u / ml ) fasting glucose ( mmol / l)/22.5 . gas6 protein concentration was measured using a sandwich elisa and a polyclonal mouse anti - human gas6 antibody ( r&d systems , lille , france ) as a catcher and a biotinylated goat antiserum as a detector ( r&d systems ) , using previously described methods . the technique has been validated by food and drug administration guidelines in a previous study ( intra- and interassay cvs of 6.5% and 8.5% , resp . ; mean recovery on 10 patients of 97% ; lower limit of quantification 0.26 ng / ml ) . dna was isolated from buffy coats using the qiaamp dna blood kit and following the manufacturer 's instruction ( qiagen , valencia , ca , usa ) . the qualities of isolated genomic dnas were quantified using agarose gel electrophoresis , and the quantities were determined using a spectrophotometer . snps rs8191973 and rs8191974 in gas6 , as well as rs4802113 and rs2304232 in axl , were genotyped using a taqman assay with allele - specific probes on the abi prism 7900ht sequence detection system ( applied biosystems , foster city , ca , usa ) according to the standardized laboratory protocols . descriptive results of continuous variables were expressed as means sd . prior to statistical analysis , the normal distribution and homogeneity of the variables were evaluated using the levene test for quality of variance , and the variables were then given a base logarithmic transformation if necessary . the parameters homa - ir , triglycerides , hscrp , il-6 , and tnf- were analyzed and tested for significance using a log scale . the studied adolescents were categorized into subgroups based on their gas6 rs8191973 genotype ( cc , cg , and gg ) , gas6 rs8191974 genotype ( gg , ga , and aa ) , axl rs4802113 genotype ( cc , ct , and tt ) , and axl rs2304232 ( aa , ag , and gg ) with gender specification . the differences between anthropometric and biochemistry data across genotypes were analyzed using a general linear model after adjusting for age , tanner stages , smoking status , drinking status , and physical activity . chi - square tests were used to determine the genotype distributions for the hardy - weinberg equilibrium and to compare the proportions of abnormal anthropometric and biochemistry variables across genotypes . we tested different genetic inheritance models , and a recessive model was applied in the final analyses for gas6 and axl . to determine whether the gas6 and axl snps are predictors of obesity and obesity - associated complications , logistic regression analysis was used to calculate the odds ratio ( or ) and 95% confidence interval ( ci ) for each genotype and combined genotypes . a two - sided p - value of < 0.05 was considered statistically significant . all statistical analyses were performed using pasw statistics 18.0 software ( spss inc . , chicago , il , usa ) . in total , 727 adolescents ( 358 boys and 369 girls ) were included in this study . the mean age of all adolescents in this study was 13.3 years ( range , 1215 ) and was similar between boys and girls . in general , boys had higher bmi ( 22.3 4.0 versus 21.2 3.3 kg / m ) , wc ( 80.0 10.1 versus 75.1 8.1 cm ) , hscrp ( 0.9 1.3 versus 0.6 0.9 mg / l ) , and glucose levels ( 93.8 6.3 versus 91.5 6.5 mg / dl ) than the girls ( all p < 0.001 ) . however , girls had higher tanner stages ( 3.2 0.5 versus 3.0 0.4 ) than the boys ( p < 0.001 ) . there was no statistically significant difference in the ages , tnf- , il-6 , and insulin levels , and homa - ir between the boys and girls . the allele frequency for the least frequent allele in boys was 12.6 , 22.1 , 41.9 , and 29.7% , and 13.4 , 19.6 , 32.7 , and 33.5% in girls for the gas6 rs8191973 , gas6 rs8191974 , axl rs4802113 , and axl rs2304232 polymorphisms , respectively . there was no significant difference in allele or genotype distribution between boys and girls at the 4 polymorphisms . no statistically significant association between anthropometric characteristics , biochemistry data , and the gas6 rs8191973 genotypes was observed in the boys and girls ( table 1 ) . however , there were significantly different hscrp levels between gg , ga , and aa genotypes of gas6 rs8191974 in boys , regardless of their age , tanner stages , smoking status , drinking status , or physical activity ( table 2 ) . moreover , boys with the gg genotype of gas6 rs8191974 had significantly higher bmi , wc , and hscrp levels than those carrying the a allele . the gas6 rs819174 genotypes were not significantly associated with any anthropometric characteristics and biochemistry in girls . the p - values of all comparisons between anthropometric and biochemistry data across gas6 genotypes were presented in supplemental tables 1 and 2 available online at http://dx.doi.org/10.1155/2014/674069 . in addition , the association between circulating gas6 protein levels and gas6 polymorphisms was investigated . we found that the gas6 rs8191973 or rs8191974 genotypes were not significantly associated with circulating gas6 protein levels in both the sexes . there were significantly different hscrp levels between tt , tc , and cc genotypes of axl rs4802113 in boys , independent of their age , tanner stages , smoking status , drinking status , or physical activity ( table 3 ) . in addition , boys with the gg genotype of axl rs2304232 had significantly higher il-6 and insulin levels and increased homa - ir than those carrying the a allele ( table 4 ) . however , in girls , axl rs4802113 or rs2304232 polymorphisms were not significantly associated with any anthropometric characteristics or biochemistry ( tables 3 and 4 ) . the p - values of all comparisons between anthropometric and biochemistry data across axl genotypes were presented in supplemental tables 3 and 4 . boys with the gg genotype of gas6 rs8191973 were 1.87-fold more likely to have higher hscrp levels than the c allele carriers . even after adjusting for age , tanner stage , smoking status , drinking status , and physical activity , a significant relationship between the gg genotype of gas6 rs8191973 and higher hscrp levels was still observed in boys ( table 5 ) . moreover , boys with the gg genotype of gas6 rs8191974 exhibited a 1.40-fold greater risk for developing high bmi , a 1.58-fold greater risk for developing high wc , and a 2.68-fold greater risk to have higher il-6 levels than the a allele carriers . even after adjusting for all possible confounding factors including age , tanner stage , smoking / drinking status , and physical activity , the relationship between the gg genotype of gas6 rs8191974 , higher bmi / wc , and higher il-6 levels still remained significant in boys . however , the axl rs4802113 or rs2304232 polymorphisms showed no significant association with abnormal adiposity , inflammatory markers , and homa - ir in boys or girls ( see supplemental table 5 ) . logistic regression analyses were applied to evaluate whether the combination of the gas6 rs8191974 and rs8191973 polymorphisms is a stronger risk factor for high adiposity , inflammatory markers levels , and homa - ir than when alone . the combined effects of the 2 gas6 gene polymorphisms in the risk of high bmi , wc , il-6 , and hscrp levels are shown in figure 1 . after adjusting for the relevant confounding factors , we still observed that boys with the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 exhibited a 47-fold higher risk of high bmi , wc , il-6 , and hscrp levels than the individuals with both the c allele of the gas6 rs8191973 and the a allele of the gas6 rs8191974 did ( or = 4.92 , 95% ci : 1.0823.6 , p = 0.018 ; or = 4.18 , 95% ci : 1.0522.5 , p = 0.016 ; or = 4.08 , 95% ci : 1.0628.56 , p = 0.015 ; or = 7.22 , 95% ci : 1.4635.72 , p = 0.010 , resp . ) . however , for girls , there was no statistically significant association between the combination of the gas6 rs8191974 and rs8191973 polymorphisms and abnormal variables . in addition , we evaluated the combined effect of the gas6 rs8191973 or rs8191974 marker with axl gene polymorphisms and its association with risk of high adiposity , inflammatory marker , and homa - ir . however , combinations of gas6 markers with axl gene polymorphisms were not found to be significantly associated with any abnormal variables in both boys and girls ( data not shown ) . in this study , a strong association between gas6 and axl polymorphisms with body adiposity , systemic inflammation , and insulin resistance was identified among boys . the risk of possessing high adiposity and inflammatory markers levels was higher in boys carrying the gg genotype with gas6 rs8191973 or rs8191974 than the noncarriers . moreover , the combination of both gas6 polymorphisms had an additive effect on the development of obesity and obesity - associated inflammation in boys . these data strongly suggest that gas6 and axl genes play a role in the pathogenesis of childhood obesity and its associated complications . gas6 was originally identified as a gene that is expressed in fibroblasts and increases with serum starvation and contact inhibition ; gas6 is also a potential growth factor for fibroblasts . maquoi and colleagues demonstrated that when fed with a high - fat diet , gas6-deficient mice had significantly less fat than their wild - type counterparts . the authors also reported the expression of gas6 and its 3 receptors ( tyro-3 , axl , mer ) in murine adipose tissues , thus suggesting that gas6 may act in an autocrine and/or paracrine manner to promote murine adipose tissue development . previous experiments in transgenic mice demonstrate that gas6 might also induce obesity - associated inflammation via recruiting immune cells into the adipose tissue to producing and secreting proinflammatory cytokines [ 8 , 16 , 17 ] . our recent clinical study found that circulating gas6 protein levels are associated with adiposity and inflammatory markers in overweight / obese adolescents . in this study , gas6 is further implicated as a candidate susceptibility gene for obesity and systemic inflammation . however , the association between gas6 genotypes and circulating gas6 protein levels was not observed among adolescents . we hypothesize that gas6 polymorphisms could affect the biology of the gas6 protein itself rather than its transcription or process rate , thus influencing adiposity regulation and systemic inflammation . to validate this , recent studies demonstrated that gas6/tam signaling is involved in releasing inflammatory cytokines ( such as il-6 and hscrp ) in diverse human diseases [ 23 , 31 , 32 ] . in addition , the gas6/tam signaling is also known to be involved in several inflammation - related systems , including maturation of immune cells , endothelial activation , and immunoregulation . our present study found that the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 are strongly associated with higher circulating il-6 and hscrp levels in boys . therefore , the gas6 polymorphisms presumably influence gas6/tam signaling and could further activate inflammatory reactions and result in releasing circulating il-6 and hscrp . however , the comprehensive effects of the gas6 polymorphisms in regulation of inflammatory cytokines still remain to be determined by more researches . interestingly , a previous study found that the a allele or the aa genotype of gas6 rs8191974 is associated with decreased risk of stroke . moreover , the a allele and the aa genotype are also thought to be related to a lower risk of developing acute coronary syndrome or type 2 diabetes , suggesting that this genotype exhibits protective activities against developing acute coronary syndrome and type 2 diabetes [ 23 , 24 ] . we also observed similar results in those with the a allele or aa genotype of gas6 rs8191974 . these subjects exhibited a lower risk for developing obesity and systemic inflammation than those with the gg genotypes . together , these findings suggest that the gas6 rs8191974 polymorphisms play an important role in the development of obesity and obesity - associated complications ( e.g. , type 2 diabetes , cerebrovascular , and cardiovascular diseases ) . the protective role of the aa genotype of gas6 rs8191974 against the developing childhood obesity and obesity - associated complications requires further study . the axl protein is a membrane - bound receptor that belongs to the tam family of receptor tyrosine kinases . axl exhibits the highest affinity for gas6 as compared to the other members of the tam family , whereas protein s predominantly binds mer and tyro-3 . gas6/axl signaling has been shown to be involved in the pathogenesis of obesity and systemic inflammation [ 1315 ] . however , our study demonstrates that axl polymorphisms are associated with systemic inflammation rather than childhood obesity . moreover , the combination of gas6 and axl gene polymorphisms is not significantly associated with any variables in adiposity among adolescents . our findings indicated that axl gene polymorphisms might not play a significant role in childhood obesity . recently , scroyen and colleagues have published similar findings indicating that deficiency in a single axl receptor did not significantly affect adipogenesis or adipose tissue development in mice . this is because an axl deficiency can be partially compensated by other tam family members ( tyro-3 and mer ) via gas6 interaction . further studies are needed to investigate the effect of tyro-3 and mer receptors on the development of childhood obesity . in addition , our present study also indicates that gender disparity exists regarding the effects of the gas6 polymorphisms on anthropometric characteristics and inflammatory markers . we found no significant difference in genotype frequencies between boys and girls ; however , the effects of the gas6 polymorphisms , individually or combined , only manifest in boys . the gas6 gene contains an estrogen response element in its promoter and is upregulated by estrogen via an activated estrogen receptor in mammary epithelial cells . therefore , we hypothesized that the gender - specific effect of the gas6 polymorphisms on childhood obesity might be due to a disparity in sex hormone distributions . this has been previously reported to be associated with gas6 expression and body composition [ 40 , 41 ] . despite these results , our study does have certain limitations . first , this was a cross - sectional study , as such we might not be able to assess gas6 polymorphisms on weight dynamics and the development of obesity - associated complications throughout life . second , because of the limitations of our questionnaire , we were not able to comprehensively estimate every adolescent 's daily intake . the impact of dietary energy intake on genetic susceptibility also requires further investigation to better understand any confounding effect . in conclusion , we indicate an association between the gas6 and axl polymorphisms with adiposity , circulating inflammatory markers , and insulin resistance of adolescents , especially in boys . moreover , the gg genotype of gas6 rs8191973 or rs8191974 strongly correlates with susceptibility to develop obesity and systemic inflammation in boys . nonetheless , these results together with those from studies in cellular and animal models encourage the study of the gas6/tam system in childhood obesity and its potential complications and further support the hypothesis that modulation of gas6 activity may indeed provide an important intervention point for future therapies . infertility is one of the crucial and critical events in sex life which engages 1015% of couples all over the world , and as one of the most distressful life experiences , exposes couples to social and psychological problems . couples , who are faced with this critical situation , are prone to depression , anxiety , loss of self - esteem and dissatisfaction with their sex life more than others . however , the intensity of psychological problems resulting from cultural - social circumstances varies in different societies , to the point that the frequency of anxiety in infertile couples has been reported in a wide range from 48% to 96% . although , the advances in assisted reproductive techniques have opened new doors for infertile couples , but studies have shown that these treatments are accompanied with stress , anxiety and depression . but the intensity of these disorders could be defined by the cause of the infertility . evaluating the related factors of depression and anxiety among iranian infertile couples showed that women who were infertile themselves showed higher levels of anxiety and depression than women with male factor infertility . however , in societies that define women as the main source of fertility , starting assisted reproductive treatments with limited successful outcomes could be harmful to these women 's mental health . utilization of assisted reproductive techniques requires processes such as daily injection to stimulate ovulation , vaginal ultrasound and painful processes such as oocyte aspiration which are all too expensive and alongside with the fear of failure could become a harmful condition . although , fertile women , who start assisted reproductive treatments due to their husband 's infertility have natural biological conditions to get pregnant , but they have to tolerate these critical processes due to male factor infertility . also , women are under more cultural and social pressures . these conditions could affect the vulnerability of fertile women during assisted reproductive treatment ; therefore the aim of this study was to evaluate the effect of assisted reproductive treatments on fertile women 's mental health . thus , the prospective study conducted on 70 fertile women who referred to receive assisted reproduction treatments ( in vitro fertilization / intracytoplasmic sperm injection ) due to male infertility . subjects had referred to the fertility and infertility center of esfahan from september 2013 to march 2014 . the number of samples was calculated based on 80% test power and 95% confidence level . inclusion criteria were primary infertility , not having any corporal and mental illnesses , not being a drug addict according to individual 's report , not having a history of mental illnesses , and not experiencing severe distress over 1-month prior to the treatment based on holmes - rahe scale . exclusion criteria included experiencing distressful conditions during the study , discontinuation of the treatment and the cancellation of egg harvesting . the study was approved by the medical ethics committee of the isfahan university of medical sciences . mental health evaluation tool was the valid 28-questioned general health questionnaire that measures mental health on likert scale ( 03 ) in hypochondriasis , anxiety , social impairment , and depression dimensions . in this method gaining the score of 5 in each subscale and 23 in all dimensions together was defined as having a mental disorder . sampling was conducted through simple sampling ; that is , all persons , who referred to fertility and infertility center of esfahan for treatment and to start the process protocol due to male factor infertility were interviewed . then by referring to their medical records and conducting holmes - rahe scale , their eligibility for the study was assessed . after being considered eligible for entering the study , their demographical features including age , educational level of couples , economic condition , and the duration of their infertility were recorded . ovulation was stimulated through the long protocol in all the participants . during the treatment period , ovarian reaction conditions and number of injections employed for ovarian stimulation were followed - up . 3 h after the pickup process , and under appropriate circumstances , the questionnaire for general health was completed again . if the circumstances were inappropriate filling of the questionnaire was delayed for 2 h. research data were analyzed using spss software version 16 [ spss inc . : chicago , il , usa ] , statistical paired t - tests and multi - variable linear regression tests . seven qualified subjects quit the study because of the unwillingness to continue ( three persons ) and ovarian hyper stimulation ( four persons ) . demographic and clinical data results showed no significant difference in the mean score of different dimension of mental health before ovulation induction and after oocyte harvesting [ table 2 ] . also , the rate of psychological disorder in the anxiety dimension showed no significant different before the induction ( 71.4% ) and after the pickup ( 66.7% ) ( p = 0.19 ) . but the level of depression after pickup ( 31.7% ) was significantly lower than before the induction ( 39.7% ) ( p = 0.007 ) . comparison of mental health dimensions b and a results showed no significant difference in the dimension of hypochondriasis before the induction ( 61.9% ) and after the harvesting ( 66.7% ) ( p = 0.07 ) . also , the level of social impairment showed no significant difference before the induction ( 84.1% ) and after the pickup ( 87.3% ) ( p = 0.08 ) . the results of multi - variable linear regression to evaluate the relation between underlying variables and mental health condition before starting the process with the level of depression and anxiety after the harvesting are shown in table 3 . the relation between mental health dimensions and background variables after ovulation stimulation the level of depression after egg harvesting was related to physical condition , depression and social impairment , independent from underlying variables . also , the level of depression after egg harvesting was related to the economic condition , it also showed a significant relation with social impairment and hypochondriasis [ table 3 ] . the present study aimed to assess the level of mental health in fertile women undergoing assisted reproductive treatments as a result of male factor infertility , at the beginning of ovulation stimulation process , and the effect of this process on their mental health . results showed that these women do not start assisted reproduction treatment under appropriate mental health conditions . but the level of depression and anxiety disorders at the beginning of ovulation stimulation was higher in the present research than other reports . this would indicate that the process of treatment in fertile women is also a critical one and might affect their mental health . in a pursuit , to achieve a conceptual model for the effect of ovulation induction on women 's health , its negative effects on women 's functioning and health have been shown . a qualitative study showed that iranian infertile couples show emotional reactions such as fear , concern , anxiety and depression as a result of fear of the failure of treatment during fertility treatment . however , these mental disorders may be harmful even for women with appropriate fertility potentials . also being angry with their husbands due to their infertility could be another explanation for women 's mental reactions . positive correlation between the level of depression and anxiety and other dimensions of experiences during infertility treatment might be accompanied with social impairment , thus affecting the levels of depression and anxiety . efforts to conceal infertility and efforts to get treatment as a result of concerns about being labeled as infertile , are common among iranian couples . this , by itself , could increase the level of anxiety among women when treatment starts . another reason for the high level of depression and anxiety among women at the beginning of the ovulation stimulation might be the negative impact of gonadotropin - releasing hormone ( gnrh ) agonists on the mental health . although , this research did not evaluate mental health before and after the beginning of gnrh agonists , but this might explain women 's psychological disorders before ovulation stimulation regarding high frequency of mental disorders at the time of ovulation stimulation . with regards to the negative impact of anxiety and depression during the treatment on the result of assisted reproduction treatment , it is necessary that this group of women would be examined before the treatment process , since they are exposed to risks of mental health disorders , and be treated in case of high levels of depression and anxiety . following the assessment of the main hypothesis of the research , that is the effect of ovulation stimulation on women 's mental health , study results showed that ovulation stimulation does not affect mental health ; the level of depression and anxiety , and the measure of psychological disorders in these two - dimensions did not show a significant difference before the ovulation and after the harvesting process . on the contrary , the depression level was decreased after the harvesting process . the negative impact of distress of infertility treatment on the level of depression has already been reported . the cessation of treatment distress with the conclusion of ovulation stimulation and harvesting processes might be a reason for the decrease of depression level in subjects under study . furthermore , results showed that fertile women 's level of depression and anxiety does not depend on the reaction of the ovary to the ovulation stimulation , and as these women have no problem in ovarian response to ovulation stimulation , their mental health depends more on other factors . in addition , all subjects , who sustained the research were those who reached the stage of the harvesting process ; the potential for ovum production was proven for these women . this would enhance the self - esteem of subjects and decrease their concern over ovarian nonresponsiveness . but direct correlation between the level of anxiety and depression in the stage after harvesting process with social impairment and hypochondriasis indicates that in this stage also the awareness of social dysfunction , independent from financial status , will have its own effect on mental health . factors other than ovulation stimulation process which might influence the individuals levels of depression and anxiety have not been assessed in the present research . depression and anxiety might result from a variety of factors , which require more extensive research . another result of this research was that financial status , independent from the quantity of follicle obtained from ovulation stimulation , showed a reverse correlation with anxiety level after harvesting process . in ozken 's research , also financial status was a factor determining the anxiety in infertile couples . regarding financial pressures resulting from high costs of assisted reproduction treatments , which lead to the success of only 35% for each cycle , women 's concerns over the success of results in fertilization as an outcome of infertility of the partner detected after harvesting process while insemination in laboratory are not far from expectation . nevertheless , although women are healthy in terms of fertility in couples undergoing male factor infertility treatment , effort to have babies not by normal methods and facing difficult , costly treatment protocols might threaten their evaluation of their feminine role , thus reducing their self - esteem . another finding of this research was the existence of correlation between depression level after treatment process and anxiety level at the first stage , while this correlation was observed between depression level after treatment process and anxiety level at the second stage . this finding indicates that women , who enter the process of treatment with a higher anxiety level are prone to a higher level of depression ; therefore necessary measures should be taken to prevent it . one of this study 's limitations was that mental health assessment was conducted after starting the primary stages of the treatment therefore , the stress of this stage could affect the mental health during the whole process . this study showed that fertile women who undergo assisted reproduction treatments do not start the treatment process in an appropriate mental health condition , and this condition would continue during the whole process until egg harvesting . therefore , it is suggested that before starting the treatment the mental health of these women should be evaluated using screening tools and counseling sessions would be applied for vulnerable women .

the present study was designed to explore the effects of gas6 and axl gene polymorphisms on adiposity , systemic inflammation , and insulin resistance in adolescents . after multistage sampling from the data of the taipei children heart study - iii , we collected 358 boys and 369 girls with an average age of 13.3 years . we genotyped the adolescents ' gas6 rs8191973 , gas6 rs8191974 , axl rs4802113 , and axl rs2304232 polymorphisms . significantly higher body mass index ( bmi ) , waist circumference ( wc ) , and hscrp levels were found in boys with the gg genotype of gas6 rs8191974 than a allele carriers ; higher il-6 and insulin levels and increased homa - ir were found in boys with the gg genotype of axl rs2304232 than the a allele carriers . there was a significant difference in hscrp levels of boys with the tt , tc , and cc genotypes of axl rs4802113 . boys with both the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 exhibited higher bmi , wc , il-6 , and hscrp levels than the boys carrying both the c allele of the gas6 rs8191973 and the a allele of the gas6 rs8191974 . in conclusion , gas6 and axl polymorphisms are associated with adiposity , systemic inflammation , and insulin resistance in adolescents , especially in boys . introduction : the process of assisted reproductive treatment is a stressful situation in the treatment of infertile couples and it would harm the mental health of women . fertile women who started infertility treatment due to male factor infertility have reported to experience less stress and depression than other women before the assisted reproductive process but considering the cultural and social factors and also the etiology of the assisted reproductive process , it could affect the metal health of these women . therefore , this study was conducted to evaluate the mental health of fertile women who undergo assisted reproductive treatment due to male factor infertility.materials and methods : this study was a prospective study on 70 fertile women who underwent assisted reproductive treatment due to male factor infertility . the exclusion criterion was to stop super ovulation induction . to assess mental health , anxiety and depression dimensions of the general health questionnaire were used . before starting ovulation induction and after oocyte harvesting , the general health questionnaire was filled by women who were under treatment . data were analyzed using multi - variable linear regression , paired t - test , and chi-square.results:the results showed that the mean score of depression and anxiety before ovulation induction and after oocyte harvesting were not significantly different ; but the rate of mental health disorder in the depression dimension was significantly decreased after oocytes harvesting ( 31.7% vs. 39.7% ) . also , there was a significant relation between the level of anxiety and depression before ovulation induction and after oocyte harvesting ( p < 0.05 ) . the anxiety level after oocyte harvesting had a positive and significant correlation with the economic situation ( p < 0.05).conclusion : this study revealed that the process of assisted reproductive treatment does not affect the mental health in fertile women independently , but these women start assisted reproductive process with high levels of depression and anxiety . therefore , prior to the assisted reproductive treatment mental health consultation is needed .

balance impairment is an important fall - risk factor , and increases in range of postural sway in the mediolateral direction in older adults are associated with increased fall - risk and rates . postural sway has been shown in older adults to be strongly related to other measures of balance . multivariate analysis reveals serum vitamin d levels as an independent variable associated with postural sway . in individuals with suboptimal levels of vitamin d , epidemiological studies have shown that vitamin d levels show seasonal variation [ 7 , 8 ] . lowest levels of serum vitamin d are recorded towards the end of winter , approximately four weeks after the shortest day of the year . overall , vitamin d supplementation did not reduce rate of falls ( rar 1.00 , 95% ci 0.90 to 1.11 ; seven trials ; 9324 participants ) or risk of falling ( rr 0.96 , 95% ci 0.89 to 1.03 ; 13 trials ; 26 , 747 participants ) but may do so in people with lower vitamin d levels before treatment . older adults are at risk for lower levels of serum vitamin d because of age - related changes in uvb absorption and skin capacity to synthesize vitamin d , reduction in activation in the kidneys , and reduced expression of vitamin d receptors in tissues . there are many factors affecting fall - risk for older individuals , and although these may be different for inside and outside falls , strength and balance remain two important physical fall - risk factors . a recently published overview of the literature supports an assertion that age - related changes in postural reactions may be related to vitamin d status - mediated through either central nervous system integration or antigravity muscles as the effectors in postural responses . despite changes in vitamin d across the seasons , muscle strength in the quadriceps muscles has been shown to remain stable . the relationship of postural sway to any potential increased winter fall rate and decreased levels of vitamin d has not been investigated . the winter season sees an increase in injuries from falls and in the number of accidental deaths from falls . fracture rates from falls in older adults also increase at the end of the winter season , following two to eight weeks after the nadir in serum vitamin d levels . some studies report an increased rate of falls , for both inside and outside falls [ 13 , 14 ] ; however , significant seasonal variation in fall rates was not found in a three - year study while , in a second study , seasonal variation in fall rates was reported in women but not in men . coincident static balance changes with any potential increased fall rates in winter have not been previously reported . the data presented here forms part of a larger study , from which two other papers with the same clinical trial registration have been published [ 12 , 16 ] . the aim of this study was to determine differences in static balance ( postural sway ) , vitamin d , incidence of falls , and type of fall serially at the end of each season over a 12-month period , in older community - living adults . we hypothesised that postural sway , falls , and vitamin d would show a seasonal variation and that there would be an inverse relationship between vitamin d and the other variables . at the end of consecutive seasons , static balance , vitamin d status , and fall rate were measured within a longitudinal study design . no intervention was implemented by study researchers , so that the study could identify natural variations that occur over the seasons . data was collected over a three - week period in each season from end of spring 2009 to the end of spring 2010 , with collection of data timed to coincide with expected peaks and troughs in serum vitamin d levels in australia at latitude 41 degrees south ( tasmania ) . after each assessment , participants were given an appointment for the next collection block in three months ' time . independently living community - dwelling adults aged between sixty and eighty - five years were recruited through local print media and community clubs . daily intake of oral supplementation of vitamin d of greater than 800 international units was also an exclusion criterion . participants were also excluded if they had a history of neurological disease and were withdrawn if they suffered a medical condition while participating in the study that would impact on their ability to perform the physical tests . liver and kidney disease both impact vitamin d metabolism , and any potential participants with either of these conditions were excluded . a priori sample size calculation was based on a previous study reporting mediolateral sway range in a sample of community - dwelling older adults , and this indicated a minimum requirement of 81 completed participants ( minimum effect size 2.5 mm sway ; standard deviation ( sd ) 8 mm ; power 0.8 , alpha 0.05 ) . ninety - eight participants were recruited with the anticipation of a 15% drop - out rate . this repeated measures cohort study was designed to be able to detect differences in postural sway . this project received ethical approval from the human research ethics committee ( tasmania ) network ( h0010561 ) . postural sway range in the medio - lateral sway direction was measured using a force platform ( amti accugait pjb 101 , massachusetts , usa ) for thirty seconds under conditions of eyes open and closed , as well as with the additional challenge of using a 6.5 cm foam cushion ( eyes open and eyes closed ) ( airex elite balance pad ag , switzerland ) . participants were asked to remain stationary with their arms by their sides and look straight ahead while standing on the force platform . foot position ( bare feet ) was standardised with heels 4 cm apart using marked placement for the feet to ensure repeatability between testing occasions . venous blood samples were collected and clotted , then centrifuged ( 1610 relative centrifugal force ) for 15 minutes . serum concentration of 25-hydroxy vitamin d was measured by direct , competitive chemiluminescent immunoassay in a commercial accredited laboratory , using liaison method ( diasorin inc . , participants received an individually coded calendar for 12 months of the study on which to report any falls and associated details by date , including information regarding the location and cause of the fall . information about the type of fall , any injuries that resulted , and if medical attention was sought was recorded . this study utilised the who definition of a fall as an event which results in a person coming to rest inadvertently on the ground or other lower level . annual cyclic trends were investigated by fitting a sine wave formula to data for postural sway and vitamin d , with the amplitude of the seasonal variation ( in percentage change ) and the annual mean values were estimated using repeated measures nonlinear regression , adjusted for age and gender . for secondary analysis the three monthly data were interpolated linearly to estimate intermediate values to correspond with the observed fall incident data . mixed - methods poisson regression was used to determine associations between falls , fall injuries , and postural balance and vitamin d. the association between postural balance and vitamin d and season was estimated using mixed - methods linear regression , adjusted for age , gender , and strength . for comparison , seasonal data for falls was grouped into autumn and winter and compared to spring and summer . physical activity using the champs questionnaire and muscle strength using the physiological profile assessment tools were recorded , and this information has been published elsewhere but forms an integral part of the meta - analysis of this paper . data from eighty - eight participants ( 70% females ) are included in the final analysis . five people did not attend appointments , and five people could not complete testing because of medical events . the participants had a mean ( sd ) age of 69.2 ( 6.5 ) years and body mass index 27.4 ( 3.9 ) kg m. all participants were living in their own homes independently , with only 10% being sole occupants . common chronic controlled health conditions included cardiovascular disease ( 39% ) and arthritis ( 14% ) . twenty - six percent of the participants reported the use of more than 4 medications . all four balance measures had the highest sway scores ( poorest balance ) at the first end of spring measurement . all other seasonal measures were significantly different from this first time point ( all p < 0.05 ) , but no subsequent significant difference was seen after any other seasonal measures , indicating a lack of seasonal variation in this outcome ( p > 0.05 ) . no associations between postural sway and vitamin d were observed ( all p > 0.05 ) . increased postural sway was associated with fall injuries ( irr 1.59 ( ci 1.14 to 2.24 ) ( p = 0.007 ) but not fall rates ( irr 1.36 ( ci 0.95 to 1.97 , p = 0.09 ) . there was 15% variation in this variable over the year , with a peak at the end of summer and the lowest values at the end of winter . seventy - five percent of fall diaries were posted on schedule while the remaining diaries were returned at the subsequent assessment appointment , resulting in a compliance of 100% . thirty - three percent of the cohort ( 29 people ) fell at least once , with 10% of the whole group falling multiple times ( 8 people ) . over the duration of the study , 48 falls were recorded : 14 of these occurred inside the house , and 34 occurred outside . six falls were due to fainting or dizziness and forty due to trip - related events , with one categorised as being pushed over ( by a horse ) and one not able to be categorised . twenty - eight falls resulted in injury , with only four requiring medical treatment ( including one fracture ) . further details on season variation in location and type of fall are provided in table 2 . there were significantly fewer falls during spring than any other season ( p = 0.01 ) , with no other differences between the seasons recorded . when falls data were combined from autumn and winter seasons and compared to the combined spring and summer seasons , there were more falls reported in the combined autumn and winter seasons ( 30 compared to 18 ) . less injuries from falls were recorded in spring than any other season ( p = 0.02 ) , with no other seasonal differences recorded . this is the first cohort study to determine that no seasonal variation of postural sway occurs across 12 months in our population . significant seasonal variation in serum vitamin d levels , with higher serum levels in summer , was recorded . there was a significant relationship between postural sway and the number of injurious falls observed , with lower values for sway range ( i.e. , better stability ) associated with less fall injuries . a significant learning effect was seen with all measures of mediolateral postural sway with time point one having larger ranges than all of the other time points ( all p > 0.05 ) . further to this , there was no seasonal variation in postural sway under any of the four static balance test conditions measured ( eyes open and closed on a firm surface or foam surface ) . postural sway range has been used to identify those people with balance impairment , and it may be useful in describing the fall - risk status of a particular individual . our data indicates that postural sway does not appear to be subject to changes across the year within a participant . it has been suggested that this measure is important in describing sensorimotor deficits or disability rather than functional abilities and hence may not be subject to changes that may occur due to altered patterns of activity or sunlight exposure seen seasonally . the stability of this measure in a cohort across a year provides important information for researchers planning interventions designed to impact postural sway and clinicians who are measuring the effectiveness of their interventions . mediolateral sway range has been shown to be an independent fall - risk factor for indoor falls . in our study , a lower proportion of falls occurred indoors ( 29% ) compared to outdoors ( 71% ) , and although this is similar to other studies where more healthy samples have been reported as having a greater proportion of falls being outdoor falls ( 74% ; ) , it may be a reason why no association between sway range and fall incidence was seen . a trend for this association was evident and a larger sample size may have found a significant relationship between these two variables , as this study was powered to determine mediolateral sway changes not fall rates . an association between increased sway range and rate of injurious falls was recorded in our study , reinforcing the importance of this measure for those most at risk of injury . overall there was no significant relationship found between postural sway and vitamin d. as increased postural sway is linked to low levels of vitamin d , it may be that the levels of vitamin d were sufficient for this parameter even at their lowest levels in participants in this study and did not influence postural sway . if this threshold situation is true , it may be that seasonal variation in postural sway may be present in a population with much lower levels of vitamin d , but this is outside the scope of the current study . annual rates of falling for adults over 65 have been reported up to 40% , and although our cohort includes some adults between the ages of sixty and sixty - five , with a mean age of 69 years and a fall rate of thirty - three percent , our population appears to be representative of older community - dwelling adults in terms of fall rate . fall rates in older - old adults ( over 75 years ) have been shown to vary seasonally , but consistent data for the general population of healthy older community - dwelling adults has not been previously reported . one previous study grouped the peak seasons of winter and autumn together and found differences in fall rates . manipulation of our data in a similar way reveals that there were more falls during the autumn / winter half year compared to spring / summer ( table 2 ) . these seasonal differences may be related to intrinsic factors that may be subject to seasonal variation ( e.g. , vitamin d , physical activity , and muscle strength ) as well as seasonally related environmental factors ( e.g. , weather , temperature ) . further research needs to investigate interventions to address potentially modifiable factors to reduce the increased falls risk in the autumn / winter period . our data indicates a higher rate of falls in summer than has previously been reported , perhaps due to activity characteristics of our cohort . summer and winter falls differ between the genders , with more men falling due to slips in winter and more women falling due to trips in summer . the high proportion of women in our study ( 79% ) may be a factor in the high rate of summer falls observed . another factor to consider is the relationship between fall status and vitamin d. although 60 nmol / l has been determined to be the cutoff for fall - risk function , 16/48 falls ( 33% ) occurred in participants in this study whose vitamin d was above that cutoff level . this may be explained by the higher proportion of summer time falls observed in our study . during summer months , with longer hours of daylight at this latitude , a large proportion of the falls occurred in the outside ( 13 of the 14 falls 93% ) ( table 2 ) . by contrast , in winter out - of - doors fall rate was reduced to 64% , indicating a higher winter time proportion of inside falls . for our generally healthy study population , it is likely that our participants were engaged in outdoor activities with higher associated risk of falls during the warmer weather ; for example , several summer falls occurred while bushwalking . fall injuries , especially fractures , have been found to increase in winter ; this includes both inside ( hip ) and out - of - doors falls ( wrist ) . studies in the area of seasonal variation in fracture rates provide good evidence for increased fracture rates from falls in winter , but these appear in populations with older participants than ours ( mean age over 75 years ) [ 8 , 13 ] . our study recorded few injuries that required medical attention , hence making it difficult to compare serious fall injury data . although this study aimed to recruit independently living community - dwelling older adults , bias in the sample may be present , as volunteers to this type of research project may be more robust than the community members at large . the lack of more frail subgroups , including those with cognitive impairment , depression , or using walking aids , limits the generalisability of this study . this study provides some evidence that measurements of postural sway may be affected by some effect of test - retest learning , and this needs to be considered in future research . this is the first study to investigate the effects of season and postural sway and found that postural sway remained stable over the 12 months . this study provides important evidence for clinicians and researchers that postural sway remains stable over an annual cycle but may be influenced by a learning effect . since the dawn of history , nature and natural sources , such as plants , animals , microbes , and minerals have remained a veritable source of bioactive compounds with medicinal values . among these sources , plants have been the most explored and exploited for their bioactive medicinal components . lead compounds or templates for the rational development of drugs are of more specific efficacies and fewer side effects ( 1 ) . one of the botanicals of interest in which the abortifacient claims in the folk medicine have been substantiated by scientific evidence ( 2 ) but with no documentation in the open scientific literature on its bioactive abortifacient agent(s ) is senna alata . senna alata ( linn . ) roxb ( leguminosae ) , also known as craw - craw plant or ringworm plant ( english ) , asunwon oyinbo ( yoruba - western nigeria ) , nelkhi ( igbo - eastern nigeria ) , filisko or hantsi ( hausa - northern nigeria ) is an erect tropical , annual herb which grows up to 0.15 m high . the fruit is a pod , while the seeds are small and square in shape . the plant has been claimed to be used in the management of several diseased conditions such as hepatitis , dermatitis , jaundice , gastroenteritis , eczema , constipation and diarrhoea ( 3 , 4 ) . previous studies have shown that s. alata have antifungal , antibacterial and antioxidant activities ( 3 , 58 ) . furthermore , yakubu et al . ( 2 ) reported that the aqueous leaf extract of the plant contained saponins ( 1.22% ) , flavonoids ( 1.06% ) , cardiac glycosides ( 0.20% ) , phenolics ( 0.44% ) , alkaloids ( 0.52% ) , cardenolides and dienolides ( 0.18% ) . aqueous leaf extract of s. alata has also been scientifically validated for its acclaimed use as an abortifacient ( 2 ) . however , there is no study in the open scientific literature that has reported on the exact bioactive abortifacient agent(s ) in s. alata leaves . therefore , the present study aimed to validate the speculation that the alkaloids in aqueous extract of s. alata leaves are responsible for its abortifacient activity . the focus on alkaloid was a follow - up from our previous study that speculated on alkaloids to be responsible for the abortifacient activity of the crude extract of s. alata leaves and from several studies that implicated the phytochemical property of colchicine , quinazoline alkaloids , e.g. vasicine and vasicinone in several botanicals such as xylopia aethiopica , peganum harmala epigeal parts , areca catchu nuts and gloriosa superba roots as abortifacient bioactive agents and/or their role in the contraction and relaxation of uterine muscles ( 912 ) . the plant leaves , obtained from herb sellers at oja tuntun , ( new market ) in ilorin , nigeria , was authenticated at the herbarium unit of the forestry research institute of nigeria ( frin ) in ibadan , nigeria . a voucher specimen ( fhi 10845 ) was deposited at the herbarium of the institute . assay kits for both glucose and cholesterol were products of randox laboratories , ltd , united kingdom , while those of progesterone , follicle stimulating and luteinizing hormones were products of inteco uk ltd , united kingdom . thin layer chromatographic ( t lc ) plates and silica gel were products of merck ( darmstadt , germany ) . para - nitrophenyl phosphate and other reagents were products of sigma- aldrich inc . , st . male and female wistar rats ( rattus norvegicus ) weighing 178.913.07 and 143.99 1.21 g , respectively , were obtained from the animal holding unit of the department of biochemistry , university of ilorin , ilorin , nigeria . the animals which were housed individually in plastic cages and placed in a well - ventilated room ( temperature : 28 - 31c ; photoperiod : 12 hr natural light and 12 hr darkness ; humidity : 50 - 55% ) were provided with unrestricted access to rat pellets ( bendel feeds and flour mills , ewu , nigeria ) and water . the animals were also handled according to the guidelines of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ets 123 ( 13 ) . the leaves of senna alata which were oven - dried at 40c for 48 hr were pulverised using a mikachi blender ( mk-1830 , china ) . alkaloids were extracted from the powder according to the procedure described by manske ( 14 ) . a known amount ( 500 g ) of the powder was extracted in 1.2 l of hexane for 72 hr and filtered with whatman no the hexane extract ( containing fats , oils , terpenes , waxes ) were discarded and the resulting residue extracted again in 1.2 l of methanol for a week and subsequently filtered . the filtrate was evaporated using a rotavapor ( r110 , gallenkamp , england , uk ) and the process was repeated two more times . the three filtrates were combined , concentrated to give a methanolic , green slime ( 90 g ) which was treated with 1 m hcl and then basified by adding 5 m naoh with continuous stirring until a cloudy precipitate appeared . a known volume ( 500 ml ) of chloroform and 200 ml of 1 m nacl were added and the process was repeated three more times . equal volumes ( 150 ml ) of 1 m nacl and 5 m naoh were added to the organic layer in a separating funnel after which the mixture was evaporated to yield brownish - black slurry ( 18 g ) of alkaloids that corresponded to a yield of 3.60% that was used for subsequent experiments . the procedure described by singh and sahu ( 15 ) was adopted for the preparation of thin layer chromatography ( tlc ) plates . furthermore , about 10 l of the test solution ( extract ) was spotted onto the thin layer plate using a micropipette after which the plates were developed in chloroform : methanol ( 10:2 ) in which 0.01 g / ml of butylated hydroxyl toluene and butylated hydroxyl anisole were added to prevent oxidation ( which may lead to increase in the number of bands with time ) ( 16 ) . the relative values related to the solvent front ( rf ) of the spots were also computed . twenty - four pregnant rats were allocated in a complete randomize design to four groups ( a , b , c and d ) consisting of six animals each . animals in group a ( controls ) , orally received 0.5 ml of distilled water with the aid of an oropharyngeal cannula while those in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration was done once daily from day 10 until day 18 of pregnancy ( period of organogenesis in wistar rats ) ( 18 ) . by the 19th day ( 24 hr after their last dose ) the following parameters were recorded / computed : number of live fetuses ; number of dead fetuses ; average weight of live foetuses ; survival ratio ( % ) = ( number of live fetuses/ number of live + dead fetuses ) 100 ; number of rats that aborted ; percentage of rats that aborted= ( number of rats that aborted / number of rats assessed ) 100 ; number of rats with vaginal bleeding ; number of implantation sites ; number of corpora lutea ; implantation index=(total number of implantation sites / number of corpora lutea ) 100 ; pre - implantation loss=(number of corpora lutea number of implantation sites / number of corpora lutea ) 100 ; post - implantation loss= ( number of implantation sites number of live fetuses / number of implantation sites ) 100 ; number of resorption sites = number of implantation sites in the control animals number of implantations in the test animals ; resorption index=(total number of resorption sites / total number of implantation sites ) 100 . the weights of the animals both before pairing and prior to sacrifice , as well as feed and water intake were also recorded . the in vivo estrogenic / anti - estrogenic response of the rats to the alkaloids was evaluated by adopting the procedure described by kanno et al . twenty - four ovariectomized , female rats ( 159.677.12 g ) were allocated to four groups ( a , b , c and d ) of six animals per group . group a ( controls ) , orally received 0.5 ml of distilled water while animals in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration which commenced from the eighth day of ovariectomy , lasted for another seven days . on day 16 , the weights of the animals were determined prior to sacrifice and uterine to body weight ratio was computed . the uterine protein , glucose , cholesterol , and alkaline phosphatase activity were determined using standard procedures ( 2023 ) . the serum and uterine homogenates were prepared according to the procedures described by yakubu and bukoye ( 24 ) . the procedures outlined in the manufacturer 's protocol were adopted for the quantitative determination of progesterone , follicle stimulating and luteinizing hormones in the serum of the animals . data which were expressed as the meansd of six independent replicates were statistically analyzed using one - way analysis of variance and duncan multiple range test . the plant leaves , obtained from herb sellers at oja tuntun , ( new market ) in ilorin , nigeria , was authenticated at the herbarium unit of the forestry research institute of nigeria ( frin ) in ibadan , nigeria . a voucher specimen ( fhi 10845 ) was deposited at the herbarium of the institute . assay kits for both glucose and cholesterol were products of randox laboratories , ltd , united kingdom , while those of progesterone , follicle stimulating and luteinizing hormones were products of inteco uk ltd , united kingdom . thin layer chromatographic ( t lc ) plates and silica gel were products of merck ( darmstadt , germany ) . para - nitrophenyl phosphate and other reagents were products of sigma- aldrich inc . , st . male and female wistar rats ( rattus norvegicus ) weighing 178.913.07 and 143.99 1.21 g , respectively , were obtained from the animal holding unit of the department of biochemistry , university of ilorin , ilorin , nigeria . the animals which were housed individually in plastic cages and placed in a well - ventilated room ( temperature : 28 - 31c ; photoperiod : 12 hr natural light and 12 hr darkness ; humidity : 50 - 55% ) were provided with unrestricted access to rat pellets ( bendel feeds and flour mills , ewu , nigeria ) and water . the animals were also handled according to the guidelines of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ets 123 ( 13 ) . the leaves of senna alata which were oven - dried at 40c for 48 hr were pulverised using a mikachi blender ( mk-1830 , china ) . alkaloids were extracted from the powder according to the procedure described by manske ( 14 ) . a known amount ( 500 g ) of the powder was extracted in 1.2 l of hexane for 72 hr and filtered with whatman no . 1 filter paper . the hexane extract ( containing fats , oils , terpenes , waxes ) were discarded and the resulting residue extracted again in 1.2 l of methanol for a week and subsequently filtered . the filtrate was evaporated using a rotavapor ( r110 , gallenkamp , england , uk ) and the process was repeated two more times . the three filtrates were combined , concentrated to give a methanolic , green slime ( 90 g ) which was treated with 1 m hcl and then basified by adding 5 m naoh with continuous stirring until a cloudy precipitate appeared . a known volume ( 500 ml ) of chloroform and 200 ml of 1 m nacl were added and the process was repeated three more times . equal volumes ( 150 ml ) of 1 m nacl and 5 m naoh were added to the organic layer in a separating funnel after which the mixture was evaporated to yield brownish - black slurry ( 18 g ) of alkaloids that corresponded to a yield of 3.60% that was used for subsequent experiments . the procedure described by singh and sahu ( 15 ) was adopted for the preparation of thin layer chromatography ( tlc ) plates . furthermore , about 10 l of the test solution ( extract ) was spotted onto the thin layer plate using a micropipette after which the plates were developed in chloroform : methanol ( 10:2 ) in which 0.01 g / ml of butylated hydroxyl toluene and butylated hydroxyl anisole were added to prevent oxidation ( which may lead to increase in the number of bands with time ) ( 16 ) . the relative values related to the solvent front ( rf ) of the spots were also computed . twenty - four pregnant rats were allocated in a complete randomize design to four groups ( a , b , c and d ) consisting of six animals each . animals in group a ( controls ) , orally received 0.5 ml of distilled water with the aid of an oropharyngeal cannula while those in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration was done once daily from day 10 until day 18 of pregnancy ( period of organogenesis in wistar rats ) ( 18 ) . by the 19th day ( 24 hr after their last dose ) the following parameters were recorded / computed : number of live fetuses ; number of dead fetuses ; average weight of live foetuses ; survival ratio ( % ) = ( number of live fetuses/ number of live + dead fetuses ) 100 ; number of rats that aborted ; percentage of rats that aborted= ( number of rats that aborted / number of rats assessed ) 100 ; number of rats with vaginal bleeding ; number of implantation sites ; number of corpora lutea ; implantation index=(total number of implantation sites / number of corpora lutea ) 100 ; pre - implantation loss=(number of corpora lutea number of implantation sites / number of corpora lutea ) 100 ; post - implantation loss= ( number of implantation sites number of live fetuses / number of implantation sites ) 100 ; number of resorption sites = number of implantation sites in the control animals number of implantations in the test animals ; resorption index=(total number of resorption sites / total number of implantation sites ) 100 . the weights of the animals both before pairing and prior to sacrifice , as well as feed and water intake were also recorded . the in vivo estrogenic / anti - estrogenic response of the rats to the alkaloids was evaluated by adopting the procedure described by kanno et al . twenty - four ovariectomized , female rats ( 159.677.12 g ) were allocated to four groups ( a , b , c and d ) of six animals per group . group a ( controls ) , orally received 0.5 ml of distilled water while animals in groups b , c and d orally received 0.5 ml of the alkaloids corresponding to 250 , 500 and 1000 mg / kg body weight , respectively . the administration which commenced from the eighth day of ovariectomy , lasted for another seven days . on day 16 , the weights of the animals were determined prior to sacrifice and uterine to body weight ratio was computed . the uterine protein , glucose , cholesterol , and alkaline phosphatase activity were determined using standard procedures ( 2023 ) . the serum and uterine homogenates were prepared according to the procedures described by yakubu and bukoye ( 24 ) . the procedures outlined in the manufacturer 's protocol were adopted for the quantitative determination of progesterone , follicle stimulating and luteinizing hormones in the serum of the animals . data which were expressed as the meansd of six independent replicates were statistically analyzed using one - way analysis of variance and duncan multiple range test . the alkaloids yielded 0.30 g which corresponds to 1.50% of the starting material of 500 g. the five different spots gave rf values of 0.28 , 0.33 , 0.39 , 0.47 and 0.55 . the spots gave positive reaction with both meyer 's and wagner 's reagents producing a creamy precipitate and reddish - brown spots on a grey background ( tlc ) . the alkaloid truncated the development of the fetuses as none of them survived in the experiment groups as against the average number of live fetuses of 10.26 in the distilled water - treated control animals ( table 1 ) . the average weight of live fetuses in the controls was 4.92 g against none in all the extract - treated animals . the percentage of fetal death in the 250 , 500 and 1000 mg / kg body weight of the alkaloid - treated animals was 6.03 , 6.00 and 6.50 , respectively . there was neither episode of abortion nor vaginal bleeding in all the alkaloid - treated animals . both the number of implantation sites and corpora lutea decreased significantly ( p < 0.05 ) . while the implantation index was similarly high in all the alkaloid - treated animals , the resorption index , as well as pre- and post- implantation losses were many fold higher than the controls ( table 1 ) . although , all the pregnant rats gained weight at the end of the experimental period ( final maternal weight ) compared with their weight prior to pairing with the males ( initial maternal weight ) ( table 1 ) , the weight gained by the alkaloid - treated animals was about 50% less than the control animals . furthermore , the feed and water intake by the animals treated by different doses of the alkaloid decreased significantly ( p < 0.05 ) . effect of the alkaloids from senna alata leaves on some abortifacient parameters of pregnant rats values are expressed as meansd of six independent determinations ; test values carrying superscripts different from the control for each parameter in the same row are significantly different ( p < 0.05 ) . maternal weights of the animals before pregnancy were compared with their corresponding weights after pregnancy for each treatment group ( p < 0.05 ) all different doses of the alkaloids significantly ( p < 0.05 ) decreased the serum concentrations of the follicle stimulating hormone , luteinizing hormone and progesterone in the pregnant animals ( table 2 ) . effect of alkaloids from senna alata leaves on some female reproductive hormones of pregnant wistar rats values are expressed as meansd of six independent determinations ; test values carrying superscripts different from the control for each parameter in the same row are significantly different ( p < 0.05 ) the alkaloids also decreased ( p < 0.05 ) the absolute weight of the uterus , computed uterine to body weight ratio , and concentrations of uterine glucose and cholesterol ( table 3 ) . in contrast , the concentration of uterine protein and the activity of alkaline phosphatase increased significantly ( p < 0.05 ) . furthermore , the alkaloids did not provoke vaginal opening or cornification in any of the animals ( table 3 ) . effect of alkaloids from senna alata leaves on some indices of oestrogenicity in pregnant rats values are expressed as meansd of six independent determinations ; test values carrying superscripts different from the control for each parameter in the same row are significantly different ( p < 0.05 ) analysis of the tl chromatogram indicated that the mixture consisted of five alkaloids as evidenced by the creamy precipitates and reddish- brown spots produced with meyer 's and wagner 's reagents , respectively . in the present study , alkaloids from s. alata leaves significantly affected the fetal and maternal parameters of the animals . for instance , the death of the fetuses in the alkaloid - treated pregnant rats may not only suggest inhibition of mitotic division of the fetuses ( 25 ) , since the animals were exposed during the period of organogenesis , but may also show its relevance to the reduction in the concentrations of gonadotropins and progesterone in the present study . ( 2 ) on the aqueous leaf extract of the plant where only the 500 and 1000 mg / kg body weight of the extract produced fetal death . furthermore , the absence of abortion and vaginal bleeding in the alkaloid - treated animals which were hitherto observed with the crude extract in our previous study suggest zero abortifacient activity for the alkaloid . therefore , the abortifacient activity of the aqueous extract of the plant leaf reported earlier ( 2 ) is not due to the alkaloidal content alone , but to some other phytochemicals such as saponins and flavonoids which may act synergistically or additively to produce the desired result . it is interesting to note that both the number of implantation sites and corpora lutea decreased in the alkaloid - treated animals when the process of implantation ought to have been completed ( implantation takes place normally within 5 - 6 days post - coitus in rats ) before the exposure of the animals to the alkaloid mixture ( treatment commenced from day 10 of pregnancy ) . the reason for this decrease is not immediately known but may not be unconnected with the consequence of some general hormonal effect ( reduced progesterone ) and/or absence of conceptuses growth ( 26 ) . it is also possible that blastocytes were not activated and well - positioned before implantation , probably due to impaired muscular activity of the uterus ( 27 ) . the implantation index and pre - implantation loss evaluates the number of blastocysts implanted in the uterus while the resorption index and post - implantation loss relate to the number of implanted blastocysts and those that have not developed ( 18 , 28 ) . therefore , the high implantation index , and pre- and post- implantation losses suggest that pregnancy was interrupted by the alkaloid , probably creating an environment that was not conducive for the fertilized eggs . normally , an abortion in most cases is accompanied by vaginal bleeding and when this is absent , the resorption will increase . therefore , the increase in the resorption index in the alkaloid- treated animals confers antifertility effects ( such as anti - implantation , anti - blastocystic and antizygotic ) of the alkaloid . the alkaloid did not exhibit complete abortifacient effect since the pregnant animals exposed to the alkaloid presented with closed vagina just like the controls . the findings in the present study is similar to the report by elbetieha et al . ( 29 ) where the administration of 200 , 400 and 800 mg / kg body weight of ethanolic extract of salvia fruticosa did not cause pregnancy failure but increased the number of resorption in the pregnant rats . the alkaloids reduced the sense of taste and appetite of the animals as evidenced by the decrease in feed and water intake . such reduction may account for the decrease in the computed percentage gain of maternal weight and may also be a consequence of impaired growth and development of the uterine contents ( 30 ) . all these findings except those of feed and water intake , as well as maternal weight gain , contrast the previous report by yakubu et al . it is well - known that for the implantation of the fetus and sustenance of pregnancy , an exact equilibrium must exist between the secretion of estrogen and progesterone ; this regulation is controlled by luteinizing and follicle stimulating hormones ( 31 ) . thus , the reduction in the gonadotropins may equally be responsible for the reduced concentration of progesterone in the present study and this may not only account for the death of the fetuses , but also for the increase in the resorption sites . the reduction in progesterone may suggest impaired endometrium function , which will adversely affect the normal secretion of special proteins required to nourish an implanted fertilized egg , and consequently , pregnancy failure . thus , it is possible that the alkaloids posses anti - gonadotropic and anti - progestogenic activities which are inimical to the continued development of the fetuses . many plant extracts with anti - fertility properties are known to exhibit estrogenic activity by increasing protein synthesis , uterine weight , water uptake and retention of fluid leading to ballooning of the uterus , uterine content of glucose , cholesterol , glycogen and alkaline phosphatase activity , thereby changing the uterine milieu and creating non - receptive conditions in the uterus ( 32 ) . therefore , the contrasting effects the alkaloids on some of these parameters in the present study suggest that estrogenic activity was not total but selective . the alkaloids exhibited more anti - oestrogenic activity ( 71.43% ) than oestrogenic ( 28.57% ) . overall , the alkaloid from s. alata leaves at the oral doses of 250 , 500 and 1000 mg / kg body weight on daily basis from days 10 until day 18 post - coitum exhibited several potential effects on the maternal and fetal outcomes of pregnant rats ( anti - implantation , anti - gonadotropic , anti - progesteronic , selective estrogenic , embryonic resorption and fetotoxic activities ) , but it could not induce abortion in the animals . therefore , the alkaloids may not alone be responsible for the abortifacient effects of the crude extract of s. alata which had been reported earlier . finally , work is in progress on isolating the other phytochemicals ( saponins and flavonoids ) and evaluating their synergistic effects in pregnant animals .

introduction . low serum vitamin d levels are associated with increased postural sway . vitamin d varies seasonally . this study investigates whether postural sway varies seasonally and is associated with serum vitamin d and falls . methods . in a longitudinal observational study , eighty - eight independently mobile community - dwelling older adults ( 69.7 7.6 years ) were evaluated on five occasions over one year , measuring postural sway ( force platform ) , vitamin d levels , fall incidence , and causes and adverse outcomes . mixed - methods poisson regression was used to determine associations between measures . results . postural sway did not vary over the year . vitamin d levels varied seasonally ( p < 0.001 ) , peaking in summer . incidence of falls ( p = 0.01 ) and injurious falls ( p = 0.02 ) were lower in spring , with the highest fall rate at the end of autumn . postural sway was not related to vitamin d ( p = 0.87 ) or fall rates , but it was associated with fall injuries ( irr 1.59 ( ci 1.14 to 2.24 , p = 0.007 ) . conclusions . postural sway remained stable across the year while vitamin d varied seasonally . participants with high values for postural sway demonstrated higher rates of injurious falls . this study provides important evidence for clinicians and researchers providing interventions measuring balance outcomes across seasons . backgroundthe abortifacient claim of senna alata ( s. alata ) was scientifically validated recently with alkaloids speculated to be the bioactive agent . this speculation is yet to be substantiated or refuted by scientific evidence . the present study was aimed to investigate the pregnancy terminating effects of the alkaloids from s. alata leaves.methodstwenty four pregnant rats ( 143.991.21 g ) allocated randomly to four groups : a , b , c and d respectively received , 0.5 ml of distilled water , 250 , 500 and 1000 mg / kg body weight of the s. alata extracted alkaloids orally , once daily from day 10 until day 18 post - coitum . the indices of abortifacient were evaluated at the end of the exposure period . the results were analyzed by both the analysis of variance and duncan 's multiple range test and p < 0.05 was considered as statistically significant.resultsthin-layer chromatographic separation produced five spots with rf values of 0.28 , 0.33 , 0.39 , 0.47 and 0.55 which gave positive reaction with meyer 's and wagner 's reagents , respectively . the number of implantation sites and corpora lutea , as well as the concentrations of fsh , lh , progesterone , weight of uterus , uterine/ body weight ratio , glucose and cholesterol decreased significantly ( p < 0.05 ) whereas the resorption index , pre- and post - implantation losses , uterine protein content and alkaline phosphatase activity increased significantly . none of the alkaloid treated animals presented with provoked vaginal opening or bleeding except fetal deaths . the alkaloid decreased the maternal weight gain , as well as feed and water intake.conclusionoverall , the alkaloids from s. alata leaves exhibited anti - implantation , anti - gonadotropic , anti - progesteronic , embryonic resorptive , feto - maternal toxic activities but not complete abortifacient . the alkaloids alone may not be the sole abortifacient bioactive agent in the leaf extract .

erg protein expression has been recently suggested to be reflective of erg gene rearrangements in prostate cancer ( pca ) documenting remarkable concordance between the two [ 16 ] . the rearrangements between the androgen receptor - regulated gene tmprss2 ( 21q22.3 ) and members of the ets family member of transcription factor gene , most commonly erg ( 21q22.2 ) , are among the most common genetic alterations detected in prostate cancer [ 711 ] . erg gene rearrangements have been detected in roughly half ( 4060% ) of pca of surgical cohorts compared to a rate of 12%15% in incidental or watchful waiting cohorts [ 7 , 1218 ] . previous studies investigating the prognostic significance of erg gene rearrangements have revealed mixed results [ 1922 ] . however , it is becoming more evident that erg gene rearrangements signify a molecular subtype of pca . some studies investigating the significance of erg protein expression in localized pca failed to show association with adverse clinical outcome [ 23 , 24 ] . however , a recent report by our group demonstrated an association of erg expression with lethal disease in patients with unsuspected and advanced / castrate resistant disease who were treated by transurethral resection of prostate . moreover , we documented significant association between erg expression and both gleason score and tumor volume . studies from our group and others have also linked erg status to responsiveness to hormonal therapy , and longer progression time to castration resistant disease , compared to men with no erg expression [ 24 , 25 ] . in the current study , we investigated the association of erg protein expression to clinical - pathological parameters in a cohort of men with localized prostate cancer . the study cohort consisted of 198 patients who were treated by retropubic radical prostatectomy for localized prostate cancer with a mean followup of 4.8 years ( range 015.8 ) . clinical progression was defined as a postoperative serum psa elevation of > 0.2 ng / ml . prostate samples were embedded onto three tissue microarray ( tma ) blocks using a manual tissue arrayer ( beecher instruments , silver spring , md , usa ) . one to nine cores ( average 3.3 ) , 0.6 mm in diameter , were sampled including benign , high grade intraepithelial neoplasia ( hgpin ) , and prostate cancer ( pca ) . after construction , 4 m sections were cut and stained with haematoxylin and eosin on the initial slides to verify the histological diagnosis . briefly , 4 m thick sections from formalin - fixed paraffin - embedded tissue blocks were stained with ventana autostainer . prior to the staining , heat - induced antigen retrieval was carried out by vegetable steamer in sodium citrate antigen retrieval buffer ( 10 mm ph 6.0 ) for 40 minutes and then cooled down to room temperature for about 20 minutes . the slides were incubated for 60 minutes at 37c with erg rabbit monoclonal antibody ( epitomics , clone epr 3864 ) at 1 : 50 dilution . a ventana iview dab detection kit ( ventana tucson , az , usa ) the diagnoses of all tma cores were confirmed by the three study pathologists ( lht , cw , and tab ) . for each patient , the two predominant patterns were sampled and included on the tmas for analysis . erg protein expression was assessed semiquantitatively using 3-tiered system ( 0 , negative ; 1 , low ; 2 , high ) . cases with either 1 or 2 intensity were considered positive based on previous correlation with erg gene rearrangement as detected by fluorescent in situ hybridization ( data not shown ) . the erg antibody was consistently strongly expressed in endothelial cells , which acted as internal control for expression and intensity level . patient characteristics were presented as frequencies and percentages for categorical variables and as means and ranges for continuous variables . chi - square tests were used to test for associations between erg protein expression and gleason score , surgical margin , and pathological stage . the kaplan - meier approach along with the log - rank test was used for the survival analyses to test the association between erg expression and serum psa relapse . in all statistical tests , mean patients ' age of this cohort was 64 years ( range 42.780.5 years ) with average follow - up time of 4.8 years ( range 0.015.8 months ) . table 1 demonstrates patients ' demographics of the study cohort with respect to erg expression . overall , there were no significant differences between the two subgroups ( erg pos & erg neg ) of patients except for pathological stage , with 37% of erg positive tumors detected in pt3 versus 24% in pt2 . to investigate erg expression in different diagnostic categories , we characterized erg expression based on individual cores sampled . when we accounted for foamy type pca morphology , the rate of erg expression was 15/84 ( 17.9% ) , compared to 302/704 ( 42.9% ) in acinar pca ( cases with no foamy type morphology ) ( p < 0.001 ) . there was no difference in high erg intensity between foamy type and acinar pca ( data not shown ) . however , mean intensity level of erg in acinar pca was significantly higher than foamy type pca , 1.01 1.27 versus 0.37 0.83 ( p < 0.001 ) . erg intensity levels in hgpin were comparable to those in foamy type pca but slightly lower ( 0.13 0.56 ) but significantly lower than those in acinar pca ( p < 0.001 ) ( figure 1 ) . erg expression was noted in 106/280 ( 37.8% ) , 175/463 ( 37.8% ) , and 37/108 ( 34.2% ) of gleason scores 6 , 7 , and 810 , respectively . figure 2 demonstrates examples of erg expression in tissue samples and the distribution of erg in relation to gleason score . when investigating relations between erg expression and pathological parameters , there was significant association between erg expression and higher disease stage . in this cohort , erg expression was present in 50/131 ( 38.1% ) patients with pt2 versus 30/55 ( 54.5% ) patients with pt3 ( p = 0.04 ) . a similar association was also noted between erg expression and extra - capsular extension . in this cohort , 52/134 ( 38.8% ) erg positive patients demonstrated organ confined disease versus 29/53 ( 54.7% ) erg positive patients showing extra - capsular extension ( p = 0.04 ) . similar trends were noted between erg expression and seminal vesicle invasion , but this was not statistically significant ( p = 0.10 ) ( data not shown ) . no significant association was noted between overall erg positivity and positive surgical margins ( table 1 ) . similarly , no association was observed with postsurgical psa levels when assessed by univariate or multivariate analysis ( figure 3 ) . although not informative due to limited patients ' numbers , the association between erg expression and higher stage disease was more pronounced in patients with higher gs . in this cohort , none of the patients with gs > 7 and erg positive ( 0/7 ) were of stage pt2 compared to 47% ( 7/15 ) of erg negative patients who were of pt2 stage ( p = 0.02 ) ( table 2 ) . this study reports on the potential significance of erg protein expression in localized prostate cancer . erg gene rearrangements and erg expression have been documented in roughly 50% of localized prostate and locally advanced castrate resistant prostate cancer compared to 12%15% in watchful waiting or incidental cohorts [ 16 , 17 , 22 , 25 , 29 ] . published reports on the significance of erg expression to patients ' outcome are conflicting with some showing association with adverse outcome , while others document no association . some suggest that it indicates a better prognosis [ 16 , 22 , 25 , 2933 ] . however , it is proposed that erg signifies a molecular class of prostate cancer and may play a role in disease progression within those tumors . this pathway is closely linked to increased rate of pten genomic deletions as well as increased erg expression or erg gene rearrangements [ 34 , 35 ] . erg gene rearrangements and erg expression are associated with adverse outcome and lethal disease in watchful waiting or expectant cohorts [ 16 , 25 ] . moreover , patients with erg overexpression demonstrated shorter progression times to castrate resistance , needing surgical intervention ( channel turp ) . in localized prostate cancer , the majority of the reported data suggest no prognostic implication for erg rearrangements or erg overexpression in relation to psa relapse or the recurrence of local disease [ 22 , 36 , 37 ] . however , two earlier reports suggested adverse association of erg rearrangements with psa relapse after radical prostatectomy [ 20 , 31 ] . in nam 's study , yashimoto 's group noted that the adverse prognostic association was linked to pten genomic deletions . of note , a study by reid et al . documented that erg gene rearrangements in addition to pten genomic deletions had actually favorable outcomes compared to pten deletion alone . these observations suggest that the method utilized in determining erg status , the inclusion or exclusion of other genomic aberration , and the cohort chosen may be the reason for the different results obtained in various studies investigating the role of erg in prostate cancer . our results support previous observations showing no prognostic relationship between erg overexpression and clinical outcome in localized prostate cancer . however , in contrast to earlier studies , we document significant association between erg expression and some pathological parameters . in this cohort , patients whose tumors were erg positive were at higher risk of exhibiting extra - prostatic extension and increased disease stage compared to patients whose tumors did not express erg . specifically , all patients with gs > 7 and erg expression showed pt3 stage disease compared to none of pt2 . however , this data is limited by the number of patients within each gs subgroup and need further confirmation . more importantly , in addition to not documenting any clinical prognostic significance for erg expression , we did not observe any association between erg expression and other pathological parameters such as gleason score or surgical margins , which further diminishes any potential prognostic significance for erg expression , at least in localized pca . another issue worth mentioning regarding erg is that , erg gene rearrangements have been previously associated with specific histopathological features and detected more frequently in some morphologic variants of prostate cancer than others . in this study , we confirm lower rates of erg expression in tumors with foamy / xanthomatous morphology which demonstrated lower mean expression intensities compared to acinar pca . the significance of this is not yet known , but suggests that those two types of tumors may be different at the molecular level . in conclusion , this study demonstrates significant association between erg expression and extra - prostatic extension and higher pathological stage in localized prostate cancer . moreover , the lack of association with any other pathological parameters also significantly diminishes any potential clinical application of erg expression , at least in men with localized prostate cancer . the three main arteries of the leg , the anterior tibial artery ( ata ) , posterior tibial artery and peroneal artery , form a dense vascular network around the distal leg , ankle , and foot , which ramifies perforators as the basis of all kinds of pedicle flaps such as lateral or medial malleolar perforator flaps . there is plenty of literature describing the anatomy and clinical application of perforators of the peroneal artery and posterior tibial artery around the distal lower leg . however , literature regarding perforator flaps of ata or anterior supramalleolar artery ( asma ) , one of the major branches of distal ata , are scant . these papers either mention the existence of the perforators from ata or asma , or describe their communication with the medial and lateral supramalleolar arteries . none of them have a detailed anatomical study on asma . the objective of this study was to identify the anatomic parameters of the asma and its perforators that would enable microsurgeons to harvest potentially multi - paddle flaps or composite flaps from the foot and ankle region . we try to preserve the ata intact after we harvest the composite flaps around the ankle using asma . we also want to use the ata as the only one pedicle and harvest multi - paddle flaps , decreasing the microsurgical risk greatly and minimizing the sacrifice of the blood supply to the ankle and foot . after the study was approved by the university of louisville irb ; 24 lower extremities of fresh human cadavers were studied . the ata was cannulated and injected with 20 ml of red latex ( microfilm , flow tech , inc . , ) at the mid - point of lower leg where ata can be identified between the extensor digitorum longus and tibialis anterior muscles . then , careful dissection along the distal 1/3 of the lower leg , the ankle , and the proximal foot ( 8 - 10 cm above the lateral malleolus and 1 - 2 cm anterior to the edge of the fibula ) was performed to observe the origin and the course of the asma . the distance between the origin of asma and the extensor retinaculum ( o - r ) was recorded . the diameters of the asma proper as well as its branches ( a set of fixed - width hooks was used for measurement ) , and the length of asma were measured . the collaterals between asma and the medial and lateral supramalleolar arteries and the number of perforators were observed . chi - square test was used for this study to analyse the variation of the o - r . fasciocutaneous flaps with multiple skin paddles were elevated based on the perforators to demonstrate its potential application . we classified the asma into four types according to the pattern of origin from the ata [ figures 1 - 4 ] . type a ( n = 10 ) asma originates from the ata with a 1 - 1.5 cm long main trunk and then gives off its lateral and medial branches [ figure 1a and b ] ; each consists of 2 - 3 perforators . in type b ( n = 7 ) , the asma does not have a main trunk , and its lateral and medial branches stem directly from ata with the sites of origin at the same level [ figure 2a and b ] . type c ( n = 6 ) is similar to type b , with the lateral and medial branches arising from the ata but at different origins [ figure 3a and b ] . type d ( n = 1 ) has 2 medial , and 1 lateral branch arising from the ata [ figure 4a and b ] . ( a ) type a the anterior supramalleolar artery originates as an artery stem . ( 1 ) short artery stem ; ( 2 ) lateral branches ; ( 3 ) medial branches ; ( 4 ) descendent perforators ; ( 5 ) ascendant perforators . ( 6 ) proximal end of anterior tibial artery ( ata ) ; ( 7 ) distal end of ata ; ( 8) blood supply to tibia ; ( 9 ) extensor retinaculum ( b ) the anterior supramalleolar artery originates as a 1 - 1.5 cm short artery stem and then ramifies lateral and medial branches . the medial branch gives off ascendant perforators and descendent perforators ( a ) type b the anterior supramalleolar artery originates as lateral and medial artery branches at the same point . ( 1 ) medial branches ; ( 2 ) ascendant perforators . ( 3 ) descendent perforators ; ( 4 ) lateral branches ; ( 5 ) proximal end of anterior tibial artery ( ata ) ; ( 6 ) distal end of ata ; ( 7 ) extensor retinaculum ; ( 8) blood supply to tibia ( b ) the anterior supramalleolar artery orients as lateral and medial artery branches at the same point . the medial branch gives off ascendant perforators and descendent perforators ( a ) type c the anterior supramalleolar artery originates as lateral and medial artery branches at the different point . ( 1 ) medial branches ; ( 2 ) ascendant perforators . ( 3 ) descendent perforators ; ( 4 ) lateral branches ; ( 5 ) proximal end of anterior tibial artery ( ata ) ; ( 6 ) distal end of ata ; ( 7 ) extensor retinaculum ( b ) the anterior supramalleolar artery orients as lateral and medial artery branches at different point . the medial branch gives off ascendant perforators and descendent perforators ( a ) type d - the anterior supramalleolar artery originates as lateral and two medial artery branches at the different point . ( 1 ) medial branches ; ( 2 ) lateral branches ; ( 3 ) proximal end of anterior tibial artery ( ata ) ; ( 4 ) distal end of ata ; ( 5 ) extensor retinaculum ( b ) the anterior supramalleolar artery orients as one lateral and two medial arteries branches at different point the mean distance between the origin of the proximal branch of asma and the superior margin of the extensor retinaculum ( o - r ) was 2.0 0.8 cm . the mean diameters of the medial branch ( d1 ) , lateral branch ( d2 ) and the main trunk of the asma ( d3 ) ( only in type a ) were 1.0 0.2 mm , 0.8 0.3 mm and 1.1 0.2 mm , respectively . the mean feasible length of the pedicles of the lateral flap ( l1 ) and medial flap ( l2 ) were 5.1 1.0 cm and 3.7 0.6 cm , respectively . bi - foliate fasciocutaneous flaps can be harvested based on the medial and lateral branches of the type a asma with preservation of the ata [ figure 5 ] . data of 24 specimens of the anterior supramalleolar artery the biofoliate flap using type a anterior supramalleolar artery huber dissected 200 feet and was the first to describe the asma as a vessel arising about 5 cm above the ankle joint . wee described that this septocutaneous branch of ata runs through the anterior crural septum between the tendons of tibialis anterior and extensor hallucis longus and gives out 3 perforators . further named the reverse - flow flap based on the perforators of this artery as anterior tibial type iii . later research showed that asma is always found in the distal 1/3 of the lower leg and appears 8 - 10 cm above the lateral malleolus , 1 - 2 cm anterior to the edge of the fibula . in 2003 , koshima dissected 4 ankles and described that the asma has 1 - 2 perforators just above the extensor retinaculum and gives off the anterolateral and anteromedial branches . however , we still lack detailed anatomical knowledge of its origin , calibre and so on . the diameters of the lateral and medial branches of asma are 0.8 0.3 mm and 1.0 0.2 mm , respectively . the feasible pedicle lengths of these two branches are 5.1 1.0 cm and 3.7 0.6 cm , respectively . thus , an island flap can be harvested based on one or both of the branches for defect coverage around the ankle . in addition , due to its collateral connection with the posterior tibial artery and peroneal artery , a reverse - flow anterolateral or anteromedial flap can also be designed as described in literature . the flaps with asma are very useful especially for patients who suffer from refractory malleolar ulcers due to paralysis , diabetes mellitus , venous stasis , or vasculitis . for patients who have cosmetic consideration , perforator - based adipofascial flap is also a good option with the use of asma . the perforators of the asma are suitable for a bi - foliate design in situations such as simultaneous defects of the volar and dorsal aspects of the hand . the flap based on asma is pliable and thin , which makes it an excellent option for hand and wrist coverage . if there is a simultaneous metacarpal deficiency at the same time , which can present especially in high - energy injuries , vascularised bone graft can also be harvested easily supplied by 1 - 2 perforators according to research by gilbert et al . an additional advantage of this perforator flap is that the ata can be preserved in type a asma , and morbidity of the donor site can be minimised . based on different types of asma , its lumen size can vary from 0.8 0.3 to 1.1 0.2 mm , which is still suitable for microvascular anastomosis . a difficult problem that a hand surgeon encounters conventional methods include inserting the hand into an abdominal pocket for several weeks followed by skin graft , using a large flap to cover all the defects together , or covering the wounds with several free flaps at the same time . often , these patients will still exhibit stiff digits and a bulky hand . and combined multiple single flaps would increase the surgical risk exponentially . in this instance , we propose a novel idea of using a multiple - foliate flap based on different perforators of the lateral and medial branches of the asma to cover multiple digits at the same time . these branches all derive from the same trunk - the ata and its terminal branch . this design provides the capability of harvesting up to 2 - 3 skin paddles based on one sizable pedicle , the ata . the terminal branches of the peroneal artery to ankle and foot , as well as the branches of posterior tibial artery to ankle and foot are also preserved . in this way , the vascular network of the ankle and foot is still preserved , and donor site morbidity is significantly decreased . there are several factors which should be kept in mind during the clinical application of asma based flaps . when the skin is opened , the lateral and medial branches of the asma are generally visible . if we want only the stem artery and their branches , we can just follow the course of these branches and easily find the origin of these branches . . however , in any situation it would be more effective to repair the extensor retinaculum carefully and immobilize the foot for 3 - 4 weeks . further study and long - term follow - up are needed to understand the blood supply of the ankle and foot after harvesting the asma - based flap . its sizable diameter and lengthy pedicle make it suitable as bi - foliate fasciocutaneous flaps or composite flaps .

background . the prognostic significance of erg expression in prostate cancer ( pca ) has generated mixed results . we sought to investigate the prognostic significance of erg expression in a localized cohort of men with pca . material and methods . we investigated erg protein expression in a cohort of 198 men with localized pca . erg expression was correlated with patients ' clinical outcome and several pathological parameters , including gleason score ( gs ) , pathological stage , surgical margin , and extra - capsular extension . results . erg expression was detected in 86/198 ( 43.4% ) patients exclusively in neoplastic epithelium . overall , erg mean expression intensity was 1.01 1.27 versus 0.37 0.83 in acinar pca compared to foamy type pca ( p < 0.001 ) . in hgpin , erg intensity levels were comparable to those in foamy type pca ( 0.13 0.56 ) but significantly lower than those in acinar pca ( p < 0.001 ) . erg expression was significantly associated with extra - prostatic extension and higher pathological stage and showed a trend toward seminal vesicle invasion . herein , erg expression was documented in 50/131 ( 38.1% ) patients with pt2 versus 30/55 ( 54.5% ) patients with pt3 ( p = 0.04 ) . erg association with higher pathological stage was more pronounced in patients with gs > 7 . grouping patients into those with gs 7 versus > 7 , there was no significant association between erg expression and gs . similarly , no association was present in relation to either surgical margins or postsurgical serum psa levels . conclusion . we report significant association between erg protein levels and extra - prostatic extension and higher pathological stage . erg expression is not associated with adverse clinical outcome and is of limited prognostic value in localized pca . purpose : a further understanding of the anterior supramalleolar artery ( asma ) and its potential applications in reconstructive surgery.materials and methods : a total of 24 fresh lower limbs from fresh cadavers were injected with red latex for dissection . the type of origin , course , diameter of the pedicle , and the distance between the origin of the asma from the anterior tibial artery to the extensor retinaculum ( o - r ) were recorded . bi - foliate fasciocutaneous flaps were harvested using the branches of the asma.results:we found four types of origin of the asma , and we have accordingly classified them into four types . 10 of them were type a , 7 were type b , 6 were type c and 1 was type d. the mean o - r ( origin of asma to retinaculum ) distance was 2.0 0.8 cm . the diameter of the medial branch ( d1 ) , the diameter of the lateral branch ( d2 ) , and the diameter of artery stem ( d3 ) ( only in type a ) were 1.0 0.2 mm , 0.8 0.3 mm , 1.1 0.2 mm , respectively . the mean pedicle length of the lateral flap ( l1 ) and medial flap ( l2 ) were 5.1 1.0 cm and 3.7 0.6 cm , respectively.conclusions:the asma exists constantly with four different types of origin . its sizable diameter and lengthy pedicle make it suitable for bi - foliate fasciocutaneous flap transfer .

ankle dorsiflexion passive range of motion ( df prom ) measurements are performed in the field of physical therapy to estimate ankle motion during functional activities1 and to prevent lower extremity injuries2 . although in the clinical setting , ankle df prom is frequently measured under non - weight - bearing ( non - wb ) conditions1 , 3 , 4 , many researchers have stated that the wb position is more appropriate for estimating the amount of ankle df motion during functional activities5 , 6 . therefore , wb ankle df prom should be measured during interventions focused on increasing ankle df prom . limited ankle df prom with knee extended may result from gastrocnemius tightness and insufficient posterior talar glide7 . thus , gastrocnemius stretching and talocrural joint mobilization have been performed as intervention strategies to increase ankle df prom3 , 8 , 9 . previous studies have reported a significant increase in ankle df prom after these interventions3 , 8 , 9 ; however , to our knowledge , no study has demonstrated the combined effect of both interventions on wb ankle df prom . therefore , the aim of the present study was to examine the influence of gastrocnemius stretching combined with joint mobilization on wb ankle df prom . in total , 11 male subjects with bilateral limited non - wb ankle df prom with knee extended ( mean age , 22.82 3.09 years ; mean height , 175.91 3.39 cm ; mean weight , 69.55 3.78 kg ; mean non - wb ankle df prom , 4.17 2.48 ) participated in this study . inclusion criteria were 1 ) ankle df prom with knee extension < 10 ; 2 ) ankle df prom with knee flexion > 10 ; and 3 ) > 5 difference in ankle df prom between knee extension and knee flexion conditions on bilateral sides in non - wb positions3 . subjects with a history of surgery on the lower extremity , fracture , or neurological diseases were excluded from this study . all participants signed an informed consent form approved by the institutional research review committee of inje university prior to participation in this study . the study protocol of this study complies with the ethical standard of the declaration of helsinki . wb ankle df prom with knee extended was measured following the procedures suggested by munteanu et al10 . subjects stood in front of a wall and placed the leg being tested behind the contralateral leg in a lunge posture . subjects were asked to place both hands on the wall and then lean forward without heel - off and knee flexion until maximum stretch was felt in the gastrocnemius on the tested leg . the force applied to the tested leg was maintained at 60 5% of the subject s weight using scales11 . an examiner determined the maximum tibial inclination using an inclinometer to measure the wb ankle df prom with knee extended . measurements of wb ankle df prom were repeated 3 times for each ankle under pre- and post - intervention conditions . the mean value of 3 trials was used for data analysis . for gastrocnemius stretching combined with joint mobilization , subjects leaned forward against the wall in the same lunge posture as that during measurement of wb ankle df prom with knee extended until the maximum gastrocnemius stretch was felt . subjects held the end - range posture while an examiner provided the talus of the tested leg with sustained anterior - to - posterior gliding force . an intervention trial was performed for 30 s , and 10 trials were repeated with 30-s rest periods for each ankle . the difference in wb ankle df prom with knee extended between pre- and post - intervention conditions was analyzed using a paired t - test . wb ankle df prom with knee extended was significantly increased in post - intervention compared with pre - intervention conditions ( 42.60 5.49 versus 38.24 4.69 , p < 0.001 ) . our findings demonstrate that gastrocnemius stretching combined with joint mobilization significantly increases wb ankle df prom with knee extended . stretching exercises increase tolerance , resulting in increased rom12 . additionally , increased displacement of the myotendinous junction ( mtj ) after gastrocnemius stretching for 5 min was found in a previous study13 . therefore , the change in tolerance and/or increase in mtj displacement might have influenced our findings . the addition of talocrural joint mobilization to gastrocnemius stretching is another possible explanation for our findings . previous research by dinh et al.3 showed a 4.25 increase in wb ankle df prom with knee extended after gastrocnemius stretching alone for 3 weeks . although gastrocnemius stretching combined with joint mobilization was applied for 5 min in the present study , the amount of increase in wb ankle df prom after intervention ( i.e. , 4.36 ) was similar to that found previously . considering this outcome , despite the relatively short period of intervention in the present study , it may be inferred that the addition of talocrural joint mobilization might maximize the effects of general gastrocnemius stretching . thus , we conclude that gastrocnemius stretching combined with joint mobilization might decrease gastrocnemius tightness and increase posterior talar gliding movement , which effectively increases wb ankle df prom with the knee extended . first , although non - wb ankle df prom was used as an inclusion criterion , changes in non - wb ankle df prom after intervention were not measured . however , we believe that wb ankle df prom is clinically more important because most functional activities are performed under the wb condition . second , our study included only male subjects , and the results can not be generalized to women . dox is one of the key chemotherapeutic drugs for cancer treatment , but its use is limited by chronic and acute toxic side effects . dox is an antibiotic anthracycline that was isolated from a pigment of streptomyces peucetius in the early 1960s and it had been employed for more than 30 years in the battle against cancer , but it is now chemically synthesized . renal dox - induced toxicity may be part of a multiorgan damage mediated mainly through free radical formation eventually leading to membrane lipid peroxidation . induction of apoptosis and modulation of nox are mechanisms that are involved in toxic adverse effects associated with dox therapy . in addition , dox has a direct renal damaging effect as it accumulates preferentially in the kidney . dox has toxic effects on other organs such as heart and liver which may lead to modulation of blood supply to the kidney and alter xenobiotic detoxification processes , respectively , thus indirectly contributing to dox - induced nephropathy . dia is a new anti - inflammatory , analgesic , and antipyretic drug that was developed specially for the treatment of osteoarthritis . it is highly effective in relieving the symptoms of osteoarthritis and may be able to modify the course of the disease . interleukin 1 is a proinflammatory and proapoptotic agent that induces cytokine production by activating nfb and mitogen activated protein kinase signaling . a major cause of dox - induced nephrotoxicity is the production of reactive oxygen species which induce cytokines , including interleukin 1 [ 6 , 9 , 10 ] . the aim of the present study was to study the effect of the interleukin 1 receptor antagonist diacerein ( dia ) on dox - induced nephropathy . dia powder was from eva pharma company and it was dissolved in 1% carboxymethylcellulose . dox hydrochloride 10 mg vial ( pharmacia italia , spa , italy ) , polyclonal rabbit / antirat caspase-3 , tnf , and nfb antibody ( lab vision , usa ) , biotinylated goat anti - rabbit secondary antibody ( transduction laboratories , usa ) , urea , gsh , sod , and catalase kits ( biodiagnostic , egypt ) , and creatinine ( humen , germany ) were purchased . adult male wistar rats weighing about 250350 g were obtained from the animal research centre , giza , egypt . animals were kept in standard housing conditions in cages and were left to acclimatize for one week . this work was conducted in the pharmacology department , faculty of medicine , el - minia university , egypt , and the animal experimental protocol was approved by the faculty board . rats were randomly assigned into 6 groups ( n = 6 each ) as follows . group i received vehicle ( 1% carboxymethylcellulose ) for 15 days and ip saline at day 11 . group ii was treated with dld ( 25 mg / kg / d orally ) for 15 days and ip saline at day 11 . group iii was treated with dhd ( 50 mg / kg / d orally ) and ip saline at day 11 . group iv was treated with vehicle for 15 days and dox ( 15 mg / kg ) at day 11 . group v was treated with dld ( 25 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . group vi was treated with dhd ( 50 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . the doses of dox and dia were based on the previous studies [ 4 , 11 ] . group i received vehicle ( 1% carboxymethylcellulose ) for 15 days and ip saline at day 11 . group ii was treated with dld ( 25 mg / kg / d orally ) for 15 days and ip saline at day 11 . group iii was treated with dhd ( 50 mg / kg / d orally ) and ip saline at day 11 . group iv was treated with vehicle for 15 days and dox ( 15 mg / kg ) at day 11 . group v was treated with dld ( 25 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . group vi was treated with dhd ( 50 mg / kg / d orally ) for 15 days + ip injection of dox ( 15 mg / kg ) at day 11 . the doses of dox and dia were based on the previous studies [ 4 , 11 ] . after 4 days of dox injection , each rat was weighed then anesthetized with ip injection of urethane ( 25% in a dose of 1.6 gm / kg ) and then sacrificed . a longitudinal section of the left kidney and one half was fixed in 10% formalin then embedded in paraffin for histopathological and immunohistochemical examinations . the rest of the kidneys were snap frozen in liquid nitrogen and kept at 80c . briefly , the method is based on the fact that sulfhydryl group of gsh reacts with 5 , 5-dithiobis ( 2-nitrobenzoic acid ) ( ellman 's reagent ) and produces a yellow colored 5-thio-2-nitrobenzoic acid which was measured colorimetrically at 405 nm using beckman du-64 uv / vis spectrophotometer , usa . assessment of renal catalase antioxidant enzyme activity was determined from the rate of decomposition of h2o2 at 510 nm after the addition of tissue homogenate as described by colorimetric kit . the assessments of sod levels were based on the ability of the enzyme to inhibit the phenazine methosulfate - mediated reduction of nitroblue tetrazolium dye and the results were expressed as unit / g tissue . the renal contents of lipid peroxides were assayed by a spectrophotometric method based on the reaction between mda and thiobarbituric acid . the absorbance values of the samples and the blank were determined at 535 nm using a ( beckman du-64 spectrophotometer , usa ) and then blank absorbance value was subtracted from the sample absorbance value . from a standard curve , mda concentration in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol / gm tissue by multiplying in the tissue dilution factor . nitric oxide ( no ) in the form of nitrite was determined with spectrophotometric method using griess reagent systems . the stable oxidation end products of no , nitrite ( no2 ) , and nitrate ( no3 ) were used as indicators of no production . nox was measured after the reduction of nitrate to nitrite by copperized cadmium granules in glycine buffer at ph 9.7 . quantification of no2 was based on the griess reaction , in which a chromophore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of n - naphthylene diamine and sulfanilamide . the absorbance of the sample and the blank were measured at 545 nm using ( beckman du-64 spectrophotometer , usa ) . the blank absorbance is then subtracted from the sample absorbance . from a standard curve , nox content in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol / g tissue . renal tissue was fixed in 10% formalin , embedded in paraffin , sectioned by a microtome at 5 m thickness and stained with hematoxylin and eosin for routine histopathological assessment . the renal tissues were examined in random microscopic areas semiquantitatively under 40 high power fields and the number of changes was assessed by the counting of 3 nonoverlapped fields for the same slide of each animal . the frequency and the severity of lesions in the kidneys were assessed semiquantitatively as follows : score : assigned normal , score + : in between normal and mild , score + + ( mild level ) : less than 25% of the examined fields revealed histological alterations , score + + + ( moderate level ) : less than 50% of the examined fields revealed histological alterations , and score + + + + ( severe level ) : less than 75% of the total fields examined revealed histological alterations . the caspase-3 , tnf , and nfb immunolabeled cells were counted . in each animal , 3 sections were examined and the cells were counted in 3 adjacent nonoverlapping fields levels . immunohistochemical staining was performed for caspase-3 , tnf , and nfb using polyclonal rabbit / antirat antibody according to previously published protocol [ 20 , 21 ] , respectively . table 1 shows the results of the effect of dia on serum creatinine and urea . rats receiving a single dose of dox ( 15 mg / kg , ip ) showed a significant increase in serum creatinine and urea levels compared to control group . both doses of dia resulted in significant decrease in serum creatinine compared to dox treated rats . dia 50 mg / kg / day but not 25 mg / kg / day resulted in significant decrease in serum urea compared to dox treated rats . renal mda was evaluated as an indicator of kidney lipid peroxidation and nitrites and nitrates as an indicator of renal nox levels ( table 1 ) . dox ( 15 mg / kg ) significantly increased renal mda and nox levels compared to control group . administrating both doses of dia to dox treated rats significantly decreased mda and nox compared to dox treated group . treatment with dox ( 15 mg / kg ) caused significant decrease in renal gsh , sod , and catalase levels compared with untreated control group ( table 2 ) . concomitant treatment of dox with dia significantly increased the levels of renal gsh , sod , and catalase compared to dox treated group . the histological study of the rat renal cortical tissue of control group ( figure 1(a ) ) , dld ( 25 mg / kg / day ) group ( figure 1(b ) ) , and dhd ( 50 mg / kg / day ) group ( figure 1(c ) ) showed normal architecture of renal glomeruli and tubules . dox treated group ( figure 1(d ) ) showed marked enlargement of some vascular glomeruli which tightly fill the renal corpuscles . dox + dld group ( figure 1(e ) ) showed amelioration of the damaging effects of dox . there were less tubular distortion , narrow bowman 's spaces , and fewer cytoplasmic vacuolations of renal corpuscle and tubular cells were also observed . dox + dhd group ( figure 1(f ) ) had more obvious decrease in the morphological changes caused by dox exposure . the severity of the morphological changes was assessed semiquantitatively ; dox exposed group showed increase in the glomerular and tubular morphological changes at the light microscopic levels when compared with control group . these changes were suppressed by the administration of both doses of dia , but the high dose showed marked improvement than the low dose ( table 3 ) . administration of dox caused significant increase in the immunoreactivity of caspase-3 , nfb , and tnf ( figures 2 , 3 , and 4 and table 4 ) respectively , which were highly expressed in both renal glomeruli and tubules cytoplasmically and in some nuclei . administration of both doses of dia concomitantly with dox decreased the expression of them , compared to dox group . administration of both doses of dia in vehicle treated rats alone and control groups showed no expression . effective anticancer therapy with anthracyclines as dox is limited because of its toxicity to various organs including kidneys . nephrotoxic action of dox is also considered to be via drug - induced free radical generation . the formation of free radicals induces the production of proinflammatory cytokines as interleukin 1 initiating the biological effects associated with inflammation . this directed our attention to investigate the role of dia which is interleukin 1 receptor antagonist as a possible nephroprotective agent against dox - induced renal damage . induction of dox nephrotoxicity was detected in our study by significant elevation of serum urea and creatinine levels which were confirmed by toxic histopathological changes compared to control group . urea and serum creatinine are the most sensitive markers of nephrotoxicity implicated in the diagnosis of renal injury [ 24 , 25 ] . the nephrotoxic effect of dox is characterized by decreasing glomerular filtration rate leading to a rise in serum urea and creatinine . our results are in good agreement with the previous studies [ 22 , 26 ] . improvement of dox - induced nephrotoxicity was previously tried by compounds that partially succeeded in preserving normal renal function and structure probably through their antioxidant and anti - inflammatory effects as caffeic acid phenethyl ester , zingiber officinale roscoe , and solanum torvum so that we investigated the role of another antioxidant and anti - inflammatory drug as dia on dox - induced nephrotoxicity . dia could significantly decrease serum urea and creatinine compared to dox treated group . that is due to the anti - inflammatory and antioxidant effects of dia which suppress dox mediated oxidative stress , inflammation , and tissue damage . our histopathological changes showed that dox treated group presented with marked damage of renal tubules . these results are in agreement with zhao et al . who detected the protective effect of rhein ( the active metabolite of dia ) on acetaminophen induced hepatotoxicity and nephrotoxicity in rats . they found that serum urea and creatinine significantly decreased in rhein and acetaminophen coadministration compared to acetaminophen group and normalization of toxic histopathological changes . the elevated levels of gsh could effectively provide thiol group for the possible gsh mediated detoxification reactions of gpx ( glutathione peroxidase ) and gst ( glutathione - s - transferase ) which is involved in the scavenging of o2 generated from the dox . our findings are consistent with the previous reports that showed that gsh concentration is significantly decreased upon dox treatment compared to control group [ 4 , 22 ] . sod extensively distributes in all cells and has a significant shielding role against oxidative injury induced by reactive oxygen species . in our study , the activities of sod and catalase significantly decreased in dox treated rats in kidney as compared to control rats . the accumulation of these highly reactive free radicals leads to the reduction of the activity of sod and catalase which in turn results in damaging effects in the form of loss of cell membrane integrity and function . the decrease in the sod and catalase activities related to the increase in the intracellular levels of h2o2 . catalase has been reported to be responsible for the detoxification of h2o2 , which is an effective inhibitor of sod . coadministration of dia significantly improved sod , gsh , and catalase levels compared to dox treated group . these results may be due to antioxidant effect of dia which was approved previously by tamura et al . who indicated the inhibitory effect of dia on indomethacin - induced gastric ulceration which could be mediated by the suppression of reactive oxygen species production based on its inhibition of neutrophil activation and antioxidant activity . in addition , hu et al . investigated the protective effects of rhein lysinate ( rhl ) , against kidney impairment in senescence - prone inbred strain 10 ( samp10 ) mice . treatment of samp10 mice with rhl significantly increased the sod and gpx levels in the kidneys . o2 reacts with lipid to form lipid peroxides followed by -oxidation to form mda . that was detected in our study which showed significant increase of mda level in dox treated group compared to control group . these results are in agreement with el - sheikh et al . and yagmurca et al . the high production of nox results in peroxynitrite formation which is a potent and aggressive cellular oxidant and is involved in dox toxicity . the current findings showed that dox administration significantly increased renal level of nox compared to control group and that is in agreement with other studies [ 26 , 37 ] . these results are in agreement with zhao et al . who detected the protective effect of rhein on acetaminophen induced nephrotoxicity in rats which was approved by significant decrease of mda and nox on coadministration of rhein plus acetaminophen group compared to acetaminophen group . our results are in agreement with martel - pelletier and pelletier who reported that no is produced through the activity of inducible nitric oxide synthase and it is a major catabolic factor involved in the pathophysiology of oa . our results are consistent with hu et al . who investigated the protective effects of rhein lysinate ( rhl ) , against kidney impairment in senescence - prone inbred strain 10 ( samp10 ) mice . induction of p53 mediates cell apoptosis through activation of caspase-3 family of proteases and apoptotic cell death . our study is showing significant increase in caspase-3 expression in dox treated group in comparison with control group . our study is in consistence with torina et al . who showed that treatment with dia once a day for 4 weeks after myocardial infarction improved ventricular remodeling by partial blockage of the proinflammatory cytokines which led to lower caspase-3 activity and nfb p65 transcription b pathway . dox - induced superoxide anion production which was reported to be responsible for tnf-induced nuclear factor ( nf ) activation that increases nf and tnf over expression . our study showed significant increase in tnf and nfb expressions in dox group compared to control group and the same results were found with al - saedi et al . . coadministration of dia significantly decreased tnf and nfb expression compared to dox treated group that is in agreement with gadotti et al . who showed that dia inhibits neuropathic pain by decreasing proinflammatory cytokines as tnf and nf. also , hu et al . the active metabolite of dia is rhein which possesses anti - inflammatory activity and may be effective in suppressing the inflammatory cytokines contributing to the pathogenesis of diabetic nephropathy . . demonstrated that rhein had protective effect in different models of nephropathy as iga induced nephropathy , obstructive nephropathy , chronic allograft nephropathy , and high glucose and angiotensin ii induced nephropathy . oral administration of rhein ( 150 mg / kg / d ) ameliorated renal lesions . rhein was capable of protecting against renal injury by decreasing the activities of nfb and caspase-3 in the early phase of glomerulosclerosis . our results are consistent with meng et al . who reported that rhein possesses various pharmacological activities , including anti - inflammatory , antioxidant , and antitumor . in their study , a model of hyperuricemia and nephropathy induced by adenine and ethambutol in mice was established . the results demonstrated that rhein significantly improved the symptoms of nephropathy through decreasing the production of proinflammatory cytokines , including interleukin 1 , prostaglandin e2 , and tnf. yu et al . aimed to explore the effect of rhein on sepsis - induced acute kidney injury by injecting lipopolysaccharide ( lps ) and cecal ligation and puncture ( clp ) in vivo and on lps - induced hk-2 cells in vitro . rhein could significantly decrease concentration of serum urea and creatinine and level of tnf , nfb , and il-1 in two different mouse models of experimental sepsis . in conclusion , dia protected against dox - induced nephrotoxicity in rats most probably due to its antioxidant and anti - inflammatory activities . however , dhd ( 50 mg / kg / day ) showed more protective effect than dld ( 25 mg / kg / day ) .

[ purpose ] the purpose of this study was to investigate the effect of gastrocnemius stretching combined with talocrural joint mobilization on weight - bearing ankle dorsiflexion passive range of motion . [ subjects ] eleven male subjects with bilateral limited ankle dorsiflexion passive range of motion with knee extended participated in this study . [ methods ] all subjects received talocrural joint mobilization while performing gastrocnemius stretching . ankle dorsiflexion passive range of motion was measured using an inclinometer under weight - bearing conditions before and immediately after intervention . a paired t - test was used to analyze the difference between weight - bearing ankle dorsiflexion passive range of motion pre- and post - intervention . [ results ] a significant increase in weight - bearing ankle dorsiflexion passive range of motion was found post - intervention compared with pre - intervention . [ conclusion ] these findings demonstrate that gastrocnemius stretching combined with joint mobilization is effective for increasing weight - bearing ankle dorsiflexion passive range of motion . nephrotoxicity is one of the limiting factors for using doxorubicin ( dox ) . interleukin 1 has major role in dox - induced nephrotoxicity , so we investigated the effect of interleukin 1 receptor antagonist diacerein ( dia ) on dox - induced nephrotoxicity . dia ( 25 and 50 mg / kg / day ) was administered orally to rats for 15 days , in the presence or absence of nephrotoxicity induced by a single intraperitoneal injection of dox ( 15 mg / kg ) at the 11th day . we measured levels of serum urea , creatinine , renal reduced glutathione ( gsh ) , malondialdehyde ( mda ) , total nitrites ( nox ) , catalase , and superoxide dismutase ( sod ) . in addition , caspase-3 , tumor necrosis factor alpha ( tnf ) , nuclear factor kappa b ( nfb ) expressions , and renal histopathology were assessed . our results showed that dox - induced nephrotoxicity was ameliorated or reduced by both doses of dia , but diacerein high dose ( dhd ) showed more improvement than diacerein low dose ( dld ) . this protective effect was manifested by significant improvement in all measured parameters compared to dox treated group by using dhd . dld showed significant improvement of creatinine , mda , nox , gsh , histopathology , and immunohistochemical parameters compared to dox treated group .

suicide is a leading cause of death among adolescents and has become a significant public health issue globally ( 1 ) . statistics korea report 2014 showed that suicide is the cause of 28.4% of adolescent deaths in 2013 , and it was the number one cause of death among adolescents ( 2 ) . furthermore , suicide is the second to third cause of mortality among adolescents in the western world and is an important concern in developing countries ( 3 ) . numerous causes of suicidal behavior during adolescence have been reported , including mental disorders , peer affiliations , and level of achievement in school ( 4 ) . additionally , weight - related concerns are a common cause of suicidal behaviors ( 56 ) . this transition often leads to confusion about personal identity , especially in the context of enduring developments in identity and cognition as well as physical growth ( 7 ) . adolescents typically enter a period of a high level of self - consciousness about their body shape and size ( 8) . they have a greater tendency to consider how they look to others at this life stage than during any other period of life ( 9 ) . these weight - related issues are influenced by many factors including friends , peers , social norms , and media ( 10 ) . the media is a particularly strong influence because it has become a general source of information and adolescents are easily exposed to it ( 11 ) . the media encourages as " ideal " slim and thin body shapes for girls and muscular bodies for boys ( 1213 ) , and provides abundant information on the newest diet methods and quick fixes for weight control ( 14 ) . this may induce adolescents , who have a particularly keen interest in their figure , to distort their body image and to engage in unneeded or inappropriate weight control behaviors ( wcb ) ( 15 ) . many adolescents engage in inappropriate wcb such as vomiting , taking nonprescription diet pills , and taking laxatives or diuretics . among korean adolescents engaging in wcb , 13.4% of boys and 18.8% of girls took inappropriate methods to control their weight ( 16 ) . according to a study using data from national surveys , similar rates were also observed in the united states , with 10% of boys and 21.3% of girls conducting at least one inappropriate wcb ( 17 ) . inappropriate wcb are a harmful influence on adolescents ' physical and psychological development ( 12 ) . for instance , a 5-year longitudinal study reported that engagement in inappropriate wcb can be a predictor of obesity and eating disorders ( 18 ) . moreover , adverse outcomes of inappropriate wcb also include the onset of depression ( 19 ) and suicide ideation ( 20 ) . due to the high prevalence of and the possible negative side effects associated with inappropriate wcb , it is a growing serious public health concern . therefore , the prevention of inappropriate wcb among adolescents is imperative to protect them from negative physical and psychosocial consequences . to date , studies about inappropriate wcb merely suggest the association between inappropriate wcb and negative psychosocial consequences ( 212223 ) . in the present study , we investigate not only the association between inappropriate wcb and suicide ideation and attempt , but also the difference in this association with body mass index , body weight perception , and body shape misperception . moreover , we investigate the top five wcb combinations used by korean adolescents and examine the association between these five wcb combinations and suicide ideation and attempt . we used a cross - sectional design ; thus , the results should be interpreted with caution due to possibility of a bi - directional effect . we used data from the 2014 korea youth risk behavior web - based survey ( kyrbs ) . the kyrbs has been conducted annually since 2005 by the korean centers for disease control and prevention ( kcdc ) , the korea ministry of education , science and technology , and the korea health and human services . the data were collected via an ongoing , anonymous web - based survey in a self - reporting format that was conducted on a nationally representative sample of middle- and high - school students . it aimed to plan and assess the korean adolescent health promotion policies by investigating health - related behaviors and status . in the 2014 survey , 799 middle and high schools were selected , including 72,060 students in grades 7 to 12 ( stratified ) on a national scale . however , we excluded 2,334 individuals due to missing data for variables used in this study ; therefore , our cohort ultimately comprised 69,726 adolescents ( 35,224 boys and 34,361 girls ) . suicide ideation and attempt were measured via responses to the following questions : " have you seriously considered suicide during the past 12 months ? " and " have you tried suicide during the past 12 months ? " the possible responses to both questions were " yes " or " no . " we characterized wcb via responses to the following multi - part question : " have you experienced the following weight control methods during the past 30 days , with the following activities listed : 1 ) did regular exercise , 2 ) fasted at least 24 hours , 3 ) ate less , 4 ) took prescription diet pills , 5 ) took nonprescription diet pills , 6 ) took laxatives or diuretics , 7 ) vomited , 8) ate only one food , 9 ) took oriental medicine , and 10 ) ate diet food . " the possible responses to all questions were " yes " or " no . " if participants responded ' yes ' to at least one of 2 ) , 5 ) , 6 ) , 7 ) , and 8) , we classified them into the " inappropriate wcb " group ( 16 ) . if participants responded ' no ' at all of 2 ) , 5)-8 ) , we classified them into the " appropriate wcb " group . if participants responded ' no ' to all activity choices , we classified them into the " nothing " group . body mass index ( bmi ) was calculated by self - reported height and weight . bmi percentiles for age and gender were calculated according to the 2007 standard growth charts for korean children and adolescents by the korean pediatric society ( 24 ) , and four categories were created : underweight ( 15th percentile ) , normal weight ( 16th to 84th percentile ) , overweight ( 85th to 95th percentile ) , and obese ( 95th percentile or bmi 25 ) . we further grouped overweight and obese groups into a single overweight group , thus using three bmi categories body weight perception ( bwp ) was split into five categories : very underweight , underweight , normal weight , overweight , and very overweight . we further grouped these categories into three groups : thin , normal , and obese . body shape misperception ( bsm ) was determined by an agreement between bmi and bwp categories . participants perceiving their weight at least one category below their actual bmi categories were designated part of the " underestimate " group , while those perceiving their weight at least one category above their actual bmi were designated part of the " overestimate " group ( i.e. , if participants whose bmi was a normal weight perceived their body weight as obese , they were placed in the " overestimate " group ) ( 25 ) . control variables included age , household economic status , parents ' presence , residential area , school level , academic achievement in school , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress level , and depression . we performed statistical analyses of the survey data using sas version 9.4 ( sas inc . , the relevant primary sampling units , sample weights , and stratification were considered in the analysis given that the kyrbs was designed as a complex sample . a pearson 's test was used to determine significant differences in the distribution of each variable . next , a logistic regression analysis was conducted to determine the association between wcb and suicide ideation and attempt . additionally , we performed a set of subgroup analyses using bwp , bmi , and bsm to determine whether body related variables led to differences in the association between wcb and suicide ideation and attempt . results were presented as adjusted odds ratios ( ors ) with 95% confidence intervals ( 95% cis ) . the ethics approval for the publicly open kyrbs data was waived by the institutional review board ( irb ) . after the purpose of the survey was fully explained to the students , written informed consent was provided . students were assured that they could refuse or withdraw from the research at any stage ( 16 ) . we used data from the 2014 korea youth risk behavior web - based survey ( kyrbs ) . the kyrbs has been conducted annually since 2005 by the korean centers for disease control and prevention ( kcdc ) , the korea ministry of education , science and technology , and the korea health and human services . the data were collected via an ongoing , anonymous web - based survey in a self - reporting format that was conducted on a nationally representative sample of middle- and high - school students . it aimed to plan and assess the korean adolescent health promotion policies by investigating health - related behaviors and status . in the 2014 survey , 799 middle and high schools were selected , including 72,060 students in grades 7 to 12 ( stratified ) on a national scale . however , we excluded 2,334 individuals due to missing data for variables used in this study ; therefore , our cohort ultimately comprised 69,726 adolescents ( 35,224 boys and 34,361 girls ) . suicide ideation and attempt were measured via responses to the following questions : " have you seriously considered suicide during the past 12 months ? " and " have you tried suicide during the past 12 months ? " the possible responses to both questions were " yes " or " no . " we characterized wcb via responses to the following multi - part question : " have you experienced the following weight control methods during the past 30 days , with the following activities listed : 1 ) did regular exercise , 2 ) fasted at least 24 hours , 3 ) ate less , 4 ) took prescription diet pills , 5 ) took nonprescription diet pills , 6 ) took laxatives or diuretics , 7 ) vomited , 8) ate only one food , 9 ) took oriental medicine , and 10 ) ate diet food . " if participants responded ' yes ' to at least one of 2 ) , 5 ) , 6 ) , 7 ) , and 8) , we classified them into the " inappropriate wcb " group ( 16 ) . if participants responded ' no ' at all of 2 ) , 5)-8 ) , we classified them into the " appropriate wcb " group . if participants responded ' no ' to all activity choices , we classified them into the " nothing " group . body mass index ( bmi ) was calculated by self - reported height and weight . bmi percentiles for age and gender were calculated according to the 2007 standard growth charts for korean children and adolescents by the korean pediatric society ( 24 ) , and four categories were created : underweight ( 15th percentile ) , normal weight ( 16th to 84th percentile ) , overweight ( 85th to 95th percentile ) , and obese ( 95th percentile or bmi 25 ) . we further grouped overweight and obese groups into a single overweight group , thus using three bmi categories body weight perception ( bwp ) was split into five categories : very underweight , underweight , normal weight , overweight , and very overweight . we further grouped these categories into three groups : thin , normal , and obese . body shape misperception ( bsm ) was determined by an agreement between bmi and bwp categories . participants perceiving their weight at least one category below their actual bmi categories were designated part of the " underestimate " group , while those perceiving their weight at least one category above their actual bmi were designated part of the " overestimate " group ( i.e. , if participants whose bmi was a normal weight perceived their body weight as obese , they were placed in the " overestimate " group ) ( 25 ) . control variables included age , household economic status , parents ' presence , residential area , school level , academic achievement in school , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress level , and depression . we performed statistical analyses of the survey data using sas version 9.4 ( sas inc . , the relevant primary sampling units , sample weights , and stratification were considered in the analysis given that the kyrbs was designed as a complex sample . a pearson 's test was used to determine significant differences in the distribution of each variable . next , a logistic regression analysis was conducted to determine the association between wcb and suicide ideation and attempt . additionally , we performed a set of subgroup analyses using bwp , bmi , and bsm to determine whether body related variables led to differences in the association between wcb and suicide ideation and attempt . results were presented as adjusted odds ratios ( ors ) with 95% confidence intervals ( 95% cis ) . the ethics approval for the publicly open kyrbs data was waived by the institutional review board ( irb ) . after the purpose of the survey students were assured that they could refuse or withdraw from the research at any stage ( 16 ) . the demographics of the 35,224 boys and 34,361 girls in this study are listed in table 1 . of the participants , 10.6% ( n = 3,709 ) of boys and 15.0% ( n = 5,172 ) of girls reported that they experienced suicide ideation , while 2.1% ( n = 722 ) of boys and 3.4% ( n = 1,151 ) of girls reported attempting suicide . 33.5% ( n = 11,827 ) of boys and 45.2% ( n = 15,489 ) of girls reported that they engaged in appropriate wcb , and 4.2% of boys ( n = 1,466 ) and 9.6% of girls ( n = 3,324 ) reported that they engaged in inappropriate wcb . boys who had engaged in inappropriate wcb were more likely to report suicide ideation than those not engaging in wcb group ( or , 1.57 , p < 0.001 ) . girls engaging in inappropriate wcb also had a high suicide ideation rate ( or , 1.32 , p < 0.001 ) . boys engaging in inappropriate wcb were also more likely to attempt suicide than those not engaging in wcb ( or , 2.49 , p < 0.001 ) . girls engaging in appropriate wcb ( or , 1.21 , p = 0.02 ) or inappropriate wcb ( or , 1.92 , p < 0.001 ) were more likely to attempt suicide than girls not engaging in wcb at all , and the association was stronger among the latter comparison group . the association between suicidal behavior and wcb stratified by bmi , bwp , and bsm is shown in table 3 . boys with an underweight bmi and that engaged in inappropriate wcb experienced the highest rate of suicide ideation ( or , 2.22 , p < 0.05 ) compared to other bmi groups . girls with an underweight bmi and that engaged in inappropriate wcb were 4.53 times more likely to attempt suicide than girls who did not engage in wcb . girls who perceived their weight as normal and engaged in inappropriate wcb were more likely to experience suicide ideation ( or , 1.50 , p < 0.001 ) and attempt ( or , 2.31 , p < 0.001 ) than girls who did not engage in wcb . considering bsm , boys who distorted their body shape and who engaged in inappropriate wcb were more likely to experience suicide ideation ( underestimate : or , 1.44 , p < 0.01 , overestimate : or , 1.91 , p < 0.01 ) and attempt ( underestimate : or , 1.96 , p < 0.01 , overestimate : or , 2.94 , p < 0.01 ) than boys who do not engaged in wcb . girls who underestimated or overestimated their body shape and engaged in inappropriate wcb showed significant association with suicide attempt ( underestimate : or , 1.89 , p < 0.001 , overestimate : or , 1.73 , p < 0.001 ) compared to girls who do not engaged in wcb . adjusted for age , sex , household economic status , parents presence , residential area , school level , academic achievement , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress status , and depression . can not be calculated due to the lack of number . table 4 lists the odds ratios of the top five wcb combinations engaged in by korean adolescents and their association with suicide behaviors . conducting regular exercise and eating less together with fasting was significantly associated with suicidal ideation ( boys : or , 1.70 , p < 0.01 , girls : or , 1.33 , p < 0.05 ) and attempt ( boys : or , 1.84 , p < 0.05 , girls : or , 2.24 , p < 0.001 ) among both boys and girls , and girls had higher rates of attempted suicide . adjusted for age , sex , household economic status , parents presence , residential area , school level , academic achievement , subjective health status , sleep satisfaction , physical activity , current alcohol consumption , current smoker , stress status , and depression . in the present study , we investigated association between inappropriate wcb and suicide ideation and attempt by korean adolescent by using nationally represented data . inappropriate wcb was significantly associated with suicidal behaviors in both boys and girls , and with a higher rate in boys . our study findings of a higher likelihood of suicidal behaviors among adolescents engaging in wcb ( especially inappropriate wcb ) , confirm results described in previous studies . adolescents using inappropriate methods to control weight are highly likely to experience psychological symptoms including anxiety , fatigue , and impaired concentration ( 26 ) . additionally , there is a prospective association between wcb and depression ( 27 ) . depression in adolescence is a serious public health concern due to the increased risk of suicide among depressed youth ( 28 ) . moreover , wcb can cause eating disorders , with fasting an especially high risk factor for development of eating disorders ( 29 ) . another study among adolescents and with a 5 year follow up reported that unhealthy wcb were the most powerful predictors of eating disorders ( 18 ) . eating disorders have adverse consequences on adolescents ' physical and mental health ( 30 ) , and can cause suicide ideation and attempt ( 31 ) . therefore , one possible interpretation of this finding is that wcb represent a risk factor for suicide ideation and attempt by causing depression or eating disorders , both of which have serious consequences on adolescents ' mental health . furthermore , our study demonstrated stronger associations among boys than girls , which is corroborated by other studies ( 623 ) . girls are more sensitive to their weight and easily engage in inappropriate wcb ( 32 ) , whereas boys are not as sensitive about their weight and have a lower likelihood of engaging in inappropriate wcb ( 7 ) . therefore , it is possible that boys who consider controlling their weight in inappropriate ways may already be suffering from severe psychological distress due to experiencing immense trauma from teasing or other adverse events . we investigated differences in association between suicide ideation and attempt and wcb by conducting subgroup analyses using bmi , bwp , and bsm . in the context of bmi , underweight boys engaged in inappropriate wcb experienced the highest rate of suicide ideation and attempt than other bmi groups , and underweight girls were considerably more likely to attempt suicide . additionally , suicidal behavior was also associated with boys and girls of a normal weight engaged in inappropriate wcb . this suggests that adolescents who do not need to control their weight but engage in unnecessary wcb nonetheless are at higher risk for experienced adverse consequences ( 12 ) . with regards to bsm , the results of this study showed that misperception of body shape is also associated with suicide ideation and attempt . the media continues to portray slender bodies for girls and muscular bodies for boys as ideal body shapes , and this could have an effect on body shape misperception among adolescents ( 33 ) . this mismatch contributes to adolescent depression , which in turn increases the risk of suicide among youth ( 34 ) . moreover , this mismatch could be related to adolescents ' unnecessary weight control behaviors to obtain the ideal body shape . eventually , the effect of inappropriate wcb aggravates adverse effects from a distorted body image , and it seems that this psychological burden causes adolescents to think about or attempt suicide . we also extracted the five most common combinations of weight control methods used by korean adolescents and investigated the association between these wcb and suicidal behaviors . only conducting regular exercise was the most frequent wcb among boys and the third most common among girls . however , this combination was not significantly associated with suicidal behaviors in boys or girls . the most common wcb engaged in by girls was ' regular exercise and eating less ' , and it also did not show any significant association with suicidal behaviors . interestingly , both boys and girls engaging in ' regular exercise , fasting , and eating less ' were more likely to experience suicidal behaviors compared to those who did not conduct any wcb . although regular exercise and eating less are both appropriate wcbs , when fasting , which is classified as an inappropriate wcb , is added , this combination could contribute to adolescents ' suicidal behaviors . according to a previous study , fasting for weight control showed an association with depression when compared with other wcbs ( 35 ) . in practice , fasting causes the blood glucose concentration to fall below normal , and this status releases the hormone cortisol , which is associated with anxiety and negative feelings ( 36 ) . therefore , it seems that this combination influences suicidal behaviors due to the strong influence of fasting on depressive symptoms . this study was designed as cross - sectional study ; therefore , we could not exclude the potential bi - directional effect . in addition , the time frames of wcb ( within the past 30 days ) and suicide ideation and attempt ( within the past 12 months ) differed . therefore , a reverse - time order could not be clearly excluded ( 6 ) . moreover , responses from the kyrbs represent self - reported data , and therefore may be inaccurate . additionally , we could not obtain detailed descriptions of each wcb including frequency or amount . however , despite the limitations , this study can be generalized by using nationally representative data . additionally , compared to previous studies that only reported associations between wcb and suicidal behavior , we further investigated differences in this association in the context of bmi , bwp , and bsm . moreover , this is the first study to investigate common wcb combinations and to determine the association between the top five such combinations and suicide ideation and attempt . considering that the high incidence of suicide and the severe negative impact of adolescents ' inappropriate wcb on their health , our findings represent an important motivation for health policy makers to identify solutions for controlling adolescents ' suicide problem . inappropriate wcb among adolescents is a serious public health concern considering its widespread prevalence and harmful influence on growth , physical health , and psychosocial growth . additionally , as shown here , wcb can be associated with health compromising behaviors including suicide ideation and attempt . therefore , to protect adolescents ' healthy psychological status in the context of weight concerns , policy makers and health professionals must endeavor to correct the distorted body perception experienced by adolescents . additionally , they should help adolescents develop skills for avoiding inappropriate wcb , and encourage appropriate methods of weight control such as physical activity , fruit and vegetable consumption , and the avoidance of fatty and sweet foods . future studies should be designed regardless of gender or weight status because boys and normal or underweight adolescents are currently low priority even though they are also at risk for adverse effects due to wcb according to the present study . therefore , broader viewpoints and approaches are needed when considering adolescents ' weight - related behaviors . in the past 15 years , green fluorescent protein ( gfp ) has changed from a nearly unknown protein to a commonly used molecular imaging tool in biology , chemistry , genetics , and medicine . in 2006 , more than 10 000 papers gfps and gfp - like proteins ( i.e. , chromoproteins and fluorescent proteins ) are particularly useful due to their stability and the fact that the chromophore ( see figure 1 ) is formed in an autocatalytic cyclization of the 65syg67 sequence that does not require a cofactor . this means that unlike most other bioluminescent reporters , gfp fluoresces in the absence of any other proteins , substrates , or cofactors . furthermore , it appears that fusion of gfp to a protein does not alter the function or location of the protein . by changing residue 66 and/or the amino acid residues around the chromophore (n1c1c2c3 ) and (c1c2c3c4 ) dihedral angles of the gfp chromophore . in the protein , r1 is gly67 and r2 is ser65 , and in hbdi , an often - used model compound , r1 = r2 = ch3 . in one - bond flips ( -obf ) the dihedral rotation occurs around the torsional angle , in a -obf it is around the dihedral angle , and in a positively correlated hula - twist ( + ht ) the and dihedral angles concertedly rotate in the same direction ( as shown above ) , while in a negatively correlated hula twist ( ht ) they concertedly rotate in opposite directions . a plot of the and dihedrals for a perfectly correlated negative ht will have a slope of 1 . if the chromophore cavity is complementary with a planar chromophore , then the and best fit line will pass through the origin and all the and angles will be centered around the origin . a nonzero intercept along the or axis ( see , for example , figure 4 ) or and dihedrals centered in quadrant ii ( > 0 ; < 0 ) or quadrant iv ( < 0 ; > 0 ; see , for example , figure 8) are indications of a cavity that is not complementary with a planar chromophore . the fluorescent emission of the chromophore within gfp occurs with high efficiency ( quantum yield fl = 0.8 ) and a respectable fluorescence lifetime ( 3 ns).(19 ) when the protein is denatured the fluorescence yield decreases by at least 3 orders of magnitude.(20 ) model compounds of the chromophore do not fluoresce in solution ( quantum yield fl < 10 ) , unless the rotation of the arylalkene bond is restrained.(21 ) fluorescence can , however , be obtained by lowering the temperature to 77 k ; this freezes the solution . it has been suggested that twisting between the phenolate and imadazolidinone groups of the chromophore is the mechanism for the ultrafast fluorescence quenching internal conversion process . neutral form ( a ) of the chromophore , with the phenolic oxygen protonated , can convert to the anionic species ( b ) by going through an intermediate state ( i ) . the change from forms a to i is solely a protonation change , while the change from i to b is a conformational change with most changes occurring at thr203 . upon excitation of the a state , an excited - state proton transfer ( espt ) occurs in which the proton is transferred from the chromophore to glu222 in a time scale of the order of picoseconds . following radiative relaxation from the excited - state intermediate ( i * ) , the systems returns to the ground state a through the ground - state intermediates i1 and i2.(29 ) excitation of the anionic b state results in direct emission from the excited state ( b * ) at 482 nm . recently , a nonfluorescent dark state , state c , has been observed that is distinct from states a and b and absorbs at higher energies.(30 ) the c state , perhaps the neutral trans form of the chromophore , may be populated by nonradiative decay from a * and it may be depopulated by excitation to the excited c * state with transcis isomerization to repopulate state a. fluorescent blinking has been ascribed to nonadiabatic crossing and conversions between the neutral and anionic states,(31 ) or a possible dark ( z ) zwitterionic state . while the ground - state minimum of the gfp chromophore is close to planar , this is not necessarily so for the excited state ; in fact , in some cases the excited state has an energy minimum with a twisted chromophore in which both rings are 90 to each other.(32 ) according to quantum mechanical calculations , the ground and excited states for the one - bond flip ( obf ) and hula twist ( ht ) in the neutral form ( a ) and the obf in the zwitterionic form come very close to each other . it has been proposed that in the absence of the protein matrix , which surrounds the chromophore and prevents twisting , this process can lead to fluorescence quenching internal crossing;(32 ) see figure 3 . recent calculations on the gfp chromophore model compound hbdi suggest that the anionic form of hbdi may also undergo a -obf that leads to a favored radiationless decay channel , which is particularly efficient in solvent.(34 ) ( a ) model compounds of the gfp chromophore in the ground state ( s0 ) can be excited to the first singlet state ( s1 ) in which a ht or obf can freely occur . upon reaching the perpendicularly twisted conformation , fluorescence quenching nac ( nonadiabatic crossing ) occurs . ( b ) in the ground state ( s0 ) , the residues surrounding the gfp chromophore exert a twisting force on the chromophore ( ) . upon excitation , the conjugation across the ethylenic bridge of the chromophore is reduced and it will twist ; however , the protein matrix prevents the chromophore from reaching the perpendicularly twisted conformation ( ) and fluorescence quenching internal crossing is prevented . recently , the results from a number of interesting experiments that provide evidence for chromophore twisting and/or fluorescence quenching internal crossing ( ic ) have been published . for example , a molecular dynamics simulation of the chromophore of cyan fluorescent protein found that the average dihedral angle was about 0 ; however , when the surrounding protein was considered in the simulation the average shifted by approximately 5 , showing that the protein matrix of cfp twists the chromophore . on the basis of their calculations , the authors concluded that the driving force for this twist comes from the strong short - range repulsion by four residues ( ile167 , val150 , phe165 , and thr203 ) surrounding the average position of the chromophore.(46 ) photoswitching fluorescent proteins can be switched back and forth between the naturally occurring green state and a dark state by 405 nm irradiation ( e.g. , dronpa , mtfp0.7 , kfp1 ) . a cistrans isomerization of the chromophore has been proposed as the structural basis for the photoswitching observed in dronpa.(47 ) this is supported by the fact that mutating either val157 or met159 with smaller residues accelerates photoswitching , presumably by decreasing steric hindrance to cistrans isomerization.(48 ) the m159 t and v157 g mutations also decrease the quantum yield of dronpa from 0.85 to quantum yields of 0.23 and 0.77 , respectively.(48 ) recently , it has been suggested that adoption of a trans configuration can not solely be responsible for the nonfluorescent form.(49 ) on the basis of nmr analyses , miyawaki et al . propose that the fluorescence of the protein is regulated by the degree of flexibility of the chromophore but is not necessarily accompanied by cistrans isomerization.(49 ) interestingly , gfp is not unique in photoactive yellow protein ( pyp ) the protein matrix also prevents the chromophore from adopting a completely planar structure . in pyp the asymmetric proteinchromophore interaction probably serves as the initial accelerant for the light induced photocycle,(50 ) which ultimately leads to a cistrans isomerization.(51 ) the chromophore in wild - type gfp is planar due its extended system ( see figure 1 ) ; however , the energy barrier to deformation is low and the protein matrix in wild - type gfp exerts some strain away from planarity . when the chromophore is computationally permitted to freely rotate , it will adopt a conformation that complements the protein matrix . recently , we have used computational methods to show that wild - type gfp is not an anomaly and that all gfp and gfp - like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore.(54 ) in most cases the freely rotating chromophore undergoes rotations of at least 20. in some cases these rotations are accompanied by an equal but opposite rotation of the dihedral angle ( a negatively correlated ht ) . none of the proteins examined have a cavity that causes a rotation solely around the dihedral angle.(54 ) these calculations were done by minimizing , with freely rotating and dihedral angles , the crystal structure of 38 gfp analogues and mutants found in the pdb . they found the energy minimum conformation of a freely rotating chromophore in the protein matrix of the gfp mutant or gfp - like protein examined ; however , they did not provide any information about the range of low - energy conformations available to a freely rotating chromophore . to get this information , we ran molecular dynamics simulations of some of the interesting gfp mutants in the protein databank.(56 ) by running molecular dynamics simulations , with freely rotating and dihedrals , we have been able to determine the range of conformations available to chromophores with complete rotational freedom . the coordinates of three crystal structures ( 1gfl,(57 ) 1myw,(58 ) 2emd(59 ) ) were obtained from the protein data bank ( pdb),(56 ) and hydrogen atoms were added to protein and solvent atoms as required . the opls_2005 force field of macromodel v9.0016(60 ) was used . starting structures for mutants for which no crystal structure has been determined were calculated by graphically mutating a known structure and undertaking a conformational search . the flexible dihedral angles of all the side chains of residues that are within 8.00 of the chromophore were randomly rotated by between 0 and 180 , and all solvent molecules in that sphere were randomly rotated and translated by between 0 and 1.00 in each monte carlo ( mc ) step.(63 ) 15 000 mc steps were taken in each search . structures within 50 kj / mol of the lowest energy minimum were kept and a usage - directed method(62 ) was used to select structures for subsequent mc steps . structures found in the conformational search were considered unique if the least - squared superimposition of equivalent non - hydrogen atoms found one or more pairs separated by 0.25 or more . the lowest energy structure obtained in the monte carlo torsional and molecular position variation search the final structures obtained from the mc search ( monte carlo torsional and molecular position variation and large - scale low mode ) or fully minimized pdb structures were used to initiate md simulations with freely rotating and dihedral angles ( v1 = v2 = v3 = 0.000 ) . the predynamics simulation was set for 100 ps and the full md simulation for 5000 ps . the cambridge structure database ( csd ) v5.29 was released in january 2008 ; it comprises 436 384 small molecule crystal structures . the area of the convex hull of the and graphs was calculated as was the smallest convex set containing the region . molecular mechanics and dynamics calculations have been used to examine the steric environment of the chromophore in gfp in its ground state . they are techniques that are based on classical physics and were designed to model structural and not electronic properties ; therefore , molecular mechanics and dynamics simulations of gfp can not examine the excited state of the chromophore . quantum calculations are an excellent technique to determine the energy profiles of the ground and excited states of the chromophore ; however , they are cpu - intensive and it would be cost prohibitive to do conformational searches on the excited state of a series of gfp and gfp - like proteins . therefore , in an attempt to supplement the quantum calculations , we have examined the conformational space available to the chromophore within gfp using a freely rotating chromophore that is an approximation ( based on qm calculations ) of the conformational space available to the chromophore in its excited state . as shown in figure 2 , gfp adopts two states , the neutral state ( a ) of the chromophore with the phenolic oxygen protonated , and the anionic species ( b ) the change from forms a to i is solely a protonation change , while the change from i to b is a conformational change with most changes occurring at thr203.(67 ) in order to see whether the gfp chromophore has different dihedral freedom in forms a , b , and i , we conducted molecular dynamical simulations with a freely rotating chromophore , as described in the . not surprisingly , table 1 and figure 4 show that forms a , b , and i of gfp have similar dihedral freedom and that all undergo similar negatively correlated ( same slopes of best fit lines in figure 4 ) hula twists . plot of the vs dihedral angles ( see figure 1 for nomenclature ) for the 2000 gfp - a ( pink ) , gfp - b ( green ) , and gfp - i ( light - blue ) structures obtained in the freely rotating molecular dynamics simulation . however , there are subtle differences ; forms a and i are closer to the planar ground - state conformation , while the b form adopts conformations further from planarity when given rotational freedom around the and dihedrals ( intercept further from = 0 ) ; and the anionic b form has the most freedom ( largest area , and and range ) , while the neutral a form has the least freedom . the a form has robust hydrogen bonds between both arg96 and gln94 and the imidazolidinone carbonyl group that remain intact throughout the simulations . in the intermediate ( i ) form these hydrogen bonds are supplemented by an additional hydrogen bond between the phenolic oxygen of the chromophore and his148 ( see table 2 ) . the main difference between the anionic b form and the intermediate form is that thr203 has to rotate in order to hydrogen bond to the phenolic oxygen in form i , otherwise they have the same hydrogen - bonding interactions throughout the simulation . the y66h mutant of gfp exhibits blue fluorescence and has therefore been termed blue fluorescent protein . the crystal structure of bfp ( not to be confused with blue fluorescent protein from aequorin bfp - aq(68 ) ) has been solved at ph 4.5(27 ) and ph 8.5;(59 ) the overall fold of the protein is identical to wild - type gfp and consists of a chromophore surrounded by an 11-stranded -barrel . while gfp has absorption maxima at 395 and 475 nm and emits at 508 nm , bfp absorbs at 382 nm and emits at 448 nm.(9 ) unfolding of bfp results in an absorption red shift of 15 nm,(27 ) and quantum mechanical calculations suggest that the 15 nm shift might be due to the protein - induced nonplanarity of the chromophore.(69 ) blue fluorescent protein ( bfp ) has a much lower fluorescence quantum yield than gfp ( fl = 0.20 vs 0.80 ) . it has been suggested that this is due to the fact that his66 ( bfp - chromophore ) forms fewer hydrogen bonds with the surrounding protein than tyr66 ( gfp - chromophore ) does , and that the smaller imadazole ring ( his66 ) in bfp may have more conformational freedom than the larger phenol ( tyr66 ) , which leads to more intersystem crossing.(27 ) in a very elegant series of experiments , boxer et al . have shown that the fluorescence quantum yield of blue fluorescent protein increases from fl = 0.20 to 0.35 when the pressure is increased from atmospheric pressure to 570 mpa.(70 ) analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes . temperature - dependent fluorescence measurements reveal two barriers ( 19 and 3 kj / mol , respectively ) for the transition into nonfluorescing states . these steps are probably linked with dissociation of the hydrogen bond between the chromophore and his148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state , respectively.(70 ) in order to establish the consequences of the y66h mutation on the flexibility of the chromophore , molecular dynamics simulations of the ph 8.5 bfp crystal structure ( pdb code 2emd with neutral imidazole rings for his66 and his148 ) with a freely rotating chromophore were run . [ we thank one of the referees for suggesting the ph 8.5 ( 2emd ) over the ph 4.5 ( 1bfp ) structure . ] figure 5 and table 1 show that the chromophore in bfp has significantly more rotational freedom than that available to the chromophore in gfp , and that the bfp cavity is less complementary to a planar chromophore than the gfp cavity is ; that is , the bfp sampled structures are further from a planar chromophore ( = = 0 ) and the intercept for the best fit line through the bfp structures is further from the origin than that of gfp . plot of the vs dihedral angles for the 2000 bfp ( blue ) and gfp - a ( green ) structures obtained in the freely rotating molecular dynamics simulation . analysis of the 2000 bfp structures revealed a hydrogen bond between the nh of the his66 imidazole ring and glu222 , which is retained through - out the entire simulation ( see figure 7 ) . the smaller imadazole ring of the bfp chromophore results in more dihedral freedom for the chromophore in bfp than phenol in gfp ; however , there seems to be no large difference in the number and stability of hydrogen bonds formed by the chromophore in the two fps . recently , two new blue fluorescent proteins ( azurite and a5 ) with enhanced brightness and photostability were created . the methodology applied to find the brighter bfp mutants was based on the concept that replacing the residues surrounding the chromophore with bulkier amino acids would constrain the chromophore s motion and thereby increase the proteins brightness . the crystal structure of a5 has yet to be solved . in order to find a starting conformation for the md simulation , the 2emd crystal structure was graphically mutated to a5 and a thorough conformational search was undertaken ( see ) . given the high structural similarity between all the solid state structures of gfp mutants in the protein databank , a thorough conformational search should find the lowest energy conformations of a5 by making the relevant mutations to the 2emd structure . figure 6 and table 1 show that the conformational space available to the chromophore in bfp is larger than that in a5 and that the bfp conformations tend to be further from planarity ; i.e. , for a5 median = 2.92 , median = 2.22 and intercept of best - fit line is 4.62 vs for bfp median = 27.59 , median = 10.18 and intercept of best - fit line 14.27 . similar results were found for md simulations carried out from all of the five lowest energy conformational families sampled in the conformational search of a5 and in a md simulation initiated from an a5 structure obtained by taking the 2emd structure , graphically mutating it to a5 , and thoroughly minimizing the structure without undertaking a conformational search . plot of the vs dihedral angles for the 2000 bfp ( dark blue ) and a5 ( pink ) structures obtained in the freely rotating molecular dynamics simulation . the n of the his66 imidazole ring hydrogen bonds with water305 ( 2emd numbering ) in all the sampled structures . this water connects his66 to the surface of the protein via a robust hydrogen - bonding network to his148 and is also hydrogen bonded to glu222 . in half the structures sampled , the n of the his66 imidazole ring hydrogen bonds to water302 which is in an extensive hydrogen - bonding network with val68 , arg224 , and gln69 . in contrast to 2emd there are no hydrogen - bonding interactions with the n hydrogen ; see figure 7 . hydrogen - bonding interactions ( --- ) for 2emd ( top ) and a5 ( bottom ) chromophore . these results seem to indicate that the enhanced brightness of a5 is at least in part due to the fact that its chromophore movement is restricted relative to bfp . this restriction can be due to both steric factors and the increased hydrogen - bonding networks in a5 . in the first rationally designed mutant based on the crystal structure of gfp - s65 t , it was decided to mutate t203 into a tyrosine so that it could stack with the phenolic group in the chromophore . ( 73 ) the resultant mutant , yfp , is red - shifted by 16 nm relative to gfp - s65 t and does indeed have a stacking interaction between the chromophore and tyr203.(74 ) on the basis of the crystal structure of yfp , remington et al . proposed that the red shift was due to the additional polarizability of the -stacked tyr203 , the hydrogen - bond pattern around the chromophore , or an out - of - plane distortion of the chromophore ( in analogy with out - of - plane distortions in porphyrin systems).(74 ) comparison with the t203v mutant revealed that the t203y substitution leads to a significant i - form population and only about 10 nm of the shift can be ascribed to the interaction.(75 ) a new varient of yfp , venus , with improved brightness and maturation properties as well as a reduced environmental sensitivity was developed and crystallized.(58 ) since some researchers have argued that the presence of tyr203 leads to a decrease in chromophore flexibility ( van der waals volume for tyr = 141 vs 93 for thr ) , and others argue that this is not necessarily so,(32 ) we have examined the conformational space available to a freely rotating chromophore in yfp ( pdb code 1myw ) with the phenol of tyr66 in both the protonated and unprotonated forms . both table 1 and figure 8 show that the chromophore ( in the phenolic form ) in gfp and yfp has differing rotational freedom . in yfp the chromophore has approximately 1.2 times more dihedral freedom than in gfp and although the dihedrals undergo negatively correlated hula - twisting in both proteins , in yfp the average conformation sampled is further from planarity than that found in gfp ; i.e. , the majority of the sampled conformations are located in quadrant iv . plot of the vs dihedral angles ( see figure 1 for nomenclature ) for the 2000 structures obtained in the freely rotating molecular dynamics simulation of gfp ( dark blue ) and yfp ( pink ) in their phenolic forms . the majority of the yfp conformations sampled are nonplanar ( i.e. , in quadrant iv < 0 and > 0 ) . during the entire simulation the hydrogen bonds between tyr66 and ser205 , arg96 and the imidazolone carbonyl , as well as between tyr203 and water354 remain stable , while those between tyr203 and gln69 were less stable . we also examined the distance between the centroid of tyr66 ( the choromophore ) and tyr203 . the distance is fairly short and does not change much during the simulation ( minimum = 3.43 , maximum = 4.18 , average = 3.75 , sd = 0.103 ) while the angle between the planes of the phenols varies between 0.0 and 22.5 ( average = 6.9 , sd = 4.11 ) . the short distance between the phenol rings is indicative of stacking , which may be responsible for the red shift observed in yfp ; however , the variability in the angle between the planes of the phenols shows that tyr203 does not significantly restrict the rotational freedom of the excited chromophore . the cambridge structural database ( csd)(77 ) v5.29 comprises 436 384 small molecules crystal structures . it has 5551 structures with two phenol rings in separate molecules that have centroids within 5.00 of each other . the closest ones are 3.16 from each other , and only 832 structures are within 3.75 ; i.e. , the average distance between the phenol centroids in the freely rotating yfp . a plot of the distance vs the angle between the phenol planes in our freely rotating simulation and the csd structures shows that the protein matrix of yfp restricts the distance between tyr66 and tyr203 from being larger than 4.18 , but that the angles between the two phenols have as much freedom in yfp as in the small molecule structures with intermolecular distances of less than 4.18 ( see figure 9 ) . it does not seem as if the increased fluorescence lifetime of yfp is due to a decrease in the dihedral freedom of the chromophore . it is more likely due to electronic effects such as those proposed by jung et al.(78 ) they suggested that the anionic form of the chromophore can be described by two mesomeric forms , the benzoidal and the quinoidal structure . the benzoidal form with the majority of its negative charge on the phenol is stabilized by hydrogen bonding to thr203 , which is not possible in yfp and therefore the t203y mutation favors the quinoidal form , which may have a longer fluorescence lifetime.(78 ) distance vs angle between the phenol planes in yfp ( ) and all structures in the csd that have two unconnected phenols separated by less than 5.00 ( + ) . some have suggested that the crystal structures of strongly fluorescent gfp and gfp - like proteins have their chromophores in a cis configuration , while those of the nonfluorescent proteins are in a trans configuration . however , the trans - planar form of eqfp611 is an important exception : it is fluorescent.(82 ) remington et al . have suggested that coplanarity might be more important than isomer configuration in determining the efficiency of fluorescence . most current theories ( see ) suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield . our calculations show that this is the case for the systems examined . for bfp , a5 , yfp , and gfp , there is an inverse correlation between the dihedral freedom of the chromophore and the quantum yield ; see table 1 . in all simulations , the protein matrix is not complementary with a planar chromophore , and in all cases the freely rotating chromophore undergoes a negatively correlated hula twist ( also known as a bottom hula twist mechanism ) .

suicide is a leading cause of death among adolescents globally , and body weight is also a recognized reason for adolescent suicide . therefore , we investigated the association between weight control behaviors ( wcb ) and suicide ideation and attempt , focusing on inappropriate weight control measures . we used data from the 2014 korea youth risk behavior web - based survey , representing a total of 35,224 boys and 34,361 girls aged 12 to 18 years . adolescents were classified into groups based on wcb : appropriate wcb , inappropriate wcb , and no wcb . we performed logistic regression models to examine associations between wcb and suicide ideation and attempt , controlling for covariates . both boys and girls with inappropriate wcb were more likely to report suicide ideation and attempt . underweight and normal weight boys with inappropriate wcb were more likely to think or attempt suicide , and underweight girls with inappropriate wcb were also more likely to attempt suicide . among five common wcb combinations , the combination of " regular exercise , fasting , eating less " was highly associated with suicide ideation and attempt . we confirmed that inappropriate wcb is associated with suicide ideation and attempt among korean adolescents . given the high incidence rate of suicide among adolescents and the adverse effect of inappropriate wcb , encouraging adolescents to control their weight in healthy ways is imperative . green fluorescent protein ( gfp ) and gfp - like fluorescent proteins owe their photophysical properties to an autocatalytically formed intrinsic chromophore . according to quantum mechanical calculations , the excited state of chromophore model systems has significant dihedral freedom , which may lead to fluorescence quenching intersystem crossing . molecular dynamics simulations with freely rotating chromophoric dihedrals were performed on green , yellow , and blue fluorescent proteins in order to model the dihedral freedom available to the chromophore in the excited state . most current theories suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield . according to our calculations , the dihedral freedom of the systems studied ( bfp > a5 > yfp > gfp ) increases in the inverse order to the quantum yield . in all simulations , the chromophore undergoes a negatively correlated hula twist ( also known as a bottom hula twist mechanism ) .

fnas represent an advantageous , effective means to obtain diagnostic cellular material as they can often sample a wide area of the target lesion , acquire tumor cells with lower contamination by background stromal connective tissue elements , and allow for immediate assessment during rapid on - site evaluation ( rose ) . during the on - site assessment , a member of the cytopathology team is present and prepares direct smears , using the contents expelled from the needle , at the location of the procedure . this is advantageous for three reasons : each needle pass can be examined to determine tumor cell adequacy ; there is an opportunity to engage the clinical care provider in a conversation regarding the preliminary diagnosis and relevant molecular diagnostic tests ; and the cytopathology team member can help ensure that the specimen is processed in a manner that optimizes judicious triage for ancillary tests , including molecular studies . fnas can be performed without rose and in this setting , the contents of the fna needles are typically expelled and rinsed in a cell preservative solution for use in liquid - based cytology ( lbc ) preparations . examples of lbc preparations include thinprep ( hologic , bedford , ma , usa ) , surepath ( becton dickinson , franklin lakes , nj , usa ) , thin - layer advanced cytology assay system ( tacas , mbl , tokyo , japan ) , and liqui - prep ( lgm international inc . , nonetheless , rose is an effective means to maximize the chances of success in acquiring adequate tumor cells for diagnosis and anticipated molecular studies . the ultimate goal is to prevent unnecessary repeat procedures to obtain additional tissue just for molecular studies which can lead to delays in treatment . for patients afflicted with lung cancer at our institution , we perform rose for cytopathologist - performed fnas and for ebus - guided fnas , performed by our clinical colleagues . the contents of each needle pass are expelled onto a slide which is utilized to prepare direct smears . the needle is then rinsed into buffered media ; we utilize rpmi media for this purpose . commonly , a pair of direct smears is prepared per needle pass ; one is air - dried and the other is immediately alcohol - fixed . the air - dried smear is stained on - site with the diff - quik ( romanowsky ) stain and the stained slide can be examined under the microscope immediately thereafter . the alcohol - fixed smear is stained later in the cytopathology laboratory with the papanicolaou stain . alternatively , the needle contents can be distributed over multiple smears allowing for flexibility in the utilization of direct smears for cytomorphologic evaluation and ancillary studies ( fig . based on the findings in the diff - quik stained smears , the determination can be made to perform additional needle passes to obtain more tumor cells for diagnosis and/or anticipated ancillary studies while the patient is still accessible . for instance , additional direct smears can be prepared and/or dedicated needle passes for the rinse solution ( i.e. , the needle contents are rinsed in the rpmi solution without the preparation of smears for those needle passes ) obtained for the cell block preparation . this overall approach is flexible , forgiving , and engages the cytopathology team in maximizing the chance of success in obtaining adequate cellular material for diagnosis and ancillary studies . this approach can help minimize the chances of encountering the scenario in which diagnostic cellular material is present on only one smear ; microdissection of the cells for molecular studies can result in sacrificing this only diagnostic slide in this context , which can have medico - legal consequences . if this scenario is encountered , however , this risk can be mitigated by digital archiving prior to microdissection either via digital slide scanning and/or obtaining photomicrographs . after immediate assessment of the cytomorphologic findings on the diff - quik stained smears , a preliminary diagnosis can be rendered by the cytopathologist and communicated to the clinical care providers . after the conclusion of the procedure , the needle rinse solution , containing a suspension of aspirated cells , is centrifuged in the cytopathology laboratory to pellet the cells . once the supernatant is removed , the cell pellet is congealed in a matrix ; an agar - like substance such as histogel ( thermo scientific , waltham , ma , usa ) can be used or the cells can be mixed with plasma and thrombin to create a clot . at our institution , we employ histogel for this purpose . commonly , this cassette is fixed in formalin and processed to ultimately create a formalin - fixed , paraffin - embedded ( ffpe ) cell block . this is analogous to a ffpe block of small biopsy tissue , which can be sectioned for evaluation via the hematoxylin and eosin stain and ancillary tests such as immunocytochemistry or molecular diagnostic assays . it should be noted that heavy metal fixatives such as zenker s fixative and acidzinc - formalin , acidic fixatives such as bouin s solution , or decalcification solutions should generally be avoided as these render specimens unusable for molecular testing . the main advantage of using cell blocks is that the majority of ancillary tests are validated for ffpe sections ; ffpe cell blocks are treated similarly to traditional surgical pathology ffpe blocks . furthermore , multiple serial sections from cell blocks can be utilized to perform a battery of ancillary studies . first , adequate cellularity in cell blocks is not guaranteed at the time of the procedure . performing dedicated needle passes , for the needle rinse solution during the fna procedure , may improve the chances of obtaining a sufficiently cellular cell block but still does not guarantee success . in addition , for hypocellular cell blocks , there is a risk of depleting the cells of interest upon deeper sectioning for molecular tests . second , the cell block is derived from a needle rinse solution which is a pooled specimen that contains contents of all the needle passes . thus , if one or more needle passes contain tumor cells but other needle passes contain abundant benign background cellular elements , the tumor cells will be diluted by these benign cells in the cell block . in addition to the above disadvantages , dna extracted from ffpe cell blocks may produce sequencing artifacts as formalin fixation leads to the crosslinking of nucleic acids and proteins and the possibility of sequence alterations . recently , given the shortcomings of cell blocks , there has been an increasing appreciation of alternative cytopreparatory platforms for molecular testing such as direct smears and lbc samples . in the subsequent sections below , the utilization of these cytopreparatory platforms will be discussed in the context of the clinically relevant mutations and translocations analyzed during molecular diagnostic testing of nsclcs . the epidermal growth factor receptor ( egfr ) gene encodes a transmembrane growth factor receptor that exhibits tyrosine kinase activity . upon activation , intracellular signaling is mediated by cytoplasmic effectors in the ras - raf - mek - erk , pi3k - akt , and stat pathways . egfr mutations are predominantly observed in lung adenocarcinomas ; the l858r substitution and small in - frame deletions in exon 19 are the most commonly observed mutations and account for up to 90% of all egfr mutations in this setting . these mutations are more commonly associated with east asian ethnicity , female gender , and non - smoking history . however , these are not absolute rules and clinical characteristics should not be used to exclude lung cancer patients from mutation testing . lung adenocarcinomas harboring these sensitizing mutations in egfr have been shown to respond to egfr tyrosine kinase inhibitors ( tkis ) . , tki therapy has been demonstrated to result in improved progression - free survival , compared to standard chemotherapy , for patients with lung adenocarcinoma harboring egfr mutations [ 11 - 16 ] . egfr mutation analysis is commonly performed via polymerase chain reaction ( pcr ) and sequencing - based approaches ; advances in the development of testing modalities have afforded a multitude of methodologies . sanger sequencing is considered the gold standard as this involves direct dna sequence acquisition and can provide information regarding the presence of all potential mutations including common , known mutations and novel mutations . nonetheless , this test requires a relatively higher enrichment of tumor cell dna content in the sample . the typical analytic sensitivity for sanger sequencing is 15%20% mutant allele , which equates to 30%40% tumor cells assuming that the genetic mutation is a heterozygous event without amplification . this can be problematic in both small biopsy and cytology specimens , especially cell blocks , in which the tumor cell population can be diluted by background benign cellular elements such as inflammatory cells , bronchial epithelial cells , and/or stromal mesenchymal cells . especially in this setting , a negative mutation result can be either due to the true absence of the mutation in the tumor cells or insufficient percent tumor cellularity that falls below the analytic sensitivity threshold thereby resulting in the failure to detect the mutation even despite the presence of the mutation . therefore , often times , there is more reliance on tumor cell enrichment by either macrodissection or microdissection to obtain a reliable result . sanger sequencing is also relatively more labor intensive and time consuming than targeted methods and can lead to longer turnaround times . in contrast to the general sanger sequencing approach , targeted mutation detection methods such as pcr - restriction fragment length polymorphism , real - time pcr , pyrosequencing , high resolution melting analysis ( hrma ) , and pcr fragment analysis can be utilized . the advantages of these approaches include their improved analytic sensitivity and less time - consuming nature leading to reduced turnaround times . at our institution , we utilize a multiplex pcr fragment analysis assay for egfr mutation testing ; this allows for the simultaneous assessment of the two most commonly observed egfr mutations ( fig . 2 ) . the analytic sensitivity of this method is better than that of sanger sequencing ; a minimum of only 10% tumor cells is required . in the past decade , myriad studies have been reported demonstrating that a variety of cytologic samples and cytopreparatory platforms can be effectively utilized for egfr mutational analysis . these have been reviewed elsewhere but salient examples will be discussed below . as mentioned previously , ffpe cell blocks represent the traditional cytopreparatory platform on which ancillary molecular diagnostic tests are performed . much of the reasoning behind this lies in that ffpe cell blocks best approximate ffpe blocks containing tissue specimens and the majority of the molecular assays are validated using ffpe material . nonetheless , in light of the inherent disadvantages of cell blocks mentioned previously , investigation into other cytopreparatory platforms for molecular diagnostic assays have been performed by our group and many others . cytopathology specimen preparation is diverse and versatile ; the consequence of this is that each type of cytologic analyte platform needs to be carefully validated for any given molecular test . cytologic direct smears may ultimately prove to be the cytologic platform best suited for pcr - based analysis due to the high quality of nucleic acids and immediate nature of specimen assessment for tumor cell adequacy . cells on direct smears are not exposed to formalin but rather undergo an alcohol - based fixation process prior to staining ; thus , the negative effects of formalin fixation on nucleic acid quality is non - contributory for direct smears . furthermore , the tumor cells of interest can be directly visualized on the direct smear and isolated for nucleic acid extraction and molecular analysis ; in essence , what you see is what you get . we have previously demonstrated that uncoverslipped and previously coverslipped / decoverslipped diff - quik stained smears are robust sources of cellular material for pcr - based mutational analysis such as egfr mutation testing of lung adenocarcinoma and braf mutation testing of metastatic melanoma fna specimens . we and others have observed that destaining the smears is not necessary for successful dna extraction and pcr . our preference in utilizing diff - quik stained smears over papanicolaou stained smears is rooted in the following observations : ( 1 ) cellular material on a diff - quik stained smear can be easily visualized without a coverslip and immediately triaged for molecular diagnostic testing ; and ( 2 ) the report by killian et al . that nucleic acids extracted from diff - quik stained smears show better preservation and integrity than dna extracted from papanicolaou stained smears . with respect to the latter point , others have pointed out that papanicolaou stained smears are just as feasible for pcr - based molecular tests . multiple groups have successfully demonstrated the utilization of cytologic direct smears for egfr mutation testing of lung adenocarcinomas [ 2,9,25 - 36 ] with low failure rates and high degree of concordance with egfr molecular testing results from corresponding histologic specimens , when available . in our experience , 100 to 200 tumor cells are sufficient for successful dna isolation and egfr mutation analysis and this is congruent with the observations from others . lbc samples have also been investigated for egfr mutation testing and have been shown to be an effective analyte for this purpose . this is valuable when a member of the cytopathology team is not available for rose . several lbc technologies have been developed including thinprep , surepath , tacas , and liqui - prep . of these , the emerging literature , to date , on the utilization of lbc samples for molecular testing of nsclc have predominantly focused on cytolyt ( cytyc corp . , marlborough , ma , usa ) cell suspensions and thinprep slides . collecting fna samples in the methanol - based cytolyt solution results in reduction of background blood as the cytolyt solution exhibits hemolytic properties . the cell suspension can be divided and an aliquot is used to prepare a thinprep slide that is stained via the papanicolaou method , similar to alcohol - fixed papanicolaou stained direct smears . tumor cells can be isolated from the thinprep slide and used for dna isolation and egfr mutation analysis . furthermore , the cell suspension aliquot that is not used to prepare a thinprep slide can be centrifuged and the cell pellet itself can be used as an analyte for mutation testing [ 38 - 40 ] . similar to the needle rinse cell suspension utilized to prepare cell blocks , the cytolyt cell suspension is a pooled specimen if multiple needle passes are expelled and rinsed into cytolyt . thus , if one or more needle passes are high in tumor cell content whereas other needle passes contain a high proportion of benign cellular elements , the tumor cells in the final pooled cell suspension will be diluted . first , targeted methods for detecting egfr mutations with improved analytic sensitivity , relative to sanger sequencing , can be utilized . second , tumor cell enrichment from the thinprep slide can be accomplished by laser capture microdissection ( lcm ) . to illustrate these points , malapelle et al . directly compared the performance of egfr mutation analysis by sanger sequencing using paired samples for each case analyzed : ( 1 ) pelleted cells from the cytolyt suspension and ( 2 ) tumor cells obtained from the thinprep slide via lcm . they observed that egfr mutations were more reliably identified in the latter rather than the former . this difference was minimized when utilizing more sensitive egfr mutation analytic approaches which included hrma and pcr fragment analysis assays . based on these studies , the authors speculated that coupling the use of highly sensitive egfr mutation detection assays and cytolyt derived cell pellets may be sufficient obviating the need for microscopy . nonetheless , it must be emphasized that any negative molecular testing result should carefully be reconciled with the analyte input utilized for the mutation assay . this is essential to determine whether the negative result represents a true negative or potentially a false - negative , necessitating possible retesting . in this regard , the utilization of microscopy still remains an essential pre - analytic quality assurance activity to best ensure that the input analyte is of sufficient tumor cellularity to maximize confidence in the results of the mutation assay . of note , a follow - up study by bellevicine et al . they observed that the direct smears exhibited significantly higher cellularity than thinprep slides , on average . accordingly , the average yield of dna extracted from direct smears was significantly higher than that from thinprep slides . as the l858r substitution and deletions in exon 19 represent approximately 90% of all egfr mutations in lung adenocarcinoma , one antibody is specific for the exon 21 l858r mutation and the other specific for the 15-base pair/5-amino acid deletion ( e746_a750del ) in exon 19 ( clone 6b6 ) . the immunohistochemical approach to detecting mutant egfr proteins using these antibodies has been examined in several studies on lung cancer tissues as well as cytologic and small biopsy samples [ 42 - 48 ] . the sensitivity of these assays range from 47%92% but their high positive predictive value and specificity supports the feasibility of utilizing this approach as a first - line screening approach . first , immunohistochemistry should be performed after careful validation and formulation of immunostain scoring criteria . the significance of how best to interpret equivocal staining results should be clarified as overinterpreting weak immunoreactivity as a positive mutation result can lead to increased false positives and decreased specificity . second , the clone 6b6 antibody best detects the 15-base pair ( e746_a750del ) egfr deletion mutant protein but demonstrates variable immunoreactivity for the egfr mutant proteins resulting from non-15-base pair deletions in exon 19 . the anaplastic lymphoma kinase ( alk ) protein is a receptor tyrosine kinase and rearrangements involving the alk gene locus is observed in approximately 5% of lung adenocarcinomas . these mutations are more commonly observed in younger age patients who are never - smokers . however , there can be exceptions to this and clinical characteristics should not be used to exclude lung cancer patients for alk rearrangement testing . most alk rearrangements in lung adenocarcinoma result from interstitial deletions and small inversions within the short arm of chromosome 2 . this results in the fusion of portions of the echinoderm microtubule - associated protein - like 4 ( eml4 ) and alk genes . other less common alk rearrangements involve fusions between alk and other genes , such as kif5b and tfg . alk rearranged lung adenocarcinoma has been recognized as a legitimate target for small molecular inhibitor therapy ; crizotinib was shown to be efficacious in treating patients with these cancers . in a phase 1 study evaluating 143 patients , an overall response rate of 61% and estimated overall survival rates of 74.8% at 12 months therefore , the evaluation of lung adenocarcinoma fna samples for alk rearrangements , in addition to egfr mutations , has become increasingly incorporated into patient management algorithms . fish is currently the preferred approach to assaying lung adenocarcinomas for alk rearrangements according to expert recommendations based on review of the literature . in the united states of america , there is only one test approved by the food and drug administration ( fda ) for this purpose . this assay utilizes a dual alk breakapart probe strategy in which orange and green labeled probes hybridize to the highly conserved translocation breakpoint region in the alk gene . alk gene loci that have not undergone rearrangement typically display fused orange and green signals ( yellow ) or juxtaposed touching orange and green signals . when an alk rearrangement occurs , the orange and green signals become separated . however , as the majority of the alk rearrangements involve a small inversion within chromosome 2p , rather than a rearrangement involving another chromosome , the extent to which the two signals are split is finite . in order to score a nucleus as positive for the alk rearrangement , the orange and green signals must be separated by a distance of > 2 signal diameters ( fig . 3 ) ; a nucleus can also be scored positive if a single orange signal without a corresponding green signal is observed . typically , up to 100 tumor cell nuclei are scored in this assay and a lung adenocarcinoma is considered positive for the alk rearrangement if at least 15% of the nuclei are scored as positive for the rearrangement . the fda has approved this dual breakapart probe set for use on paraffin embedded tissue sections . as mentioned previously , paraffin embedded cell blocks are processed similarly to paraffin embedded tissue blocks ; thus , cell blocks are traditionally used for the performance of alk rearrangement fish assays . nonetheless , there is an observed noticeable failure rate when using and relying on cell blocks for these assays due to insufficient tumor cell material in a significant proportion of cases . thus , we and others have investigated alternative cytopreparatory platforms for alk fish . in our study , we demonstrated the effective use of diff - quik stained direct smears for this purpose and observed that the performance of this assay using direct smears was better than the performance using cell blocks . . also examined the use of papanicolaou stained direct smears and observed that the failure rate due to insufficient cellularity was significantly higher for cell blocks than for direct smears . therefore , the utilization of both diff - quik and papanicolaou stained smears are feasible analytes for alk fish . recently , thinprep slides have also been shown to be a feasible platform for alk fish . the advantage of utilizing cytologic preparation platforms such as direct smears and thinprep slides for this purpose is that entire tumor cell nuclei are being analyzed . fish evaluation on paraffin sections derived cell blocks and other ffpe blocks are prone to signal loss in some of the tumor cells due to section truncation artifacts . in addition , alternative methods to fish have been the subject of recent investigation . immunohistochemistry utilizing antibodies directed against alk is an attractive alternative as it is simpler , quicker , and less expensive . the challenge associated with this approach is that the alk protein is expressed at much lower levels in alk rearranged lung tumors than in anaplastic large cell lymphoma , the prototypical alk rearranged tumor . fortunately , monoclonal anti - alk antibodies ( clones d5f3 , d9e4 , and 5a4 ) have been shown to exhibit high sensitivity and specificity . immunohistochemistry using the d5f3 and 5a4 monoclonal antibodies on cytologic specimens have been recently described and shown to exhibit a high degree of concordance with alk fish testing . this supports the feasibility of utilizing an immunohistochemical approach as a first - line screening methodology to select specimens for alk fish testing . of note , the mouse monoclonal anti - alk1 antibody ( cd246 ) typically used for the diagnosis of anaplastic large cell lymphoma is less reliable for identifying alk rearrangements in lung adenocarcinoma ; this is most likely attributable to the limited sensitivity of this particular antibody in detecting the lower expression levels of the alk fusion proteins in lung adenocarcinoma relative to anaplastic large cell lymphoma . finally , the application of reverse transcriptase pcr ( rt - pcr ) to cytologic direct smears for alk rearrangement analysis has been recently reported . when evaluating a cohort of paired cytologic - histologic specimens by rt - pcr , mitiushkina and nonetheless , as a cautionary note , there is a high degree of variability in eml4-alk fusion events with at least 13 variants of eml4-alk being reported . furthermore , other fusion partners to alk such as tfg and kif5b can be observed . therefore , an rt - pcr approach may not capture all clinically relevant alk rearrangements . while egfr mutations and alk rearrangements represent the two best characterized , clinically actionable molecular alterations in nsclc , other molecular markers are becoming increasingly appreciated and investigated . ros1 encodes a receptor tyrosine kinase and is rearranged in approximately 2% of lung adenocarcinomas . met encodes another receptor tyrosine kinase , hepatocyte growth factor receptor , and is amplified in a subset of nsclc ; a significant proportion of these cases are seen in context of acquired resistance to egfr tkis . as crizotinib also targets this receptor , studies are under way to determine whether this agent will be effective in met amplified lung cancers . therapeutic agents targeting braf , such as dabrafenib , are currently being investigated in clinical trials . in addition , gene rearrangements involving the ret tyrosine kinase gene ( e.g. , kif5b - ret ) have been observed in approximately 2% of lung adenocarcinomas ; ret specific tkis such as sunitinib , sorafenib , and vandetanib may be useful in treating patients with these lung cancers . finally , amplification of fgfr1 and mutations in pik3ca have been observed in some lung squamous cell carcinomas and agents targeting these gene products are also under investigation . the above mentioned molecular genetic markers highlight the distinct molecular profiles that are becoming increasingly appreciated for different subtypes of nsclc , especially adenocarcinomas and squamous cell carcinomas . therefore , efforts to correctly subtype cases of nsclc are important , including challenging cases of poorly - differentiated nsclc . at our institution , if the subtyping of nsclc is deemed to be challenging based on cytomorphologic evaluation alone , immunocytochemistry is utilized ; we have demonstrated that this can be accomplished using smears as well as cell blocks . for example , napsin - a and thyroid transcription factor-1 are often positive in lung adenocarcinomas and can be utilized for confirming this subtype of nsclc . in contrast , p63 and p40 are useful markers for confirming a diagnosis of squamous cell carcinoma . ultimately , utilizing fna samples of nsclc for immunocytochemistry must be judiciously leveraged to ensure that sufficient material still exists for molecular ancillary testing . given the continuously evolving landscape in our understanding of the genetic events responsible for lung cancer pathogenesis , the integration between anatomic pathology , particularly cytopathology , and molecular diagnostics will become even more essential . emerging molecular technological advances , such as next generation sequencing ( ngs ) , that allow for high - throughput , high - sensitivity molecular analyses will likely play an important role in the management of patients with nsclc . cytologic specimens , based on recent reports , represent a robust source of cellular material for ngs [ 62 - 65 ] . these tumors are classified as typical net ( tnet ) , goblet cell carcinoid ( gcc ) , and atypical gcc histologies . ex - goblet or composite - goblet are further classified into signet ring cell carcinoid ( srcc ) and poorly differentiated adenocarcinoid of the appendix . these tumors have a distinctive morphology showing tight clusters of cells with compact nuclei and abundant intracytoplasmic mucin resembling goblet or signet ring cells often with admixed enterochromaffin cells . pathologic features of gcc include presence of large mucin filled cells with crescent nuclei arranged in small clumps or rosettes mixed with cells of typical carcinoid appearance that stain positive for chromogranin a . patients with tnet have a 5-year survival ranging from 60% to 84% , with the most common site of metastasis being the liver . on the other hand , atypical gcc have a more aggressive clinical course with an increased incidence of lymph node and distant metastases , along with a lower 5-year survival ranging from 36% to 56% [ 5 - 7 ] . current management of gcc and atypical gcc is based on very limited data from small single institutional experiences . the rarity of appendiceal net , gcc , and srcc limits the ability to conduct appropriate randomized clinical trials to explore the optimal management . to assess the role of clinicopathologic features in survival of net , gcc , and srcc patients , the outcome of cases reported to the national cancer institute s surveillance epidemiology and end results ( seer ) program was evaluated . furthermore , to characterize the management of net , gcc , and srcc , a treatment strategy based on results of current analysis , published literature and institutional experience is suggested . seer data collected between 1973 and 2011 was used to identify cases with appendiceal tnet , gcc , and srcc . the seer registry data collection began in the early 1970s and gradually expanded from the original nine to the current 18 registries that now account for over a quarter of the united states population . eligibility criteria included international classification of diseases for oncology third edition ( icd - o-3 ) codes for primary site of appendix and histologic types of tnet ( 8240 , 8241 , 8242 , 8246 , and 8270 ) , gcc ( 8243 , 8244 , 8245 , and 8249 ) , and srcc ( 8490 ) . information regarding age at diagnosis , sex , race , year of diagnosis , grade , histology , tumor location , type of surgery , vital status and duration of follow - up , was extracted from the seer database . the patients characteristics were compared among three histologic types : tnet , gcc , and srcc by chi - square test for race , sex , and stage , and kruskal - wallis test for age . survival functions were estimated by the kaplan - meier method and log - rank test was used to assess the difference in overall survival ( os ) between three histologic types : tnet , gcc , and srcc . univariate survival analysis for each covariate was carried out using the cox proportional hazards model . the multivariate survival analysis of histology was conducted after adjusting for age , race , and sex using a backward variable selection method with an alpha level of removal of 0.1 . the model was stratified by stage since there was an interaction effect between histology and stage on os . 9.3 ( sas institute inc . , cary , nc ) was used for data analyses . null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 . seer data collected between 1973 and 2011 was used to identify cases with appendiceal tnet , gcc , and srcc . the seer registry data collection began in the early 1970s and gradually expanded from the original nine to the current 18 registries that now account for over a quarter of the united states population . eligibility criteria included international classification of diseases for oncology third edition ( icd - o-3 ) codes for primary site of appendix and histologic types of tnet ( 8240 , 8241 , 8242 , 8246 , and 8270 ) , gcc ( 8243 , 8244 , 8245 , and 8249 ) , and srcc ( 8490 ) . information regarding age at diagnosis , sex , race , year of diagnosis , grade , histology , tumor location , type of surgery , vital status and duration of follow - up , was extracted from the seer database . the patients characteristics were compared among three histologic types : tnet , gcc , and srcc by chi - square test for race , sex , and stage , and kruskal - wallis test for age . survival functions were estimated by the kaplan - meier method and log - rank test was used to assess the difference in overall survival ( os ) between three histologic types : tnet , gcc , and srcc . univariate survival analysis for each covariate was carried out using the cox proportional hazards model . the multivariate survival analysis of histology was conducted after adjusting for age , race , and sex using a backward variable selection method with an alpha level of removal of 0.1 . the model was stratified by stage since there was an interaction effect between histology and stage on os . 9.3 ( sas institute inc . , cary , nc ) was used for data analyses . null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 . the seer database yielded 1,021 tnet patients , 1,582 with gcc , and 534 srcc patients . between 1973 and 2011 , the incidence of tnet , gcc , and srcc increased . in 2011 , the incidence of tnet reached 6.7 , gcc was 0.3 , and srcc was two patients per 100,000 persons . baseline characteristics are compared between appendiceal tnet , gcc , and srcc in table 1 . significant differences in age at presentation ( p < 0.001 ) , sex distribution ( p < 0.001 ) , surgery ( p < 0.001 ) , type of surgery ( appendectomy , right hemicolectomy , and surgery not otherwise specified ) ( p < 0.001 ) , and stage ( p < tnet patients presented younger ( median age of 41 vs. 54.5 in gcc and 57 years in srcc ) . female patients constituted a higher proportion among tnet cases ( 66.2% vs. 61.4% of srcc and 48.5% of gcc ) . advanced stage disease was more common in srcc patients ( 60.9% vs. 14.1% in tnet and 10.4% in gcc ) . a higher proportion of white patients was observed in all three histologies , but the difference was not statistically significant ( p=0.11 ) . surgery was performed in 839 ( 82.6% ) of the tnet patients , 1,365 ( 86.4% ) of the gcc , and 419 ( 78.6% ) of the srcc patients , and these differences were statistically significant ( p < 0.001 ) . appendectomy was performed in 52 of patients with tnet ( 5.9% ) as compared to 54 of the gcc ( 3.9% ) , and 9 of the srcc ( 2.1% ) . right hemicolectomy was performed in 484 of the tnet ( 54.9% ) , 1,208 of the gcc ( 86.4% ) , and 301 of the srcc patients ( 70.7% ) . the differences in type of surgery were significantly different among the three histologies ( p < 0.001 ) . the mos for gcc and tnet were not reached at the time of this analysis . comparing survival among the different histologies , tnet had a survival advantage over gcc ( hazard ratio [ hr ] , 0.56 ; 95% ci , 0.45 to 0.69 ; p=0.005 ) , which had a better survival over srcc histology ( hr , 0.22 ; 95% ci , 0.19 to 0.26 ; p < 0.001 ) ( table 3 ) . in each stage , tnet and gcc had significant survival advantages over srcc ( figs . 1 - 3 ) . in the localized stage , mos was not reached for any of the three histologies . comparing srcc , the hr for survival was 0.26 ( 0.15 - 0.46 ; p < 0.001 ) for tnet and 0.42 ( 0.26 - 0.69 ; p < 0.001 ) for gcc . for regional stage disease , the mos for srcc was 35 months ( 95% ci , 30 to 45 ) , while mos for gcc and tnet were not reached . the tnet regional disease stage patients had survival advantage over gcc ( hr , 0.37 ; 95% ci , 0.24 to 0.59 ; p < 0.001 ) . gcc had better survival compared to srcc patients with regional disease stage ( hr , 0.29 ; 95% ci , 0.21 to 0.40 ; p < 0.001 ) . for distant disease stage , mos was 32 ( 95% ci , 13 to not reached ) for tnet , 23 ( 95% ci , 18 to 28 ) for gcc , and 15 months ( 95% ci , 13 to 18 ) for srcc . in this group , tnet patients had survival advantage over gcc ( hr , 0.61 ; 95% ci , 0.44 to 0.83 ; p=0.002 ) . gcc patients survived better compared to srcc patients with distant disease ( hr , 0.73 ; 95% ci , 0.60 to 0.90 ; p=0.003 ) . after adjusting for age , stage , and histology , there was a statistically significant difference in survival for srcc patients treated with hemicolectomy compared to appendectomy ( p=0.01 ) . there was no significant difference in survival for tnet and gcc patients based the type of surgery ( p=0.21 and p=0.94 , respectively ) . in the multivariate analysis stratified by stage and age , there was a statistically significant difference in survival favoring tnet ( hr , 0.41 ; 95% ci , 0.31 to 0.55 ) and gcc ( hr , 0.59 ; 95% ci , 0.48 to 0.72 ) over srcc . the seer database yielded 1,021 tnet patients , 1,582 with gcc , and 534 srcc patients . between 1973 and 2011 , the incidence of tnet , gcc , and srcc increased . in 2011 , the incidence of tnet reached 6.7 , gcc was 0.3 , and srcc was two patients per 100,000 persons . baseline characteristics are compared between appendiceal tnet , gcc , and srcc in table 1 . significant differences in age at presentation ( p < 0.001 ) , sex distribution ( p < 0.001 ) , surgery ( p < 0.001 ) , type of surgery ( appendectomy , right hemicolectomy , and surgery not otherwise specified ) ( p < 0.001 ) , and stage ( p < tnet patients presented younger ( median age of 41 vs. 54.5 in gcc and 57 years in srcc ) . female patients constituted a higher proportion among tnet cases ( 66.2% vs. 61.4% of srcc and 48.5% of gcc ) . advanced stage disease was more common in srcc patients ( 60.9% vs. 14.1% in tnet and 10.4% in gcc ) . a higher proportion of white patients was observed in all three histologies , but the difference was not statistically significant ( p=0.11 ) . surgery was performed in 839 ( 82.6% ) of the tnet patients , 1,365 ( 86.4% ) of the gcc , and 419 ( 78.6% ) of the srcc patients , and these differences were statistically significant ( p < 0.001 ) . appendectomy was performed in 52 of patients with tnet ( 5.9% ) as compared to 54 of the gcc ( 3.9% ) , and 9 of the srcc ( 2.1% ) . right hemicolectomy was performed in 484 of the tnet ( 54.9% ) , 1,208 of the gcc ( 86.4% ) , and 301 of the srcc patients ( 70.7% ) . the differences in type of surgery were significantly different among the three histologies ( p < 0.001 ) . the mos for gcc and tnet were not reached at the time of this analysis . comparing survival among the different histologies , tnet had a survival advantage over gcc ( hazard ratio [ hr ] , 0.56 ; 95% ci , 0.45 to 0.69 ; p=0.005 ) , which had a better survival over srcc histology ( hr , 0.22 ; 95% ci , 0.19 to 0.26 ; p < 0.001 ) ( table 3 ) . in each stage , tnet and gcc had significant survival advantages over srcc ( figs . 1 - 3 ) . in the localized stage , mos was not reached for any of the three histologies . comparing srcc , the hr for survival was 0.26 ( 0.15 - 0.46 ; p < 0.001 ) for tnet and 0.42 ( 0.26 - 0.69 ; p < 0.001 ) for gcc . for regional stage disease , the mos for srcc was 35 months ( 95% ci , 30 to 45 ) , while mos for gcc and tnet were not reached . the tnet regional disease stage patients had survival advantage over gcc ( hr , 0.37 ; 95% ci , 0.24 to 0.59 ; p < 0.001 ) . gcc had better survival compared to srcc patients with regional disease stage ( hr , 0.29 ; 95% ci , 0.21 to 0.40 ; p < 0.001 ) . for distant disease stage , mos was 32 ( 95% ci , 13 to not reached ) for tnet , 23 ( 95% ci , 18 to 28 ) for gcc , and 15 months ( 95% ci , 13 to 18 ) for srcc . in this group , tnet patients had survival advantage over gcc ( hr , 0.61 ; 95% ci , 0.44 to 0.83 ; p=0.002 ) . gcc patients survived better compared to srcc patients with distant disease ( hr , 0.73 ; 95% ci , 0.60 to 0.90 ; p=0.003 ) . after adjusting for age , stage , and histology , there was a statistically significant difference in survival for srcc patients treated with hemicolectomy compared to appendectomy ( p=0.01 ) . there was no significant difference in survival for tnet and gcc patients based the type of surgery ( p=0.21 and p=0.94 , respectively ) . in the multivariate analysis stratified by stage and age , there was a statistically significant difference in survival favoring tnet ( hr , 0.41 ; 95% ci , 0.31 to 0.55 ) and gcc ( hr , 0.59 ; 95% ci , 0.48 to 0.72 ) over srcc . the analysis of the seer registry sample confirms the differences in presentation and outcome of these diseases . tnet tend to occur at a younger age and has a less aggressive clinical course , early stage at presentation and significantly improved os . at the other end of the disease spectrum , the aggressive nature of srcc is reflected by the high risk of distant metastasis at diagnosis , and worse survival outcomes even when controlled for stage . the reported sex distribution in tnet ranges from a female preponderance ( 63% to 73% ) to evenly distributed . similarly for gcc , literature reports range from female gender predominance , to equal distribution . most of these series are based on small numbers of patients from single institution studies . the current report confirms , using a large national database , that there are gender differences in distribution of tnet , gcc , and srcc ( p < 0.001 ) . for srcc histology , we confirmed a higher prevalence amongst women in this seer analysis as compared to gcc histology , consistent with a previous report . additionally , appendiceal srcc appear to be clinically similar to right - sided , microsatellite unstable , signet ring cell colon adenocarcinoma with respect to gender distribution , age of diagnosis and outcome . this raises the question whether appendiceal srcc and right colon adenocarcinomas behave more like a single disease entity distinct from other colorectal cancer ( crc ) adenocarcinoma and appendiceal gcc . this apparent difference in clinical behavior will require further characterization through genomic analysis of crc srcc , appendiceal srcc and typical gcc . hence , a molecularly based classification may facilitate prognostication and perhaps target identification for future clinical trials . the analysis of the seer data confirms that histology and stage at presentation are the major determinants of outcome . the high risk of metastatic disease in the srcc patients confirms the aggressive clinical behavior of this tumor . the rate of metastasis at diagnosis in srcc histology ranges from 14% to 63% . the survival advantage for tnet and gcc over srcc persisted after controlling for the difference in stage distribution . similar observations were reported with 5-year survival of patients with advanced stage gcc , srcc and poorly differentiated adenocarcinoid tumor being 100% , 38% , and 0% , respectively . given their rarity , there is no consensus on the management of appendiceal tnet , gcc , or srcc . the treatments employed included surgical resection and cytoreduction of primary tumor and metastatic sites , intraperitoneal chemotherapy and systemic fluorouracil - based chemotherapy . debulking surgery and hyperthermic intraperitoneal chemotherapy treatment is not well - established in appendiceal net , gcc , and srcc tumors , although it improves appendiceal mucinous adecarcinoma . clinically , the management decisions are usually based on histologic subtype and stage , as well as the patient s performance status . early stage tnet , gcc , or srcc should be managed surgically , although the extent of surgery is an open question . historically , the accepted surgical dogma was that simple appendectomy is sufficient for resection of tnet less than 2 cm . recent reports demonstrated that appendiceal tnet measuring less than 2 cm may have regional nodal involvement , raising the question whether a more extensive surgery than simple appendectomy is required irrespective of size . these observations are further supported by a seer analysis where 32.8% of patients with net less than 2 cm in size ( 20/61 ) had lymph node metastasis . the north american neuroendocrine tumor society ( nanets ) and european neuroendocrine tumor society ( enets ) guidelines suggest right hemicolectomy for all tumors > 2 cm or presence of deep mesoappendiceal invasion , positive or unclear margins , higher proliferative rate ( grade 2 ) , angi olymphatic invasion , and mixed histology , irrespective of tumor size . while simple appendectomy may be adequate for early stage gcc , cecal involvement or high mitotic count index should be an indication for right hemicolectomy . surgery for srcc should always involve a right hemicolectomy due to the high likelihood of lymph node metastasis , irrespective of the size of the primary appendiceal mass . this is supported by our analysis of the seer data that indicates superior survival for patients with srcc who undergo right hemicolectomy . the role of surgical resection of the ovaries in female patients with localized srcc or gcc remains controversial . a summary of the nanets and enets guidelines is provided in table 4 . unlike crc adenocarcinoma , the role of adjuvant therapy is not established for tnet , gcc , or srcc . based on our analysis , patients with tnet have an excellent os and therefore adjuvant therapy is not indicated . on the other hand , given the high risk of systemic metastasis in srcc , we recommend offering adjuvant therapy for all patients undergoing resection . in our analysis of the seer sample , the outcome of patients with early stage gcc appears favorable , suggesting that patients with gcc should be considered for adjuvant therapy only if they have high risk features such as cecal invasion , perforation , or lymph node involvement . for gcc patients with advanced stage disease , options of treatments include peritoneal debulking with intraperitoneal chemotherapy for patient with limited peritoneal disease or systemic fluorouracilbased chemotherapy . for srcc patients , our recommendations include treating with systemic fluorouracil - based chemotherapy initially and only consider peritoneal resection for patients with good response to initial therapy . a summary of the proposed treatment algorithm is provided in fig . predictive and prognostic biomarkers are not well defined for these diseases . in an attempt to identify gene variations between appendiceal mucinous adenocarcinoma and gcc , we profiled nine gcc samples using second - generation gene sequencing . only one patient with gcc ( 11% ) had kras mutation , and two had tp53 mutation ; myc , smad4 , and apc mutations were absent in gcc . proved the absence of kras mutations in 16 and 14 gcc samples . in the light of its rarity , molecular differentiation markers for appendiceal tnet , gcc , and further confirmation with larger studies is needed to evaluate pathologic genomic sequencing for the three histologies in order to detect differences in survival and possible future biomarkers that predict response to specific target treatments . the seer registry findings confirm the distinct clinical entities of tnet , gcc , and srcc with respect to presentation and outcome .

in this era of precision medicine , our understanding and knowledge of the molecular landscape associated with lung cancer pathogenesis continues to evolve . this information is being increasingly exploited to treat advanced stage lung cancer patients with tailored , targeted therapy . during the management of these patients , minimally invasive procedures to obtain samples for tissue diagnoses are desirable . cytologic fine - needle aspirates are often utilized for this purpose and are important not only for rendering diagnoses to subtype patients lung cancers , but also for ascertaining molecular diagnostic information for treatment purposes . thus , cytologic fine - needle aspirates must be utilized and triaged judiciously to achieve both objectives . in this review , strategies in utilizing fine - needle aspirates will be discussed in the context of our current understanding of the clinically actionable molecular aberrations underlying non - small cell lung cancer and the molecular assays applied to these samples in order to obtain treatment - relevant molecular diagnostic information . purposeappendiceal tumors are a heterogeneous group of diseases that include typical neuroendocrine tumors ( tnet ) , goblet cell carcinoids ( gcc ) , and atypical gcc . atypical gcc are classified into signet - ring cell cancers ( srcc ) and poorly differentiated appendiceal adenocarcinoids . the prognosis and management of these diseases is unclear because there are no prospective studies . the aim of this study is to assess the characteristics and outcome of appendiceal tnet , gcc , and srcc patients.materials and methodsappendiceal tnet , gcc , and srcc patients diagnosed between 1973 and 2011 were identified in the surveillance epidemiology and end results ( seer ) database . demographics , type of surgery , and clinicopathologic characteristics were collected . survival functions were estimated by the kaplan - meier method , and log - rank test was used to assess the difference in overall survival ( os ) among the three histologies.resultsthe seer database yielded 1,021 tnet patients , 1,582 with gcc , and 534 srcc patients . tnet presented at a younger age ( p < 0.001 ) . patients with srcc presented with advanced stage disease ( p < 0.001 ) . the median os ( mos ) for gcc and tnet patients was not reached ; mos for srcc was 24 months . multivariate analysis stratified for stage revealed significantly longer survival for tnet and gcc than srcc ( p < 0.001).conclusionthis is the largest report to date for appendiceal neuroendocrine tumor patients , suggesting a spectrum of diseases with different characteristics and outcomes . in this report , we present a treatment approach for this complex spectrum of disease , based on the experience of ohio state and emory universities investigators .

igg4-rd is a newly recognized fibroinflammatory condition of unknown etiology characterized by tumefactive lesions , dense lymphoplasmacytic infiltration rich in igg4-positive plasma cells , storiform fibrosis and an elevated serum igg4 level [ 13 ] . the kidney is one of the organs commonly affected by igg4-rd , and tubulointerstitial nephritis ( tin ) with infiltration of numerous igg4-positive plasma cells is the predominant type of kidney lesion [ 47 ] . high levels of igg and igg4 are striking and characteristic features of renal involvement such as multiple lesions with low attenuation demonstrated by contrast - enhanced computed tomography are often evident [ 47 ] . although tin is the main feature of the kidney lesion in igg4-rd , some glomerular lesions have also been described , and membranous nephropathy ( mn ) has been recognized as the most common form [ 48 ] . in previously reported cases of mn associated with igg4-rd , mn was diagnosed together with , or after the onset of igg4-rd [ 912 ] . here , we report a case of idiopathic mn as a preceding disease before the onset of igg4-rd . he had previously been well and had no significant medical history . his serum total protein level was 46 g / l ( 4.6 g / dl ) , serum albumin 17 g / l ( 1.7 g / dl ) , urinary protein excretion 4.3 g / day without occult blood and the 24-h creatinine clearance was normal at 1.51 ml / s ( 90.5 ml / min ) ( table 1 ) . the anti - nuclear antibody was negative , and serum immunoglobulins g , a and m were all within normal limits at 11.98 g / l ( 1198 mg / dl ) , 11.37 complement levels were also within normal limits : c3 1.12 g / l ( 112 mg / dl ) , c4 0.314 g / l ( 31.4 mg / dl ) and ch50 30 u / ml . contrast - enhanced computed tomography ( ct ) demonstrated small , non - specific nodules in the right lung , and a hemangioma of the liver . table 1.clinical course and laboratory findings in the present casemay 2008january 2009july 2009july 2010december 2010march 2011august 2011eventdiagnosed as mnworsening of nsdiscontinuation of psladmission to our hospitaltreatmentdiureticspsl , furosemidefurosemide , olmesartanfurosemide , olmesartanfurosemide , olmesartanfurosemide , olmesartanfurosemide , olmesartantp ( g / l)46566071778592alb ( g / l)17152931303124igg ( g / l)12nanananana39urinalysis ( protein)3 + 3 + 3 + 3 + 3 + 3 + 3+up ( g / day)4.33.03.1nanana2.124 h ccr ( ml / s)1.511.121.37nanana1mn , membranous nephropathy ; ns , nephritic syndrome ; psl , prednisolone ; tp , total protein ; alb , albumin ; up , urinary protein excretion ; ccr , creatinine clearance ; na , data not available . clinical course and laboratory findings in the present case mn , membranous nephropathy ; ns , nephritic syndrome ; psl , prednisolone ; tp , total protein ; alb , albumin ; up , urinary protein excretion ; ccr , creatinine clearance ; na , data not available . percutaneous kidney biopsy was performed , and the histology revealed diffuse membranous changes on the glomerular basement membrane without tubulointerstitial lesions ( figure 1a and b ) . an immunofluorescence study showed diffuse , fine - granular staining of igg , c3 , fibrinogen , kappa- and lambda - chain in the glomerular capillary loops . there were no deposits in other areas including mesangeal matrix , subendothelial aspects and tubular basement membrane . an additional immunofluorescence study of igg subclasses showed predominant positive staining for igg4 and igg1 and weak positive staining for igg2 and igg3 in the glomeruli ( figure 1c ) . an electron microscopy examination revealed diffuse , homogenous , small electron - dense deposits in the subepithelial aspects of the glomerular basement membrane , which corresponded to ehrenreich and churg stage 2 . subendothelial deposits were not present , and endothelial cells did not contain any tubuloreticular structure . the mesangial matrix and tubular basement membrane did not contain electron - dense deposits . 1.histopathologic findings of kidney biopsy at the previous hospital at the time of onset of idiopathic mn . ( a ) light microscopy shows no significant mesangial cell or matrix proliferation in glomeruli , and no interstitial lesions . ( b ) diffuse , small deposits are evident in the subepithelial aspects of the glomerular basement membrane . ( c ) an immunofluorescence study of igg subclasses shows predominant positive staining for igg1 and igg4 and weak positive staining for igg2 and igg3 in glomeruli . histopathologic findings of kidney biopsy at the previous hospital at the time of onset of idiopathic mn . ( a ) light microscopy shows no significant mesangial cell or matrix proliferation in glomeruli , and no interstitial lesions . periodic acid - schiff stain , 200 . ( b ) diffuse , small deposits are evident in the subepithelial aspects of the glomerular basement membrane . ( c ) an immunofluorescence study of igg subclasses shows predominant positive staining for igg1 and igg4 and weak positive staining for igg2 and igg3 in glomeruli . a systemic work - up revealed no other disease or malignancy , and the patient prednisolone was administered at 40 mg daily , but the patient suffered repeated cerebral infarction attacks after starting the therapy . prednisolone was tapered and discontinued 6 months after the start of the therapy , without any significant effect . the patient 's proteinuria persisted at around 3 g / day and he was followed up while receiving diuretics and the angiotensin receptor blocker , olmesartan . two years after discontinuation of glucocorticoid therapy , the patient developed epigastric discomfort and was hospitalized again . laboratory examinations revealed elevated serum levels of ast , alt , alp , total bilirubin and -gtp [ 64 iu / l , 106 iu / l , 1948 iu / l , 111 mol / l ( 6.5 mg / dl ) and 1544 iu / l , respectively ] accompanied by a slight increase in serum creatinine at 100 mol / l ( 1.13 mg / dl ) and a normal serum amylase level of 111 iu / l . abdominal ct revealed swelling of the pancreatic head , dilatation of the main pancreatic duct and stenosis of the common bile duct . tumor markers including cea and ca19 - 9 were all within the normal ranges , while hypergammaglobulinemia was observed and the serum igg level was elevated at 39.21 g / dl ( 3921 mg / dl ) with marked elevation of igg4 at 19.2 hypergammaglobulinemia persisted , and serum iga , igm and complement studies showed that all were within normal limits . serum anti - nuclear antibody was negative , whereas the rheumatoid factor was positive at 120.6 iu / ml . urinary protein excretion was 2.1 g / day without occult blood and 24-h creatinine clearance was mildly deteriorated at 1 ml / s ( 60.0 ml / min ) ( table 1 ) . excretion of urinary 2-microglobulin ( 2-mg ) and n - acetyl--d - glucosaminidase was markedly elevated at 11 636 g / day and 25.0 iu / day , respectively . contrast - enhanced ct showed bronchial thickening in the bilateral lungs , pancreatic swelling with stenosis of the main pancreatic duct and bile duct , bilateral swollen kidneys with multiple lesions exhibiting low attenuation , periaortic and periarterial mass lesions , diffuse swelling of the prostate gland and systemic lymphadenopathy , compatible with the radiologic abnormalities characteristic of igg4-rd ( figure 2 ) . 2.contrast-enhanced ct at our hospital at the time of onset of igg4-rd . marked swelling of pancreatic head and bilateral swollen kidneys with multiple lesions exhibiting low attenuation were observed . contrast - enhanced ct at our hospital at the time of onset of igg4-rd . marked swelling of pancreatic head and bilateral swollen kidneys with multiple lesions exhibiting low attenuation were observed . percutaneous kidney biopsy was not performed since we could not obtain the patient 's agreement . instead , percutaneous needle biopsies from the pancreas and prostate gland specimens obtained by biopsy from the prostate gland and pancreas revealed dense lymphoplasmacytic infiltration with storiform fibrosis and infiltration of numerous igg4-positive plasma cells ( igg4/ igg - positive plasma cell ratio > 50% ) without malignancy ( figure 3 ) . 3.histopathologic features of a specimen obtained from the prostate gland by percutaneous needle biopsy at our hospital at the time of onset of igg4-rd . there is no evidence of cancer cells and there is a typical swirling pattern of fibrosclerosing inflammation with infiltration of numerous lymphocytes , the so - called storiform fibrosis. ( b ) immunohistochemical staining for igg in a needle biopsy specimen from the prostate gland , 150 . immunohistochemical staining for igg4 in a needle biopsy specimen from the prostate gland , 150 . the igg4-positive plasma cell / igg - positive plasma cell ratio was over 50% in tissue from the prostate gland . histopathologic features of a specimen obtained from the prostate gland by percutaneous needle biopsy at our hospital at the time of onset of igg4-rd . there is no evidence of cancer cells and there is a typical swirling pattern of fibrosclerosing inflammation with infiltration of numerous lymphocytes , the so - called storiform fibrosis. ( b ) immunohistochemical staining for igg in a needle biopsy specimen from the prostate gland , 150 . immunohistochemical staining for igg4 in a needle biopsy specimen from the prostate gland , 150 . the igg4-positive plasma cell / igg - positive plasma cell ratio was over 50% in tissue from the prostate gland . western blot analysis of the patient 's serum for the detection of serum autoantibodies against the m - type phospholipase a2 receptor ( anti - pla2r ) was performed , as described previously , and was negative . oral administration of prednisolone was initiated at 40 mg daily , and the patient 's clinical symptoms including jaundice and anorexia improved rapidly . the laboratory data and multiple organ lesions also improved , while proteinuria persisted at around 3 g / day . it has been reported that mn is present in 7% of kidney biopsy specimens in igg4-rd . most cases of mn associated with igg4-rd are accompanied by igg4-related tin [ 46 , 9 , 10 ] , although recent reports have also documented mn without tin but with other features of igg4-rd ( 8 , 11 , 12 ) . in mn associated with igg4-rd , positive deposition of igg4 in the glomerular basement membrane has been observed , either predominantly or together with other igg subclasses [ 812 ] . originally , the immunofluorescent examination of igg subclasses was known as a very useful tool to distinguish the primary mn from the secondary mn . predominant deposition of igg4 , often accompanied by other subclasses such as igg1 , was one of the well - known features of idiopathic mn . moreover , idiopathic mn and mn associated with igg4-rd share an essential immunologic feature known as th2 cytokine - mediated immune reaction [ 14 , 15 ] , suggesting that these two disorders share common pathophysiologic processes for increased production of th2 cytokines and subsequent increase of igg4 . on the other hand , these two disorders differ in many other features , including clinical symptoms , laboratory parameters and radiographic findings . an anti - pla2r antibody , which has been described as a major target antigen involved in idiopathic mn and seen in 7080% of patients with idiopathic mn [ 13 , 1618 ] , has been reported to be negative in all cases of igg4-rd and also cases of mn associated with igg4-rd [ 8 , 10 , 12 ] , suggesting no involvement of this antibody in the development of igg4-rd . in the present case , there was no evidence of any association of igg4-rd based on the clinical symptoms , serological data and imaging findings when the patient developed nephrotic syndrome . therefore , it was reasonable to have diagnosed the present case as idiopathic mn at the time of onset of nephrotic syndrome . the anti - pla2r antibody is described as negative in all cases evaluated in mn associated with igg4-rkd and was also negative in this patient , although the result was obtained from the serum 3 years after the diagnosis of idiopathic mn and after corticosteroid therapy . however , 2025% of patients with idiopathic mn are reported to be negative for the antibody [ 1618 ] . the absence of the anti - pla2r antibody is thus only partly helpful to distinguish from idiopathic mn . interestingly , the patient 's total serum protein level increased gradually without any significant improvement of proteinuria or elevation of the serum albumin level ( table 1 ) , and eventually the clinical symptoms and laboratory abnormalities of igg4-rd became evident 3 years after the onset of idiopathic mn . accordingly , it is possible that some conditions underlying the persistent predominance of th2-type immune responses during the 3 years of follow - up might have led to the development of igg4-rd . currently , idiopathic mn is defined as mn without any other disorder that could cause secondary mn , with predominant deposition of igg4 in the glomerular basement membrane . however , mn associated with igg4-rd has also been revealed to be igg4-predominant and , interestingly , the igg subclass of the anti - pla2r antibody seen in idiopathic mn is predominantly igg4 . although the pathogenesis of igg4-rd is poorly understood , it may share some common mechanisms with idiopathic mn , and thus a comparative study of the two diseases might be helpful for clarifying their pathogenesis . consideration for igg4-rd in mn , including systemic evaluation or measurement of serum igg4 level , would be necessary in addition to the evaluation of the anti - pla2r antibody and igg subclasses in the immune complex deposited on the glomerular basement membrane .

we have prepared two new diastereoisomeric 2-aza-5-phosphabicyclo[2.2.1]heptanes from naturally occurring trans-4-hydroxy - l - proline in six chemical operations . these syntheses are concise and highly efficient , with straightforward purification . when we used these chiral phosphines as catalysts for reactions of -substituted allenoates with imines , we obtained enantiomerically enriched pyrrolines in good yields with excellent enantioselectivities . these two diastereoisomeric phosphines functioned as pseudoenantiomers , providing their chiral pyrrolines with opposite absolute configurations . we report a case of igg4-related disease ( igg4-rd ) diagnosed after 3 years of follow - up for idiopathic membranous nephropathy ( mn ) . mn has been considered as glomerular lesion of igg4-related kidney diseases in recent years and was diagnosed simultaneously with or after a diagnosis of igg4-rd in previously reported cases . in the present case , igg4-rd developed 3 years after the diagnosis of idiopathic mn , indicating a possible relationship between idiopathic mn and igg4-rd through common underlying mechanisms of development .

an estimated 170 million people worldwide have hepatitis c virus ( hcv ) infection and over 20,000 new cases emerge every year . hcv infection frequently persists and may cause cirrhosis and hepatocellular carcinoma . in the us and most developed nations , where the prevalence of infection is 1%2% , hcv is the leading cause of chronic liver disease.1 hcv infection is a serious public health problem in egypt , and is the country with the highest hcv prevalence , where 10%20% of the general population is infected.2 interferons are a group of glycoproteins that modulate the activity of the immune system . recombinant alpha - interferon ( ifn ) has recently been approved as a therapy for chronic hepatitis c , for its antiviral effects and inhibition of cell proliferation.3 although interferon - based therapy is widely used for the treatment of chronic hcv , it is not without its disadvantages . treatment duration lasts for months , and is often fraught with potentially dangerous side effects . some of these side effects , such as influenza - like illness , myalgias , depression , and cytopenias can be intolerable resulting in premature treatment cessation , but have no lasting effects . ocular complications are among the complications of interferon therapy and include retinal hemorrhages , cotton wool spots , loss of color vision , cataracts , glaucoma , and occasionally retinal artery or vein obstruction . although the incidence of ophthalmological disorders while on interferon therapy is low , this can result in loss of vision.411 in this prospective case series study , one hundred patients with chronic hepatitis - c under interferon ( inf ) therapy were consecutively recruited from patients attending the outpatient ophthalmic clinic at the university hospital , faculty of medicine , al - azhar university , assuit . the study protocol was approved by the ethics committee at al - azhar university hospital . written informed consent was obtained from all patients included in the study , and then in addition to the medical and laboratory examination , all patients were subjected to full ophthalmological assessment to evaluate the retinopathy associated interferon therapy . inclusion criteria were : ( 1 ) seropositivity for anti - hcv antibodies determined using elisa ; ( 2 ) first use of interferon therapy . exclusion criteria were : ( 1 ) previous interferon therapy ; ( 2 ) patients with hepatitis b ; ( 3 ) patients with decompensated liver ; ( 4 ) patients with renal failure ; ( 5 ) patients with cardiovascular disorders such as coronary insufficiency , congestive heart failure , and ischemic stroke ; ( 6 ) patients with a dense cataract , visual field abnormality , glaucoma , or any other ocular abnormalities . evaluation before treatment : history of visual loss , hazy vision , and ocular disease or ocular surgeries ophthalmological examination : ( 1 ) best corrected visual acuity ( bcva ) measured using landolt s broken rings ; ( 2 ) pupillary reaction ( direct and consensual ) and if there is afferent pupillary defect ; ( 3 ) full slit - lamp examination ; ( 4 ) intraocular pressure ( iop ) measured by goldmann s applanation tonometer ; ( 5 ) fundus biomicroscopy ( carried out after maximum pupillary dilation by a combination of tropicamide 1% eye drops and phenylephrine 2.5% eye drops using both slit - lamp biomicroscopy with noncontact + 90 d volk lens and/or goldmann contact lens ) ; ( 6 ) examination of the retinal periphery using an indirect ophthalmoscope . medical examinations and abdominal ultrasonography investigations : ( 1 ) ocular , both colored fundus photography and fundus fluorescein angiography ( ffa ) ; ( 2 ) laboratory ( including enzyme - linked immunosorbent assay ( ellsa ) test for hepatitis surface ag and ab ; polymerase chain reaction ( pcr ) for hepatitis c virus rna ; full blood count ; prothrombin time ; direct and indirect bilirubin ; fasting blood sugar ; serum creatinine ; liver enzymes ( serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase ) ; alanine aminotransferase . patients were treated with pegylated interferon -2a at a dose of 180 mcg injected subcutaneously once weekly for 48 weeks . all patients underwent clinical and laboratory assessments , ophthalmic examination , at 15 days , 1 month , 2 months , 3 months , 6 months , 8 months , and 9 months after the start of ifn treatment . patients were treated with pegylated interferon -2a at a dose of 180 mcg injected subcutaneously once weekly for 48 weeks . all patients underwent clinical and laboratory assessments , ophthalmic examination , at 15 days , 1 month , 2 months , 3 months , 6 months , 8 months , and 9 months after the start of ifn treatment . initially , 120 patients fulfilled the criteria for ifn therapy , but 20 of them were excluded due to non - compliance with the follow - up visits and due to the presence of diabetic or hypertensive retinopathy . also , two patients died during the study time , due to a disease not related to hepatitis , so these patients are not included . therefore , a total of 100 patients ( 68 male and 32 female ) , with chronic hcv were selected . after the start of interferon therapy , 16 out of 100 patients ( 16% ) developed retinopathy which was bilateral in 12 ( 75% ) patients and unilateral in 4 ( 25% ) patients . retinopathy was initially diagnosed by the appearance of a cotton wool spot in 14 patients . in four of the 14 patients , retinal hemorrhage two of the 16 patients who developed retinopathy were diagnosed by retinal hemorrhage without cotton wool spots . one patient ( not diabetic or hypertensive ) had cystoid macular edema , this was the only patient who complained of the visual disturbance due to interferon . all patients had bcva of ( 6/6 ) during ifn treatment except for three patients : the one who had cystoid macular edema ( bcva was 6/12 and returned to 6/6 after cessation of treatment ) and two patients with ametropic amblyopia . in addition , we confirmed that all patients with hypertension had no retinopathy using the standard classification . to exclude the possibility of the development of diabetic or hypertension retinopathy after ifn treatment , we deleted the data obtained from patients who had diabetes or hypertension and any retinopathy 2 months after the cessation of ifn treatment . retinopathy was first diagnosed 214 weeks after the start of treatment and disappeared in 11 of the 16 patients despite the continuation of therapy . a few cotton wool spots , retinal hemorrhages , or both were observed in the remaining five patients at the end of treatment but resolved within 1 month after treatment was stopped . no other ocular complications ( ie , cataracts , glaucoma , retinal artery , or vein obstruction ) were observed . the mean hemoglobin values , white blood cell , red blood cell , and platelet counts decreased significantly ( p < 0.0001 ) during ifn treatment . the clinical profiles and laboratory data of the patients are shown in tables 1 and 2 , respectively . interferon - associated retinopathy was first recognized in 1990 when ikebe and associates reported a 39-year - old patient who developed retinal hemorrhages and cotton wool spots following intravenous administration of interferon.12 the underlying mechanisms of interferon - induced retinopathy are not completely understood . guyer et al13 had proposed an immune complex deposition causing occlusion of retinal capillaries leading to cotton wool spot formation . on the other hand , an ischemic insult , similar to that seen in hypertensive and diabetic patients could be another potential mechanism by which these changes develop.14 some studies15,16 have shown a relatively higher incidence of retinopathy ( symptomatic or asymptomatic ) ranging from 19% to 64% during treatment with interferon . panetta and gilani17 and cuthbertson et al14 show relatively lower incidences of retinopathy ( 3.8% and 6.25% , respectively ) . similar to other studies,4,5,9 our study also shows that retinal changes usually develop within the first 3 months of interferon treatment , a fact that may favor an immune etiology rather than a cumulative effect of interferon leading to the development of retinopathy . retinopathy developed by week 14 in most of the patients ( 13/16 , 81.25% ) after the start of therapy and disappeared in the majority of the patients ( 11/16 , 68.75% ) during the 48 week period in which the patients were receiving the treatment . this suggests that treatment can be continued despite the development of retinopathy in many patients . however , two patients who developed cotton wool spots early in the therapy ( 2 weeks ) thereafter suffered from retinal hemorrhage in a prolonged manner . therefore , patients who develop cotton wool spots early on during therapy should be carefully monitored . however , as reported in previous studies,4,13,14 most of the patients with retinopathy in this study were asymptomatic . this study confirms previous reports that retinopathy is a temporary and asymptomatic complication of interferon therapy . therefore there is no rule for suspending the use of interferon in patients who develop retinopathy or any other ocular side effects . it s commonly agreed among authors that careful monitoring should be performed in the presence of any ocular sign , even without symptoms.18 nathan , as an oncology fellow , knew well that white blood cells fought infections . this was an experiment repeated in front of us all the time , he says : chemotherapy lowered his patients ' white blood cells and increased their risk of infections . mackaness had shown that macrophage activation did not depend on direct contact with t cells ( 1 ) , suggesting the possibility of a secreted factor . when nathan tested the supernatant from activated t cells , he saw that it did indeed induce macrophage activation ( 2 ) . nathan got a rough idea of the molecular weight ( 3 ) , but that was the best anyone could do , he says . protein separation methods were primitive , and cloned proteins and monoclonal antibodies would only become available a decade later . henry murray , one of nathan 's collaborators , sums up the feeling of frustration : we were all nibbling at the edges of the same problem . nathan therefore changed tack to take a closer look at the activated macrophages . short - lived neutrophils were known to produce hydrogen peroxide , and nathan found the same was true of longer - lived activated macrophages ( 4 ) . unlike previous signs of macrophage activation increased spreading , phagocytosis , and glucose metabolism this so - called respiratory burst ifn had been on the cover of time magazine , and recombinant murine ifn was found to induce macrophages to kill tumor cells ( 5 ) . nathan , now a faculty member in zanvil cohn 's macrophage factory at rockefeller university ( new york , ny ) , thought ifn might also activate macrophages to kill intracellular parasites . consistent with this idea , ifn was made by antigen - stimulated t cells and was associated with defense from infection . now the respiratory burst gave nathan an assay , berish rubin ( down the street at the new york blood center ) supplied an ifn monoclonal antibody , and a phone call to genentech yielded recombinant ifn. in a seminal paper published in the journal of experimental medicine in 1983 , nathan was thus able to show that depleting ifn from unpurified t cell supernatants decreased the respiratory burst activity and the killing of intracellular protozoa in human macrophages . adding back recombinant ifn into this mix restored macrophage activation ( 6 ) . i had an assay , a hunch , a history of purifying proteins that did this , and the serendipity of meeting with people nearby who had the antibody . nathan next showed that ifn worked in people . injecting recombinant ifn directly into cutaneous lesions of lepromatous leprosy patients induced macrophage infiltration , hydrogen peroxide production , and killing of the causative pathogen , mycobacterium leprae ( 7 ) . in the 1990s , the macrophages of children with ifn receptor deficiencies were shown to be defective in killing mycobacteria ( 8) . tracing the pathway from t cells to macrophages to bacteria started , for nathan , in 1967 , and he says we still haven't finished making the molecular links .

purposeto evaluate retinopathy associated with interferon therapy in patients with chronic hepatitis c.methodsone hundred patients with chronic hepatitis c undergoing interferon therapy were examined for the presence of cotton wool spots , retinal hemorrhages , cystoid macular edema , capillary non - perfusion , and arteriolar occlusion . complete ophthalmological examination including indirect ophthalmoscopic fundus examination was carried out for all patients and colored fundus photography and fluorescein angiography were carried out for the patients with positive fundus findings . the follow - up period was 9 months.resultssixteen percent of patients developed retinopathy in the form of cotton wool spots , retinal hemorrhages , cystoid macular edema , and capillary non-perfusion.conclusioninterferon therapy can lead to retinopathy which is mostly reversible and dose related . periodic fundoscopic examinations help in early detection and prevent progression to permanent visual loss . t cells tell macrophages when to start making the toxic soup of lysosomal enzymes , reactive oxygen species , and nitric oxide that destroys intracellular pathogens . in 1983 , carl nathan proved that this start signal comes in the form of the secreted cytokine ifn.

women require special need - based health care programs as they pass through the menopausal transition . with this vision , a multisystem multispecialty health check - up and follow - up care program ( maitreyi health care program [ hcp ] ) was initiated by our team about a decade ago , and this has been continued at the medical research center , kasturba health society , mumbai . the comparative effects of three plant products on perimenopausal symptoms , quality of life ( qol ) and clinical biochemistry have been reported by our group earlier . our observation of frequent mood changes and sleep disturbances in women in the premenopausal group prompted us to undertake a study of cyclic symptoms in maitreyi hcp . in this communication , we describe the preliminary findings from the analysis of premenstrual symptoms in 200 consecutive women who underwent a comprehensive check - up in the maitreyi hcp . diagnosis of premenstrual tension syndrome ( pmts ) is based on the following criteria as per the american association of obstetricians and gynecologists : a 30% increase in the intensity of symptoms of premenstrual syndrome ( measured using a standardized instrument ) from cycle days 510 as compared with the 6-day interval before the onset of menses and documentation of these changes for at least two consecutive cycles . at least one of the following affective and somatic symptoms during the 5 days before menses in previous cycles . affective symptoms : depression , angry outbursts , irritability , anxiety , confusion , social withdrawal . somatic symptoms : breast tenderness , abdominal bloating , headache , swelling of extremities ; symptoms relieved from days 4 through 13 of the menstrual cycle . to determine the prevalence of premenstrual cyclic symptoms in women who attended the maitreyi hcp . to determine the prevalence of premenstrual cyclic symptoms in women who attended the maitreyi hcp . maitreyi 's hcp for women above 35 includes comprehensive health questionnaire , gynecological and physician 's check - up , biochemical investigations of complete blood count , glucose tolerance test , lipid profile , liver functions , serum creatinine and serum thyroid stimulating hormone . pap smear , pelvic sonography and urine and stool examination for occult blood are also included . a multidisciplinary trans - system team offers this community - oriented programme to women with voluntary services by the consultants . bone mineral density and mammography this analysis is a preliminary report restricted to the premenstrual cyclic symptomatolgy in 200 consecutive women who attended this program over 2 years . a special case record form has been used and a detailed medical and surgical history is included in the same . twelve women who continued to menstruate beyond 50 , up to 55 years , have been included in the analysis . only those who had symptoms at least during two consecutive cycles or more were included in the analysis . the american society of obstetricians and gynecologists and the american psychiatric society have now brought out the consensus criteria for inclusion of the condition in pmts or premenstrual dysphoric disorder ( pmdd ) , the latter usually requiring treatment for neuropsychiatric symptoms . accordingly , pmts is said to be present when the woman reports three or more of the following symptoms in two cycles or more[46 ] : there are 36 symptoms as listed below in the sf-36 mental component . in the present analysis , the symptoms were classified as per the medical outcomes study short form-36 for assessment of premenstrual symptoms , and the presence of three of these occurring over two or more cycles was labelled as pmts : anxiety , irritability , depression , tension , mood swings , loss of self - control , difficulty in concentration , insomnia , confusion , headache , crying attacks , fatigue , aches , breast tenderness , cramps , bloatedness , food craving , visual disturbances , tiredness , fatigue , puffiness of the face , weight gain , altered libido , aggression , nausea , vomiting , acne , skin rashes , constipation , diarrhea , joint stiffness , backache , abdominal cramps / pain , anger attacks , aggravation of epilepsy , aggravation of asthma . of 107 women , 26 ( 24.3% ) did not report any premenstrual symptoms at all and 81 ( 75.7% ) reported at least one symptom [ table 1 ] . number of symptoms experienced by women in different age groups in two or more cycles maximum frequency was seen in women from 35 to 50 years of age . forty women ( 37.4% ) who reported premenstrual symptoms had three or more cyclic symptoms for more than two cycles and could be classified as those with pmts . fifteen ( 14% ) had five or more symptoms cyclically in at least two cycles . almost 10% reported five or more symptoms inclusive of anger attacks and could be labelled as pmdd . mastalgia was the most common symptom ( 50.5% ) , followed by mood changes ( 46.7% ) , depression ( 7.5% ) and anger attacks ( 6.5% ) . pmdd was present in 10% of the cases who were referred to a clinical psychologist or a psychiatrist . many women have premenstrual cyclic symptoms of psychological or physical nature and , sometimes , these limit their functional capacity . the symptoms due to neurohormonal imbalance or ageing may start as early as 35 years of age . pmts is an entity that causes considerable morbidity , and in about 3% of the women , it may disrupt the woman 's life when severe symptoms recur . when there are severe symptoms like anger attacks , depression and suicidal thoughts , special attention including psychiatric consultation may be required , i.e. the pmdd . the prevalence of pmts in general is about 210% for disabling symptoms , while minor symptoms may be present in up to 85% of the women.[468 ] in the present series , 75.7% reported at least one symptom cyclically . in the present series halbreich has reported that in about 20% of the women , the symptoms are severe enough to warrant treatment . dean et al . have reported a prevalence of 1930% when all women from 18 to 45 years of age were screened . in a recent survey of 3,913 women of 15 - 54 years of age , tschudin et al . observed that about 10% reported pmts and 3.1% reported pmdd . when the indian scenario is considered , chaturvedi et al . in the 1990s have reported a prevalence of 20% in a general population and severe symptoms in 8% . the same authors have , in a later study , reported suicidal ideas and/or death wish during the premenstrual period in 10% of the subjects , more so among college students and industrial working women as compared with housewives . banerjee et al . , in a group of 62 women , reported pmdd in 6.4% . there is a close association between mood disorders and the sleep rhythm and disturbances in sleep pattern in premenopausal age.[31314 ] many women and their physicians regard pmts as a physiological phenomenon and do not think treatment is necessary . however , it does affect qol in some and it is therefore essential to identify which women will benefit from lifestyle management and which will require pharmacological intervention . those who want to reach their full functional potential and who find that the symptoms are hampering their progress or interfering with normal daily routine should be certainly offered safe pharmacological interventions provided that they come for routine follow - ups . because there are many etiological factors that have been described as probably causative , and multiple causes may be present in an individual woman , there is a wide variety of treatment options and combinations depending on specific symptoms and their severity . the postulated causes range from hormonal imbalances like progesterone deficiency , prolactin excess , thyroid hypofunction , fluctuation in circulating level of estrogen ; electrolyte disturbances like rennin - angiotensin alterations , antidiuretic hormone excess , decreased colloidosmotic pressure ; neurotransmitter disturbances with serotonin or gamma - amino - butyric - acid , or -endorphin activity alternations , serotonin metabolism alternations or prostaglandin imbalance ; cytokine imbalances like excess of interleukin ( il)-6 , il-1 and tumor necrosis factor ( tnf)- ; nutritional deficiencies like vitamin b or vitamin d deficiency ; lack of exercise or psychosocial disturbances.[8101315 ] the treatment therefore will depend on the particular symptom complex in the individual woman . in concordance with other authors , lifestyle measures like regular exercise , balanced nutritious diet and regular hours of sleep will benefit all , and may be adequate for some.[1921 ] for others , pharmacological interventions may be helpful if required . the therapy is multipronged and has to be individualized . while simple measures like the institution of physical exercises , relaxation techniques like yoga and vitamin and mineral supplements are adequate for most women,[1924 ] some require hormonal therapy with combined oral contraceptives or regulation of fluid and electrolytes . as a matter of fact , combined oral contraceptives are used frequently in the more severe cases and , drosperidone , containing newer combinations , is relatively free from side - effects . selective estrogen modulators , including nonsteroidal agents like centchroman , also could be useful , particularly in women with contraindications to hormones . few women need psychiatric consultation and will benefit from antidepressants , particularly sertraline or fluoxetine , while some respond to prolactin inhibitors like bromocriptine or cabergoline . complementary and alternative therapies like soy , hypericum perforatum and gingko biloba have a definite role to play.[242931 ] evening primrose oil does not have significant efficacy . we have observed relief of mild to moderate premenstrual symptoms with soy , gycyrrhiza glabra and withania somnifera . we have also observed complete relief of pmts in two women treated with an extract rich in saraca asoka , prescribed for menorrhagia , which was also controlled . there is need for more research in this area because commonly used drugs may have side - effects that may prevent regular long - term use [ table 2 ] . common side - effects of existing pharmacologic therapies for pmts recently , two well - designed studies have shown that pmts leads to loss of work hours and economic losses , and does not allow women to reach their full potential.[3436 ] effective and safe therapy is indicated . it is also important to bear in mind that other chronic diseases like migraine or depression or irritable bowel syndrome could be exaggerated during the premenstrual phase , and a multidisciplinary team should evaluate and manage such cases . many women and their physicians regard pmts as a physiological phenomenon and do not think treatment is necessary . however , it does affect qol in some and it is therefore essential to identify which women will benefit from lifestyle management and which will require pharmacological intervention . those who want to reach their full functional potential and who find that the symptoms are hampering their progress or interfering with normal daily routine should be certainly offered safe pharmacological interventions provided that they come for routine follow - ups . because there are many etiological factors that have been described as probably causative , and multiple causes may be present in an individual woman , there is a wide variety of treatment options and combinations depending on specific symptoms and their severity . the postulated causes range from hormonal imbalances like progesterone deficiency , prolactin excess , thyroid hypofunction , fluctuation in circulating level of estrogen ; electrolyte disturbances like rennin - angiotensin alterations , antidiuretic hormone excess , decreased colloidosmotic pressure ; neurotransmitter disturbances with serotonin or gamma - amino - butyric - acid , or -endorphin activity alternations , serotonin metabolism alternations or prostaglandin imbalance ; cytokine imbalances like excess of interleukin ( il)-6 , il-1 and tumor necrosis factor ( tnf)- ; nutritional deficiencies like vitamin b or vitamin d deficiency ; lack of exercise or psychosocial disturbances.[8101315 ] the treatment therefore will depend on the particular symptom complex in the individual woman . in concordance with other authors , lifestyle measures like regular exercise , balanced nutritious diet and regular hours of sleep will benefit all , and may be adequate for some.[1921 ] for others , pharmacological interventions may be helpful if required . the therapy is multipronged and has to be individualized . while simple measures like the institution of physical exercises , relaxation techniques like yoga and vitamin and mineral supplements are adequate for most women,[1924 ] some require hormonal therapy with combined oral contraceptives or regulation of fluid and electrolytes . as a matter of fact , combined oral contraceptives are used frequently in the more severe cases and , drosperidone , containing newer combinations , is relatively free from side - effects . selective estrogen modulators , including nonsteroidal agents like centchroman , also could be useful , particularly in women with contraindications to hormones . few women need psychiatric consultation and will benefit from antidepressants , particularly sertraline or fluoxetine , while some respond to prolactin inhibitors like bromocriptine or cabergoline . complementary and alternative therapies like soy , hypericum perforatum and gingko biloba have a definite role to play.[242931 ] evening primrose oil does not have significant efficacy . we have observed relief of mild to moderate premenstrual symptoms with soy , gycyrrhiza glabra and withania somnifera . we have also observed complete relief of pmts in two women treated with an extract rich in saraca asoka , prescribed for menorrhagia , which was also controlled . there is need for more research in this area because commonly used drugs may have side - effects that may prevent regular long - term use [ table 2 ] . recently , two well - designed studies have shown that pmts leads to loss of work hours and economic losses , and does not allow women to reach their full potential.[3436 ] effective and safe therapy is indicated . it is also important to bear in mind that other chronic diseases like migraine or depression or irritable bowel syndrome could be exaggerated during the premenstrual phase , and a multidisciplinary team should evaluate and manage such cases . it is very important to ensure that the first option is nonpharmacological as many women may respond to it . pharmacotherapy can be added in recalcitrant cases and must be individualized as per the woman . great care should be taken to avoid side - effects and drug interactions because multiple therapies may be used . medicinal plants may be a safe alternative in some women . because pmts could be a predictor for menopausal syndrome , it will be useful to warn women with pmts and their families of a flare up or the need for long - term follow - up . have reported pmts to be predictive of menopausal syndrome in a long - term follow - up study and have shown the association of hormonal imbalance with depressive symptoms in women . according to the korean national statistical office , the main cause of death in korea was cancer in 2012 . the total number of deaths due to cancer in 2012 was 267,221 , which was increased by 9,825 ( 3.8% ) compared to 2011.1 this was the all - time high since 1983 . currently , the treatment of cancer patients in the medical field consists mainly of surgery , radiation therapy , and chemotherapy . seventy to eighty percent of cancer patients in chemotherapy experienced side effects such as anemia , the decreased number of white blood cells and/or platelets , oral mucositis , vomiting , diarrhea , hair loss , and disabled generative functions as chemotherapy also affects the rapidly proliferating normal cells.2 most side effects are temporary and patients recover fully ; but , some side effects may take months or years to completely disappear . to decrease side effects from non - specific cytotoxicity , new drugs , such as targeted agents , are being developed to selectively destroy specific cancer cells . however , because of problems , such as tolerance manifestation , partly critical toxicity , decrease in sensitivity , and restriction in subjects , new drugs take only a small portion of the cancer treatment realm.3 chemoprevention is defined as the use of nontoxic or relatively safe chemical substances , such as vitamins , plant extracts , and pharmaceuticals to prevent cancer . it is effective in decreasing cancer risk and is helpful in early stages of cancer.4 hence , regular consumption of anticancer foods that display no or little toxicity to the body and boost efficacy of anti - tumor drugs or stop the development of tolerance would be beneficial in preventing cancer . thus , consumption of plant - derived natural foods to prevent cancer with few side effects and toxicity is crucial.5 recently , there has been a growing interest in probiotics due to the discovery of multidrug - resistant organisms that are tolerant to antibiotics . probiotics is derived from a greek word meaning for life and holds the opposite meaning from antibiotics which means against life. probiotics consist of microorganisms and substances , which modify the intestinal microflora , and when used as dietary supplement they have positive effects on health such as fortification of host immune function , suppression of diarrhea , and inhibition of carcinogenesis.6 kimchi , a korean traditional food , is fermented by probiotic lactic acid bacteria.7 kimchi lactic acid bacteria are known to suppress the activity of carcinogen - activating enzymes , such as azoreductase , nitroreductase , 7--dehydrogenase , -glucosidase , and -glucuronidase and inactivate or neutralize the cancer - causing agents and pathogenic microbes.8 in addition , the intake of lactic acid bacteria as part of kimchi improves bowel movement , strengthens immunity , ameliorates hepatocirrhosis , and decreases serum cholesterol levels.9,10 among lactic acid bacteria identified during the fermentation of kimchi , weissella , lactobacillus , leuconostoc and pediococcus species are known to play an important role in kimchi fermentation.1113 this commentary deals with anticarcinogenic effects of kimchi and lactic acid bacteria , weissella cibaria and lactobacillus plantarum separated from kimchi , which are known to be predominant in kimchi . kimchi , a fermented cabbage indispensable to korean cuisine , was once considered to be a risk factor for stomach cancer . however , unless one consumes kimchi that contains excess salt , kimchi , in general , is a healthy food recognized for its antioxidant , antiobese , cancer preventive , and other health beneficial effects.7 cancer preventive / anticarcinogenic activity of kimchi is associated with the type of ingredients and products formed during fermentation.14 the kimchi s main ingredient , chinese cabbage , has been considered to be effective in preventing stomach cancer according to epidemiologic studies and is known to inhibit colorectal carcinogenesis due to its abundance in dietary fiber.15 garlic , in particular , has been acknowledged for its anticancer effect due to high contents of organosulfur compounds ( 11 to 35 mg / g ) . thirty - three types of organosulfur compounds have been identified in garlic.16 organosulfur compounds in garlic have been shown to facilitate the detoxification of carcinogens by glutathione - s - transferase , modulate the activity of metabolizing enzymes such as cytochrome p450s , and inhibit the formation of dna adducts in several target tissues.1719 antiproliferative activity of organosulfur compounds has been demonstrated in tumor cell lines including colon , prostate , and breast , which is mediated by induction of apoptosis via the caspase-3 signaling pathway and cell - cycle arrest.20,21 one possible mechanism of cancer preventive effects of garlic in stomach cancer development involves its antimicrobial activities against helicobacter pylori , a major risk factor of stomach cancer.22 hot red chili pepper powder , one of the main ingredient of kimchi , contains capsaicin ( trans-8-methyl - n - vanillyl-6-nonenamide ) . although the role of capsaicin in carcinogenic processes has been controversial , there is accumulated evidence that capsaicin is capable to induce apoptosis of cancer cells by generating excess reactive oxygen species.23 thoennissen et al.24 demonstrated that capsaicin caused cell - cycle arrest and apoptosis in breast cancer cells by modulating the egfr / her-2 pathway and inhibited the development of pre - neoplastic breast lesions by up to 80% without toxicity . in addition to each ingredient of kimchi , kimchi extract was used to investigate cancer preventive / anticarcinogenic activity of kimchi . extract of kimchi fermented for 3 weeks inhibited the proliferation of human cancer cell lines ( gastric adenocarcinoma and acute promyelocytic leukemia ) . leukemia cells treated with kimchi extract showed increased apoptosis and decreased mitochondrial transmembrane potential.25 the juice of 3-week fermented kimchi suppressed the growth of k-562 human leukemia cells and mg-63 human osteosarcoma cells but no toxicity of kimchi juice was found in normal cells.26 sarcoma180 cells transplanted mice were treated with methanol extract of 3-week fermented kimchi . animals provided kimchi extract showed in smaller tumor weight and decreased malondialdehyde formation compared to those of the control group.27 although there has been no general acceptance on how to define probiotic microorganisms , lactobacillus species , bifidobacterium sp , saccharomyces boulardii , and some other microbes have been thought and used as probiotic strains.28 these probiotic microorganisms have been used as food supplement in order to achieve health benefit effects of them . there have been many studies reported on the functionality of lactic acid bacteria in the fermentation process of kimchi and their importance in the health beneficial effects of kimchi , including prevention of cancer.29 according to pyrosequencing analysis of commercial kimchi samples to identify kimchi lactic acid bacteria , the genus weissella was predominant at 44.4% . w. koreensis was 27.2% and w. cibaria was 8.7%.7 weissella is a newly separated lactic acid bacteria of lactobacillus family that was identified by a recent dna technique . it is a gram - positive and catalase negative bacteria included in geneally recognized as safe.30 the main lactic acid bacteria involved in kimchi fermentation has long been known as lactobacillus genus and leuconostoc genus , but more recently , weissella genus lactic acid bacteria has been newly separately identified.30 weissella is an early dominant in kimchi fermentation and produces lactic acid , acetic acid , alcohol , dextran , and co2 , which account for the unique fresh taste and texture of kimchi.31,32 weissella species found in kimchi are w. cibaria , w. koreensis , and w. hanii.12 sources of each weissella species are shown in table 1.33 w. cibaria , newly named by bjorkroth and others , has been separated from korean kimchi and various other sources , including fermented foods , greek salami , spanish sausages , and animal and human excrements . w. cibaria is gram - positive , non - pore formulating , non - motile , hetero lactic acid fermented , and catalase negative bacillus , and can not produce dextran from sucrose.34,35 studies have reported that microorganisms , including lactic acid bacteria , secreted exopolysaccharides ( eps ) with the anticancer , anti - inflammatory , immune modulating , and blood cholesterol declining functions.36 it was demonstrated that w. cibaria , leuconostoc mesenteroides and w. confusa are able to produce eps . among these lactic acid bacteria , w. cibaria exhibited higher production of eps which indicates more acid resistance ( table 2).37 w. cibaria was reported to have anticancer activity , immune modulating activity , anti - inflammatory activity , and antioxidant activity . based on the patent related to the w. cibaria s anticancer activity registered by cha et al.,38 the anticancer activity in w. cibaria w. cibaria bacteria was incubated for 24 hours in deman rogosa sharpe agar badge , diluted with phosphate buffered saline , and 10% concentration of bacteria samples were provided to normal cell strains and colorectal cancer cell strains for 72 hours . cell growth was suppressed by treatment of w. cibaria in colorectal cancer cells but not in normal cells . ahn et al.33 presented that the immune control effect of w. cibaria was stronger than the well - known probiotic bacterium , l. rhammosus gg ( lgg ) . w. cibaria produced higher levels of nitric oxide , nuclear factor ( nf)- b , cytokines ( e.g. , interleukin-1 and tumor necrosis factor- ) than lgg , suggesting that w. cibaria is more effective in immune control compared to lgg . furthermore , w. cibaria is known to have an antiviral function against the avian influenza virus.39 ornithine , a type of amino acid produced from its arginine precursor , is reported to accelerate growth hormone excretion and has an antiobesity effect . during the kimchi fermentation process , the level of arginine , an ornithine precursor , has decreased while the ornithine level increased rapidly , which suggested that kimchi is a source of ornithine . moreover , a correlation between the amount of w. cibaria and the amount of ornithine in kimchi was observed.40 use of w. cibaria as a starter for food fermentation promoted the formation of ornithine from arginine , which in turn provide health beneficial effects , such as antiobesity due to high levels of ornithine in fermented food.41,42 lactobacillus genus is a microorganism which does not form spores and is an anaerobic and facultative anaerobic gram - positive bacterium . this bacterium is not only widely dispersed in nature but is also found in human oral cavity and digestive organs . this bacterium is a beneficial microorganism that is widely used as a starter for various fermented dairy products.43 the main kimchi lactic acid bacteria of lactobacillus is l. plantarum . the rapid increase of l. plantarum in the late stage of kimchi fermentation produces large amounts of organic acid , which is known as the main substance that acidifies kimchi . acid tolerance of l. plantarum allows us to use this bacterium for natural antibacterial and antifungal products . l. plantarum s cancer preventive potential has been tested by the ames mutation assay and the sos chromotest . when cabbage was brined with 3% salt concentration and kimchi was fermented at 5c for 3 weeks , kimchi extract exerted an antimutagenic effect against aflatoxin b1.44 l. plantarum , isolated from kimchi , also exhibited a strong antimutagenic effect against n - methyl - n-nitro - n - nitrosoguanidine , 4-nitroquinoline-1-oxide.45 furthermore , l. plantarum , isolated from kimchi , had stronger antimutagenic effects compared to lactic acid bacteria originated from fermented milk.46 the macrophagocytes provided with l. plantarum separated from kimchi strengthened phagocytosis , and displayed anticancer effects in aseites carcinoma and solid tumor due to the polysaccharide chains of muramic acid in the l. plantarum s cell well . among lactic acid bacteria s cell wall substances , polysaccharide types rather than glycopeptide play a pivotal role in cancer suppression.47 to evaluate the possible use of l. plantarum as a probiotic , lee et al.48 measured the survival rate in artificial gastric fluid and intestinal fluid , adhesion compatability with human intestinal caco-2 cells , and antibacterial activity . as a result , l. plantarum strains showed more than 90% survival rate in artificial gastric fluid and intestinal fluid , and more than 400 times the survival rate of the control strain , l. casei . when pathogenic bacteria and lactic acid bacteria were incubated together , l. plantarum suppressed the adhesion of intestinal crypt cells with the pathogenic bacteria , and also suppressed pathogenic bacteria growth of escherichia coli , stapylococcus aureus , and salmonella typhimurium by 93.4% , 75.9% , and 69.3% respectively . weissella is a newly separated lactic acid bacteria of lactobacillus family that was identified by a recent dna technique . it is a gram - positive and catalase negative bacteria included in geneally recognized as safe.30 the main lactic acid bacteria involved in kimchi fermentation has long been known as lactobacillus genus and leuconostoc genus , but more recently , weissella genus lactic acid bacteria has been newly separately identified.30 weissella is an early dominant in kimchi fermentation and produces lactic acid , acetic acid , alcohol , dextran , and co2 , which account for the unique fresh taste and texture of kimchi.31,32 weissella species found in kimchi are w. cibaria , w. koreensis , and w. hanii.12 sources of each weissella species are shown in table 1.33 w. cibaria , newly named by bjorkroth and others , has been separated from korean kimchi and various other sources , including fermented foods , greek salami , spanish sausages , and animal and human excrements . w. cibaria is gram - positive , non - pore formulating , non - motile , hetero lactic acid fermented , and catalase negative bacillus , and can not produce dextran from sucrose.34,35 studies have reported that microorganisms , including lactic acid bacteria , secreted exopolysaccharides ( eps ) with the anticancer , anti - inflammatory , immune modulating , and blood cholesterol declining functions.36 it was demonstrated that w. cibaria , leuconostoc mesenteroides and w. confusa are able to produce eps . among these lactic acid bacteria , w. cibaria exhibited higher production of eps which indicates more acid resistance ( table 2).37 w. cibaria was reported to have anticancer activity , immune modulating activity , anti - inflammatory activity , and antioxidant activity . based on the patent related to the w. cibaria s anticancer activity registered by cha et al.,38 the anticancer activity in w. cibaria was tested with colorectal cancer cells . w. cibaria bacteria was incubated for 24 hours in deman rogosa sharpe agar badge , diluted with phosphate buffered saline , and 10% concentration of bacteria samples were provided to normal cell strains and colorectal cancer cell strains for 72 hours . cell growth was suppressed by treatment of w. cibaria in colorectal cancer cells but not in normal cells . ahn et al.33 presented that the immune control effect of w. cibaria was stronger than the well - known probiotic bacterium , l. rhammosus gg ( lgg ) . w. cibaria produced higher levels of nitric oxide , nuclear factor ( nf)- b , cytokines ( e.g. , interleukin-1 and tumor necrosis factor- ) than lgg , suggesting that w. cibaria is more effective in immune control compared to lgg . furthermore , w. cibaria is known to have an antiviral function against the avian influenza virus.39 ornithine , a type of amino acid produced from its arginine precursor , is reported to accelerate growth hormone excretion and has an antiobesity effect . during the kimchi fermentation process , the level of arginine , an ornithine precursor , has decreased while the ornithine level increased rapidly , which suggested that kimchi is a source of ornithine . moreover , a correlation between the amount of w. cibaria and the amount of ornithine in kimchi was observed.40 use of w. cibaria as a starter for food fermentation promoted the formation of ornithine from arginine , which in turn provide health beneficial effects , such as antiobesity due to high levels of ornithine in fermented food.41,42 lactobacillus genus is a microorganism which does not form spores and is an anaerobic and facultative anaerobic gram - positive bacterium . this bacterium is not only widely dispersed in nature but is also found in human oral cavity and digestive organs . this bacterium is a beneficial microorganism that is widely used as a starter for various fermented dairy products.43 the main kimchi lactic acid bacteria of lactobacillus is l. plantarum . the rapid increase of l. plantarum in the late stage of kimchi fermentation produces large amounts of organic acid , which is known as the main substance that acidifies kimchi . acid tolerance of l. plantarum allows us to use this bacterium for natural antibacterial and antifungal products . l. plantarum s cancer preventive potential has been tested by the ames mutation assay and the sos chromotest . when cabbage was brined with 3% salt concentration and kimchi was fermented at 5c for 3 weeks , kimchi extract exerted an antimutagenic effect against aflatoxin b1.44 l. plantarum , isolated from kimchi , also exhibited a strong antimutagenic effect against n - methyl - n-nitro - n - nitrosoguanidine , 4-nitroquinoline-1-oxide.45 furthermore , l. plantarum , isolated from kimchi , had stronger antimutagenic effects compared to lactic acid bacteria originated from fermented milk.46 the macrophagocytes provided with l. plantarum separated from kimchi strengthened phagocytosis , and displayed anticancer effects in aseites carcinoma and solid tumor due to the polysaccharide chains of muramic acid in the l. plantarum s cell well . among lactic acid bacteria s cell wall substances , polysaccharide types rather than glycopeptide play a pivotal role in cancer suppression.47 to evaluate the possible use of l. plantarum as a probiotic , lee et al.48 measured the survival rate in artificial gastric fluid and intestinal fluid , adhesion compatability with human intestinal caco-2 cells , and antibacterial activity . as a result , l. plantarum strains showed more than 90% survival rate in artificial gastric fluid and intestinal fluid , and more than 400 times the survival rate of the control strain , l. casei . when pathogenic bacteria and lactic acid bacteria were incubated together , l. plantarum suppressed the adhesion of intestinal crypt cells with the pathogenic bacteria , and also suppressed pathogenic bacteria growth of escherichia coli , stapylococcus aureus , and salmonella typhimurium by 93.4% , 75.9% , and 69.3% respectively . this paper describes the cancer preventive / anti - carcinogenic potential of kimchi lactic acid bacteria , focusing on w. cibaria and l. plantarum . the antibiotic tolerant probiotic presented in kimchi suppresses expression of carcinogen activating enzymes and possesses many other health benefits such as suppression of growth and development of pathogenic bacteria , intestinal regulation , and immune boosts . especially , the w. cibaria and l. plantarum found in kimchi have many effects , like anti - inflammatory , immune modulating , and blood cholesterol reducing activity , which may account for their cancer preventive / anticancer potential . these probiotic lactic acid bacteria in kimchi can not only be used as an antimicrobials in foods but also be implemented in developing functional foods to reduce the risk of colon cancer .

objective : to determine the prevalence of premenstrual cyclic symptoms in perimenopausal age.subjects and methods : women attending bhavan 's sparc maitreyi 's health care programme ( hcp ) for women around 40 years of age were included in the study . last 200 women who attended from april 2002 to october 2004 are included for analysis . out of these 107 qualified for final analysis as others were post hysterectomy or post menopausal . thirty five symptoms listed under premenstrual tension syndrome were analysed.results:forty one women ( 38.3% ) had 3 or more symptoms whilst 15 ( 14.0% ) had 5 or more cyclic symptoms . five women ( 4.7% ) reported that the symptoms were severe . eleven women had seeked treatment for premenstrual tension syndrome ( pmts ) . the commonest symptom was mastalgia or heaviness of breasts . next was whilst also was reported by several women . women reported anger attacks and reported depression.conclusion:pmts was common between 36 and 55 years . about half of them have experienced 3 more symptoms and 1 in 20 may require treatment . the number of death due to cancer has been increasing in korea . chemotherapy is known to cause side effects because it damages not only cancerous cells but healthy cells . recently , attention has focused on food - derived chemopreventive and anti - tumor agents or formulations with fewer side effects . kimchi , most popular and widely consumed in korea , contains high levels of lactic acid bacteria and has been shown to possess chemopreventive effects . this review focuses on weissella cibaria and lactobacillus plantarum , the representatives of kimchi lactic acid bacteria , in terms of their abilities to prevent cancer . further studies are needed to understand the mechanisms by which lactic acid bacteria in kimchi prevent carcinogenic processes and improve immune functions .

a glomangioma is a benign vascular tumor derived from the glomus body , a specialized neuromyoarterial structure involved in thermal regulation . it is a subtype of the more generalized category of glomus tumors and should not be confused with the head and neck paragangliomas , such as the glomus tympanicum or glomus jugulare . it should also be distinguished from the glomangiopericytoma , the sinonasal type hemangiopericytoma , which displays different ultrastructural and histochemical characteristics.1 glomangiomas are most commonly found subungually and are exceedingly rare in the head and neck.2 they account for only 0.6% of all nonepithelial tumors of the nasal cavity , nasopharynx , and paranasal sinuses.3 glomangiomas that induce paraneoplastic osteomalacia are even more uncommon , with only one case reported to date.4 while there are several documented cases of oncogenic osteomalacia ( oo ) caused by glomangiopericytomas,5 we present the second reported case of glomangioma - induced osteomalacia and the first case documented in english . a 42-year - old man with a history of unexplained hip , rib , scapula , and metatarsal fractures ; left foot pain with exercise ; and weakness presented to our clinic for evaluation of a paranasal sinus tumor . the endocrinology service initially diagnosed him with tumor - induced osteomalacia caused by fibroblast growth factor 23 ( fgf23 ) , which prompted an octreotide scan revealing a questionable area of enhancement in the pituitary . a subsequent magnetic resonance imaging ( mri ) showed an ethmoid mass extending through the cribriform intracranially , measuring 3.9 1.9 2.4 cm at its largest dimensions ( fig . the patient reported occasional sinus congestion , decreased sense of smell during the preceding 2 to 3 years and occasional blurry vision in the morning . his laboratory workup revealed a significantly decreased 1,25-vitamin d level of less than 8 ng / ml , a decreased phosphate level to 1.6 mg / dl , a low to normal calcium level of 8.7 mg / dl , and a significantly elevated alkaline phosphatase level at 65.3 g / l . preoperative mri , t1 with contrast , showing an ethmoid mass extending through the cribriform intracranially . the patient underwent a combined endoscopic endonasal approach to the anterior skull base with tumor resection . the cribriform defect and intraoperative cerebrospinal fluid ( csf ) leak pathology revealed a vascular neoplasm with a uniform cluster of ovoid cells arranged around the vessels and moderate focal nuclear enlargement ( fig . tumor cells stained positive for cd31 and smooth muscle actin ( sma ) but negative for cd34 , s100 , and pan - cytokeratin on immunohistochemistry ( fig . the patient reported near - complete resolution of bone pain and improvement in smell , and he had normalization of phosphate , alkaline phosphatase , vitamin d , and other laboratory values . repeat mri showed gross total resection of the mass and no detectable recurrence ( fig . 4 ) . postoperative mri , t1 with contrast , showing gross total resection of the mass with nasoseptal flap reconstruction of the skull base . osteomalacia is a disease of the bone characterized by defective mineralization of osteoid from decreased levels of available phosphate and calcium or increased bone resorption . it often presents with diffuse joint and bone pain , easy fracturing , difficulty walking , weakness , and other nonspecific symptoms . oo is a rare , disabling , and curable form of osteomalacia that affects both sexes equally and usually presents around 40 years of age.6 it is not well described in glomangiomas but has been detailed several times in relation to glomangiopericytomas and other soft tissue and bone tumors , with more than 300 reported cases7 since its debut in 1947.8 it predominantly occurs in the context of mesenchymal tumors and is thought to be due to neoplastic overexpression of fgf23 . this protein inactivates the sodium - phosphate pump in the proximal tubule ( prohibiting phosphate reabsorption and inducing renal phosphate wasting ) and reduces 1-hydroxylation of 25-hydroxy vitamin d.9 accordingly , common oo laboratory abnormalities include hypophosphatemia , normal or decreased calcium , decreased 1,25-dihydroxy vitamin d3 with resistance to vitamin d supplementation , and elevated alkaline phosphatase , which our patient exhibited . tumors causing oo tend to be small , occult , and slow growing , making diagnosis remarkably difficult . when other causes of osteomalacia have been ruled out and oo is suspected , clinicians should consider measuring a serum fgf23 level . elevated fgf23 in this setting should then prompt full body imaging ( including hands and feet ) to expose the lesion . the current imaging standard for investigation of oo is whole - body mri short tau inversion recovery ( stir).10 another commonly used modality is octreotide scintigraphy ( octreoscan ) , which utilizes a radiotracer that binds to somatostatin receptors overexpressed on the causative tumor.11 once the neoplasm is localized , surgical resection with wide margins is the definitive treatment . it is curative in essentially all cases and typically leads to rapid normalization of laboratory values and reversal of most clinical symptoms . the nonspecific presentation of osteomalacia , the obscure nature of the tumors that cause oo , and the rarity of glomangiomas in the head and neck make this patient 's diagnosis challenging . we believe this to be the second reported case of paranasal sinus glomangioma - induced osteomalacia and the first reported in english . many head and neck surgeons may not be aware of its existence or consider it in their differential diagnoses of sinus tumors . this report serves to increase the awareness of this uncommon pathology so that it may be considered and treated in future cases . gestational trophoblastic disease describes a number of gynecologic tumors that originate in trophoblastic layer including hydatidiform mole ( complete or partial ) , invasive mole , choriocarcinoma , placental site trophoblastic tumor and epitheloid trophoblastic tumor . invasive mole may arise from any pregnancy event although in most cases is diagnosed after molar pregnancy . overall cure rate in low risk patients is nearly 100% and in high - risk patient 90% . in rare cases , molar tissue traverses thickness of myometrium and leads to perforation and acute abdomen and invasive mole infrequently metastasis . the best treatment option is chemotherapy ( according to stage and score with single or multiple agent ) and in patients that fertility is not the matter , hysterectomy can be done . a 41 years old g3p2ab1 woman referred to firouzgar hospital 2 months after curettage of molar pregnancy with vaginal bleeding and acute abdomen . in workup , hcg 224000 miu / ml and evidence of metastasis was detected . chemotherapy due to stage 3 and score 9 and surgery due to acute abdomen was done . this case reported about ovarian metastasis and uterine rupture with acute abdomen and involvement of omentum in metastatic invasive mole . management of this patient was successful . in follow up , she was free of disease without sequel of any kind for five years now . invasive mole is penetration of molar tissue ( complete or partial mole ) into myometrium or uterine vasculature ( 1 , 16 , 19 ) . pathologists mention the existence of villi in trophoblastic tissue ( 8 , 12 ) . locally invasive gestational trophoblastic neoplasia develops in 15% of patients and metastatic form in 4% of patients after evacuation of complete mole and infrequently after partial mole ( 2 ) . hcg level ( > 100000 miu / ml ) , excessive uterine enlargement , theca lutein cyst size 6 cm are considered as high risks for developing post molar tumors ( high risk mole ) ( 3 ) . the most common symptom of invasive mole is persistent vaginal bleeding after evacuation of molar pregnancy ( sub involution of uterus and persistent theca lutein cyst is another symptom ) . the rise in hcg titer is a laboratory test for diagnosis of invasive mole in follow up of molar pregnancy . although definite diagnosis of invasive mole is based on pathology ( 8) , with hcg or radiologic diagnosis ( 9 ) , invasive mole is diagnosed as well . in rare cases , metastasis occurred and the most common sites were lungs ( 80% ) ( 4 ) , vagina ( 30% ) , pelvis ( 20% ) , liver ( 10% ) , brain ( 10% ) , bowel and kidney and spleen ( < 5% ) were other metastatic sites . metastasis can occur from direct extension of another pelvic neoplasm or by hematogenous or lymphatic spread or by transcelomic dissemination . possibility of metastasis to ovary is extremely rare which is even much lower than that of nongestational primary ovary choriocarcinoma with an incidence of 1 in 3.710 ( 5 ) . invasive mole is curable with chemotherapy but hysterectomy decreases the need for multiple courses of chemotherapy and in patients with heavy bleeding or sepsis for control of complication and stabilization , chemotherapy is needed ( 6 , 11 ) . here a case with invasive mole after evacuation of molar pregnancy , presented with acute abdomen and in surgery metastasis to ovary and omentum and parametrium was detected . a 41- year- old housewife woman , gravid 3 para 2 live child 2 ( all nvd ) with a last 10 years old child referred to firouzgar hospital on 24 august 2010 with a history of a dilation and curettage and 2 months of cough . on examination , she was febrile ( 38c . , tachycardia , leukocytosis ( 16000 ) and uterus was tender and enlarged . vaginal bleeding persisted and second curettage was done in another hospital . at that time , chest x - ray was normal . in our hospital , report of sample pathology given from first and second curettage was complete mole . after molar evacuation , the patient was not monitored for weekly determination of hcg level . she received antibiotics ( due to fever ) and work up for malignant trophoblastic disease or persistent gtn was done because of high hcg level ( 224000 miu / ml ) and much too enlarged uterus and involvement of the whole uterine parenchyma , which was detected on sonography ( largest tumor size in uterus was 6 cm ) . thyroid function test demonstrated hyperthyroidism . in the metastatic work up , ct scan of chest and abdomen and brain was carried out . ct scan of the abdomen and pelvis showed involvement of the left adnexa and pelvic lymphadenopathy . the patient s chest ct scan showed multiple bilateral round pulmonary metastatic lesions that were 2 cm in diameter , illustrated in figure 1 . pulmonary metastasis in invasive mole once the disease was diagnosed , the patient chemotherapy with ema started ( table 1 ) . because of acute abdomen and severe abdominal pain and unstable vital signs with diagnosis of perforative peritonitis , the patient was taken up for emergency laparotomy . due to hyperthyroid state , in induction of anesthesia , blocker agent was administered . in this patient , subtotal hysterectomy and left salpingo oophorectomy and resection of omentum were done . in gross uterus , uterine wall was perforated by tumoral invasion on the left side of posterior wall ( figure 2 ) . enlarged uterus , ovarian metastasis , perforation of uterus , and myometrium invasion the patient was iii:9 according to figo staging and who scoring ( 7 ) the bilateral uterine parameters , the external surface of the endocervical canal and the omentum were invaded by tumors . villous formation in myometrium villosity with necrosis in invasive mole this patient was treated with 4 courses of chemotherapy with ema - co . after negative hcg ( < 5 ) , she received an additional 3 courses of chemotherapy to reduce the relapse . hcg titer was later tested monthly for1 year . when the first year of testing hcg terminated , hcg testing on a bimonthly basis for 2 years was done . the patient was followed up to 5 years and after that the patient was free of tumor and the hcg was negative . invasive mole with rupture of uterus and metastasis to ovary and omentum and manifestation of acute abdomen was rare ( 5 ) . according to epidemiological retrospective survey , invasive mole secondary to hydatidiform mole occurred in six months after evacuation ( 5 , 3 ) . partial and complete hydatidiform mole are distinct disease processes with characteristic cytogenetic , histologic , and clinical features ( 8) . ( 13 ) reported a case of invasive mole presenting as acute hemoperitoneum that was similar to the present case . molar tissue can penetrate myometrium ( 9 ) and lead to uterine perforation ( 13 ) and cause vaginal bleeding due to erosion of uterine vessels ( 14 , 15 ) . similar to atala et al.s case report in 1991(14 ) , in our case , uterine perforation with acute intra peritoneal hemorrhage was seen . due to ease of entry of molar tissue into large venous lake present in myometrium and pelvis in pregnancy or trophoblastic disease metastasis of invasive mole commonly in lungs and after lungs in vagina , cervix , broad ligament ( 16 , 19 ) , in our case metastasis to lung and ovary and omentum although theca lutein cysts in high - risk mole due to high serum hcg level are seen normally , they regress spontaneously within 24 months ( 17 ) . non gestational choriocarcinoma of ovary is differential diagnosis ( 5 ) but due to occurrence of metastasis to ovary in our case after molar pregnancy , this differential diagnosis was not a matter . as in a report of invasive mole in fallopian tube ( 21 ) , the first misdiagnosis of our case in another hospital , repeat curettage was done , although the first diagnosis could be persistent gtn . in another case report ( 15 ) from bruner , similarity with our case was seen . combination chemotherapy should be continued in such cases as far as toxicity permits until the patient achieves 3 consecutive normal hcg levels ( 9 , 18 ) . as normal hcg levels are attained , 3 additional courses of chemotherapy are administered to reduce the risk of relapse . in low risk patients , one or two courses are adequate ( 19 ) . in our case , 3 courses of chemotherapy after normalization of hcg were done . hysterectomy may be required in invasive mole in order to control vaginal bleeding and in unstable patient or in sepsis ( 6 , 20 ) . hysterectomy is a reasonable option for patients who do not wish to preserve their fertility , but it does not prevent metastasis ( 20 ) . furthermore , in patients with extensive uterine tumor , hysterectomy may substantially reduce the trophoblastic tumor burden ( 20 ) and thereby limit the need for multiple courses of chemotherapy . in our case , due to unstable and uterine rupture , hysterectomy was done but the need for chemotherapy courses was not removed and after several courses of chemotherapy with ema - co courses , it was cured . however , final diagnosis of our patient was invasive mole with metastasis to ovary and omentum and lung ( stage 3 ) and uterine rupture . the patient responded to the treatment well and in follow up this patient was still fine for 5 years after definite care for metastasis invasive mole with surgery and chemotherapy . an invasive mole in uterus with ovarian and omental metastasis was diagnosed and the patient was successfully treated by hysterectomy and chemotherapy and she was fine for 5 years in follow up .

oncogenic osteomalacia ( oo ) is an uncommon but treatable cause of osteomalacia related to tumor production of fgf23 , usually caused by benign mesenchymal neoplasms . paranasal sinus glomangiomas are a rare cause of oo , with only one previously reported case . here we describe a second case ( first reported in english ) of paranasal sinus glomangioma - induced osteomalacia in a 42-year - old man . he presented with weakness and multiple spontaneous fractures , and was found to have an ethmoid sinus glomangioma with intracranial extension . the tumor was removed via endoscopic endonasal approach to the anterior skull base , which resulted in complete resolution of symptoms and no further evidence of disease 1 year postoperatively . background : invasive mole is responsible for most cases of localized gestational trophoblastic neoplasia . gestational trophoblastic disease describes a number of gynecologic tumors that originate in trophoblastic layer including hydatidiform mole ( complete or partial ) , invasive mole , choriocarcinoma , placental site trophoblastic tumor and epitheloid trophoblastic tumor . invasive mole may arise from any pregnancy event although in most cases is diagnosed after molar pregnancy . overall cure rate in low risk patients is nearly 100% and in high - risk patient 90% . in rare cases , molar tissue traverses thickness of myometrium and leads to perforation and acute abdomen and invasive mole infrequently metastasis . the best treatment option is chemotherapy ( according to stage and score with single or multiple agent ) and in patients that fertility is not the matter , hysterectomy can be done.case presentation : a 41 years old g3p2ab1 woman referred to firouzgar hospital 2 months after curettage of molar pregnancy with vaginal bleeding and acute abdomen . in workup , hcg 224000 miu / ml and evidence of metastasis was detected . chemotherapy due to stage 3 and score 9 and surgery due to acute abdomen was done . this case was reported for its rarity.discussion:this case reported about ovarian metastasis and uterine rupture with acute abdomen and involvement of omentum in metastatic invasive mole . lack of surveillance led to extensive morbidity . management of this patient was successful . in follow up , she was free of disease without sequel of any kind for five years now .

an expanded research design and methods section and supplementary figures can be found in an online data supplement available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1293/dc1 . details on glucose and insulin tolerance testing , plasma insulin level determination , lipid metabolite measurement , metabolomics , exercise capacity studies , whole - body in vivo metabolic assessment , and immunoblot analysis are provided in the online data supplement . all animals received care according to the canadian council on animal care and the university of alberta health sciences animal welfare committee . twelve - week - old c57bl/6 mice were placed on a standard chow / low - fat diet ( 4% kcal from lard ) or high - fat diet ( 60% kcal from lard , research diets ; d12492 ) for a 12-week period . at the end of week 12 , animals were injected intraperitoneally every other day with the spt1 inhibitors , myriocin ( 0.5 mg / kg ) suspended in 1x pbs , l - cycloserine ( 25 mg / kg ) suspended in 1x pbs , or vehicle control for a 4-week period . at the end of the 4-week treatment protocol , animals were killed via intraperitoneal injection of sodium pentobarbital ( 12 mg ) in the fed state in the middle of the dark cycle . tissues were excised and immediately frozen in liquid n2 . in another study , 6-week - old db / db mice and their heterozygous controls ( db/+ ) ( jackson laboratories ) as expected , mice fed a high - fat diet for 12 weeks became obese as indicated by a significant increase in weight gain , ( supplementary fig . d ) . diet - induced insulin - resistant and lean mice were placed in a comprehensive lab animal - monitoring system ( clams ) for whole - body metabolic assessment , which demonstrated a high - fat diet induced shift in fuel preference toward fatty acids as an oxidative energy source , indicated by the large drop in the respiratory exchange ratio ( rer ) ( supplementary fig . further support for an increase in fatty acid oxidation in obese mice is seen with the increase in gastrocnemius -hydroxyacyl - coa dehydrogenase ( had ) activity ( table 1 ) . contrary to previous findings ( 16,17 ) , we also report here that obesity induced by chronic high - fat feeding impairs whole - body oxygen consumption rates ( supplementary fig . 2c and d ) . -hydroxyacyl - coa dehydrogenase ( had ) activity in gastrocnemius muscle of lean and obese mice treated with vehicle control or myriocin values reported are mol / g wet weight / min of n = 5 mice . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart . after 12 weeks of low- or high - fat diet , mice were treated with either myriocin ( 0.5 mg / kg every other day ) or vehicle control . after 2 weeks of treatment , we demonstrated that inhibition of spt1 with myriocin reverses diet - induced insulin resistance , as determined by glucose tolerance and insulin tolerance testing ( fig . if these protective effects took place at the skeletal muscle level , a group of animals were killed at 30 min after insulin injection during the insulin tolerance test , and muscles were excised and harvested for immunoblot analysis of the insulin - signaling pathway . we demonstrate that insulin stimulation of both akt and glycogen synthase kinase 3 ( gsk3 ) phosphorylation were significantly improved in the gastrocnemius muscle of obese mice treated with myriocin ( fig . phosphorylation of 5amp activated protein kinase ( ampk ) , another key signaling molecule regulating glucose metabolism , did not differ in gastrocnemius muscle of control and myriocin - treated obese mice ( data not shown ) . induced insulin resistance and improves insulin signaling . a : glucose tolerance test in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . c : insulin tolerance test in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . e : insulin - stimulated akt phosphorylation at serine 473 , and ( f ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of obese insulin - resistant mice treated with either vehicle control or myriocin . values represent mean se ( n = 812 for a d ; n = 4 for e and f ) . differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from all other groups . myriocin treatment was without effect on food intake , body weight , and plasma insulin levels , but did reduce both postprandial and fasted plasma glucose levels in obese , insulin - resistant mice ( table 2 ) . although fasting plasma insulin levels did not differ between diet - induced obesity ( dio ) mice treated with vehicle - control or myriocin , more sophisticated studies monitoring the changes in plasma insulin in response to a meal tolerance test in the obese jcr : la cp rat illustrate a significant improvement in plasma insulin control after treatment with the spt1 inhibitor , l - cycloserine ( supplementary fig . 3 ) . interestingly , indirect calorimetry revealed that the improved insulin sensitivity in dio mice treated with myriocin was not associated with a decrease in fatty acid oxidation and an increase in carbohydrate oxidation , as similar rer values were observed between the dio control and myriocin - treated animals ( fig . 2 ) . effect of myriocin on body and tissue weight , and plasma glucose and insulin levels in lean and obese mice values reported are from n = 511 mice . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart ; significantly different from high - fat control . twenty - four - hour ( a ) , dark cycle ( b ) , and light cycle respiratory exchange ratio ( c ) in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart . in a parallel series of experiments , after 12 weeks of high - fat feeding , mice were treated with either the spt1 inhibitor , l - cycloserine ( 25 mg / kg every other day ) or vehicle control . although not as dramatic as the results observed with myriocin , at 2 weeks after treatment , we report improvements in glucose and insulin tolerance in mice treated with l - cycloserine ( supplementary fig . obese , insulin - resistant mice were run on an exercise treadmill to determine exercise capacity . as expected , obese mice showed a dramatic reduction in both their treadmill time and distance when compared with their lean counterparts ( fig . interestingly , treatment of obese mice for 2 weeks with myriocin reversed this reduction in exercise capacity ( fig . 3a and b ) . this improvement in exercise capacity observed in obese mice treated with myriocin can be explained by enhanced whole - body oxygen consumption rates compared with their control counterparts ( fig . c ) . in addition , we observed greater citrate synthase activity in gastrocnemius muscle of obese mice treated with myriocin ( fig . protein expression of peroxisome proliferator - activated receptor- coactivator-1 ( pgc1 ) , a transcriptional coactivator that plays a key role in regulating a number of genes involved in energy metabolism ( 18 ) , showed a trend toward a reduction in control - treated dio mice ( p = 0.077 ) that was not apparent in myriocin - treated dio mice ( fig . furthermore , we also demonstrate that pretreatment with myriocin increases citrate synthase activity in c2c12 myotubes exposed to 1.0 mmol / l palmitate for 16 h ( fig . these observations illustrate improvements in mitochondrial function , possibly explaining why exercise capacity and whole - body oxygen consumption rates were enhanced in this group . time ( a ) and distance ( b ) during an exercise capacity challenge on a running treadmill . differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart . myriocin treatment reverses the impairment in whole - body oxygen consumption rates caused by dio . c : twenty - four hour ( a ) , dark cycle ( b ) , and light cycle ( c ) whole - body oxygen consumption assessment in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . d : gastrocnemius muscle citrate synthase activity in vehicle control and myriocin - treated dio mice . e : pgc1 expression in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . f : citrate synthase activity in vehicle control and myriocin - pretreated c2c12 skeletal muscle myotubes exposed to 1.0 mmol / l palmitate for 16 h. values represent mean se ( n = 512 ) . differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the high - fat diet control mice . auc , area under the curve . metabolic profiling of mice provided further insight with regards to mitochondrial function in obese , insulin - resistant mice , as control - treated dio mice had a significant increase in long - chain acyl carnitine esters versus their lean counterparts ( table 3 ) , indicative of mitochondrial overload and the incomplete oxidation of fatty acids ( 19 ) . however , the accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice was even greater ( table 3 ) . this suggests that incomplete fatty acid oxidation rates were even more pronounced in the myriocin - treated dio mice , but these animals also had a significant reduction in short - chain acyl carnitine ester content ( table 3 ) , which is consistent with long - chain acyl - coa dehydrogenase inhibition and reduced oxidation of long - chain fatty acids . metabolic profiling of gastrocnemius muscle from lean and obese mice treated with either vehicle control or myriocin values reported are in pmol / mg protein from n = 6 mice . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the lf counterpart . after 3 weeks of treatment with myriocin , in vivo heat production and ambulatory activity were assessed in our clams apparatus . paralleling our observations with regard to whole - body oxygen consumption rates , obesity caused a decline in whole - body heat production that was reversed by myriocin treatment ( supplementary fig . moreover , obesity - induced insulin resistance was associated with reductions in physical activity that were not altered by myriocin treatment ( supplementary fig . investigation of the lipid metabolite profile in gastrocnemius muscle demonstrated that chronic high - fat feeding increased long - chain acyl - coa , ceramide and dag content , but only a trend to an increase in tag content was observed ( fig . d ) . treatment with myriocin in obese mice increased gastrocnemius tag content in comparison to their low - fat counterparts , but did not change the dio - associated rise in long - chain acyl - coa and dag content , and as expected , resulted in a dramatic reduction in ceramide content ( fig . d ) . these results suggest a key role for ceramide in mediating skeletal muscle insulin resistance , and indicate that the other lipid metabolites possibly may not be as important in the insulin - resistance development . further support for this statement is seen with the positive correlation between ceramide content and the area under the curve during the glucose tolerance test , whereas no correlation was observed with any of the other lipid metabolites ( fig . interestingly , in a previous study , we showed that mice deficient for malonyl coa decarboxylase ( mcd/ ) are protected from obesity - induced glucose intolerance and insulin resistance , which was associated with a reduction in incomplete fatty acid oxidation rates ( 19 ) . in this study we show that these same mcd/ mice do not accumulate ceramide in their gastrocnemius muscle after 12 weeks of high - fat feeding ( fig . 6 ) , although they did accumulate other lipid metabolites such as long - chain acyl - coa ( 19 ) . inhibition of spt1 reduces skeletal muscle ceramide levels with no effect on other lipid metabolites . d : gastrocnemius triacylglycerol ( tag ) ( a ) , long - chain acyl - coa ( b ) , ceramide ( c ) , and diacylglycerol ( d ) levels in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet control mice . h : correlation between the respective areas under the curve during the glucose tolerance test and ceramide ( e ) , tag ( f ) , long - chain acyl - coa ( g ) , and diacylglycerol ( h ) content of ( n = 1418 ) samples . malonyl coa decarboxylase - deficient mice ( mcd/ ) do not accumulate skeletal muscle ceramide after 12 weeks of high - fat feeding . a : area under the curve during a glucose tolerance test after 12 weeks of high - fat feeding in wild - type and mcd/ mice . b : corresponding gastrocnemius ceramide levels in mcd/ mice after 12 weeks of high - fat feeding . differences were determined using a two - way anova followed by bonferroni post hoc analysis . p < 0.05 , significantly different from the high - fat diet wild - type mice . auc , area under the curve . to determine if ceramides may also be involved in genetic forms of insulin resistance and type 2 diabetes , we treated leptin receptor deficient ( db / db ) mice with myriocin to see if we could prevent the progression of insulin resistance in these animals . we split db / db mice at 6 weeks of age into two groups , and ensured that there were no differences in glucose tolerance before initiating treatment with myriocin ( fig . both the db / db control and myriocin - treated groups experienced similar body weight increases after 2 weeks of treatment ( data not shown ) , however , although the db / db control group became glucose intolerant , the db / db group treated with myriocin did not ( fig . fasting blood glucose levels were also significantly lower in the db / db mice treated with myriocin , and although their response to insulin was delayed , myriocin - treated db / db mice demonstrated lower blood glucose levels at nearly all time points during an insulin tolerance test ( fig . 7d f ) . placing these animals in the clams apparatus yielded a profile similar to that of the dio mice . the db / db controls had a lower rer in the dark cycle than db/+ lean mice , and had lower whole - body oxygen consumption rates and ambulatory activity , but no change in overall heat production ; interestingly , myriocin treatment of db / db mice did not restore any of these altered parameters in db / db controls , except for a restoration of whole - body oxygen consumption rates during the light cycle ( fig . examination of the lipid metabolite profile revealed that tag and long - chain acyl - coa levels were elevated in gastrocnemius muscle of db / db controls versus db/+ lean mice , whereas , unexpectedly , dag and ceramide levels were similar between the two groups ( fig . h ) . myriocin treatment of db / db mice had no effect on tag , long - chain acyl - coa , or dag levels in gastrocnemius muscle versus db / db control mice , but did lead to a dramatic reduction in ceramide levels ( fig . insulin - stimulated akt and gsk3 phosphorylation were also depressed in db / db control versus db/+ lean mice , but showed an improvement in db / db mice treated with myriocin ( fig . prevention of insulin resistance in db / db mice via myriocin treatment . a : pretreatment glucose tolerance test ( gtt ) in db / db mice at 6 weeks of age . c : respective areas under the curve for the post - treatment gtt in db / db mice . d : insulin tolerance test ( itt ) in db / db mice treated with vehicle control or myriocin . f : fed and fasted plasma glucose levels in db / db mice treated with vehicle control or myriocin . differences were determined using either a two - tailed student t test , or a one - way or two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the db / db control mice . in vivo metabolic parameters , intramyocellular lipid metabolite profile , and insulin signaling in db / db mice treated with myriocin . rer ( a ) , whole - body oxygen consumption ( b ) , heat production ( c ) , and ambulatory activity ( d ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin . gastrocnemius triacylglycerol ( e ) , long - chain acyl - coa ( f ) , diacylglycerol ( g ) , and ceramide levels ( h ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin . i : insulin stimulated akt phosphorylation at serine 473 , and ( j ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of db/+ heterozygous mice and db / db mice treated with vehicle control or myriocin . differences were determined using either a one - way or two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the db / db control mice . as expected , mice fed a high - fat diet for 12 weeks became obese as indicated by a significant increase in weight gain , ( supplementary fig . d ) . diet - induced insulin - resistant and lean mice were placed in a comprehensive lab animal - monitoring system ( clams ) for whole - body metabolic assessment , which demonstrated a high - fat diet induced shift in fuel preference toward fatty acids as an oxidative energy source , indicated by the large drop in the respiratory exchange ratio ( rer ) ( supplementary fig . further support for an increase in fatty acid oxidation in obese mice is seen with the increase in gastrocnemius -hydroxyacyl - coa dehydrogenase ( had ) activity ( table 1 ) . contrary to previous findings ( 16,17 ) , we also report here that obesity induced by chronic high - fat feeding impairs whole - body oxygen consumption rates ( supplementary fig . 2c and d ) . -hydroxyacyl - coa dehydrogenase ( had ) activity in gastrocnemius muscle of lean and obese mice treated with vehicle control or myriocin values reported are mol / g wet weight / min of n = 5 mice . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart . after 12 weeks of low- or high - fat diet , mice were treated with either myriocin ( 0.5 mg / kg every other day ) or vehicle control . after 2 weeks of treatment , we demonstrated that inhibition of spt1 with myriocin reverses diet - induced insulin resistance , as determined by glucose tolerance and insulin tolerance testing ( fig . if these protective effects took place at the skeletal muscle level , a group of animals were killed at 30 min after insulin injection during the insulin tolerance test , and muscles were excised and harvested for immunoblot analysis of the insulin - signaling pathway . we demonstrate that insulin stimulation of both akt and glycogen synthase kinase 3 ( gsk3 ) phosphorylation were significantly improved in the gastrocnemius muscle of obese mice treated with myriocin ( fig . phosphorylation of 5amp activated protein kinase ( ampk ) , another key signaling molecule regulating glucose metabolism , did not differ in gastrocnemius muscle of control and myriocin - treated obese mice ( data not shown ) . inhibition of serine palmitoyl transferase 1 ( spt1 ) reverses high - fat diet induced insulin resistance and improves insulin signaling . a : glucose tolerance test in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . c : insulin tolerance test in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . e : insulin - stimulated akt phosphorylation at serine 473 , and ( f ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of obese insulin - resistant mice treated with either vehicle control or myriocin . values represent mean se ( n = 812 for a d ; n = 4 for e and f ) . differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from all other groups . myriocin treatment was without effect on food intake , body weight , and plasma insulin levels , but did reduce both postprandial and fasted plasma glucose levels in obese , insulin - resistant mice ( table 2 ) . although fasting plasma insulin levels did not differ between diet - induced obesity ( dio ) mice treated with vehicle - control or myriocin , more sophisticated studies monitoring the changes in plasma insulin in response to a meal tolerance test in the obese jcr : la cp rat illustrate a significant improvement in plasma insulin control after treatment with the spt1 inhibitor , l - cycloserine ( supplementary fig . 3 ) . interestingly , indirect calorimetry revealed that the improved insulin sensitivity in dio mice treated with myriocin was not associated with a decrease in fatty acid oxidation and an increase in carbohydrate oxidation , as similar rer values were observed between the dio control and myriocin - treated animals ( fig . 2 ) . effect of myriocin on body and tissue weight , and plasma glucose and insulin levels in lean and obese mice values reported are from n = 511 mice . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart ; significantly different from high - fat control . twenty - four - hour ( a ) , dark cycle ( b ) , and light cycle respiratory exchange ratio ( c ) in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart . in a parallel series of experiments , after 12 weeks of high - fat feeding , mice were treated with either the spt1 inhibitor , l - cycloserine ( 25 mg / kg every other day ) or vehicle control . although not as dramatic as the results observed with myriocin , at 2 weeks after treatment , we report improvements in glucose and insulin tolerance in mice treated with l - cycloserine ( supplementary fig . obese , insulin - resistant mice were run on an exercise treadmill to determine exercise capacity . as expected , obese mice showed a dramatic reduction in both their treadmill time and distance when compared with their lean counterparts ( fig . interestingly , treatment of obese mice for 2 weeks with myriocin reversed this reduction in exercise capacity ( fig . 3a and b ) . this improvement in exercise capacity observed in obese mice treated with myriocin can be explained by enhanced whole - body oxygen consumption rates compared with their control counterparts ( fig . c ) . in addition , we observed greater citrate synthase activity in gastrocnemius muscle of obese mice treated with myriocin ( fig . protein expression of peroxisome proliferator - activated receptor- coactivator-1 ( pgc1 ) , a transcriptional coactivator that plays a key role in regulating a number of genes involved in energy metabolism ( 18 ) , showed a trend toward a reduction in control - treated dio mice ( p = 0.077 ) that was not apparent in myriocin - treated dio mice ( fig . furthermore , we also demonstrate that pretreatment with myriocin increases citrate synthase activity in c2c12 myotubes exposed to 1.0 mmol / l palmitate for 16 h ( fig . these observations illustrate improvements in mitochondrial function , possibly explaining why exercise capacity and whole - body oxygen consumption rates were enhanced in this group . time ( a ) and distance ( b ) during an exercise capacity challenge on a running treadmill . differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart . myriocin treatment reverses the impairment in whole - body oxygen consumption rates caused by dio . c : twenty - four hour ( a ) , dark cycle ( b ) , and light cycle ( c ) whole - body oxygen consumption assessment in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . d : gastrocnemius muscle citrate synthase activity in vehicle control and myriocin - treated dio mice . e : pgc1 expression in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . f : citrate synthase activity in vehicle control and myriocin - pretreated c2c12 skeletal muscle myotubes exposed to 1.0 mmol / l palmitate for 16 h. values represent mean se ( n = 512 ) . differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet control mice . auc , area under the curve . metabolic profiling of mice provided further insight with regards to mitochondrial function in obese , insulin - resistant mice , as control - treated dio mice had a significant increase in long - chain acyl carnitine esters versus their lean counterparts ( table 3 ) , indicative of mitochondrial overload and the incomplete oxidation of fatty acids ( 19 ) . however , the accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice was even greater ( table 3 ) . this suggests that incomplete fatty acid oxidation rates were even more pronounced in the myriocin - treated dio mice , but these animals also had a significant reduction in short - chain acyl carnitine ester content ( table 3 ) , which is consistent with long - chain acyl - coa dehydrogenase inhibition and reduced oxidation of long - chain fatty acids . metabolic profiling of gastrocnemius muscle from lean and obese mice treated with either vehicle control or myriocin values reported are in pmol / mg protein from n = 6 mice . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the lf counterpart . after 3 weeks of treatment with myriocin , in vivo heat production and ambulatory activity were assessed in our clams apparatus . paralleling our observations with regard to whole - body oxygen consumption rates , obesity caused a decline in whole - body heat production that was reversed by myriocin treatment ( supplementary fig . moreover , obesity - induced insulin resistance was associated with reductions in physical activity that were not altered by myriocin treatment ( supplementary fig . investigation of the lipid metabolite profile in gastrocnemius muscle demonstrated that chronic high - fat feeding increased long - chain acyl - coa , ceramide and dag content , but only a trend to an increase in tag content was observed ( fig . d ) . treatment with myriocin in obese mice increased gastrocnemius tag content in comparison to their low - fat counterparts , but did not change the dio - associated rise in long - chain acyl - coa and dag content , and as expected , resulted in a dramatic reduction in ceramide content ( fig . d ) . these results suggest a key role for ceramide in mediating skeletal muscle insulin resistance , and indicate that the other lipid metabolites possibly may not be as important in the insulin - resistance development . further support for this statement is seen with the positive correlation between ceramide content and the area under the curve during the glucose tolerance test , whereas no correlation was observed with any of the other lipid metabolites ( fig . h ) . interestingly , in a previous study , we showed that mice deficient for malonyl coa decarboxylase ( mcd/ ) are protected from obesity - induced glucose intolerance and insulin resistance , which was associated with a reduction in incomplete fatty acid oxidation rates ( 19 ) . in this study we show that these same mcd/ mice do not accumulate ceramide in their gastrocnemius muscle after 12 weeks of high - fat feeding ( fig . 6 ) , although they did accumulate other lipid metabolites such as long - chain acyl - coa ( 19 ) . inhibition of spt1 reduces skeletal muscle ceramide levels with no effect on other lipid metabolites . d : gastrocnemius triacylglycerol ( tag ) ( a ) , long - chain acyl - coa ( b ) , ceramide ( c ) , and diacylglycerol ( d ) levels in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet control mice . h : correlation between the respective areas under the curve during the glucose tolerance test and ceramide ( e ) , tag ( f ) , long - chain acyl - coa ( g ) , and diacylglycerol ( h ) content of ( n = 1418 ) samples . malonyl coa decarboxylase - deficient mice ( mcd/ ) do not accumulate skeletal muscle ceramide after 12 weeks of high - fat feeding . a : area under the curve during a glucose tolerance test after 12 weeks of high - fat feeding in wild - type and mcd/ mice . b : corresponding gastrocnemius ceramide levels in mcd/ mice after 12 weeks of high - fat feeding . differences were determined using a two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from low - fat diet counterpart . p < 0.05 , significantly different from the high - fat diet wild - type mice . to determine if ceramides may also be involved in genetic forms of insulin resistance and type 2 diabetes , we treated leptin receptor deficient ( db / db ) mice with myriocin to see if we could prevent the progression of insulin resistance in these animals . we split db / db mice at 6 weeks of age into two groups , and ensured that there were no differences in glucose tolerance before initiating treatment with myriocin ( fig . both the db / db control and myriocin - treated groups experienced similar body weight increases after 2 weeks of treatment ( data not shown ) , however , although the db / db control group became glucose intolerant , the db / db group treated with myriocin did not ( fig . fasting blood glucose levels were also significantly lower in the db / db mice treated with myriocin , and although their response to insulin was delayed , myriocin - treated db / db mice demonstrated lower blood glucose levels at nearly all time points during an insulin tolerance test ( fig . f ) . placing these animals in the clams apparatus yielded a profile similar to that of the dio mice . the db / db controls had a lower rer in the dark cycle than db/+ lean mice , and had lower whole - body oxygen consumption rates and ambulatory activity , but no change in overall heat production ; interestingly , myriocin treatment of db / db mice did not restore any of these altered parameters in db / db controls , except for a restoration of whole - body oxygen consumption rates during the light cycle ( fig . examination of the lipid metabolite profile revealed that tag and long - chain acyl - coa levels were elevated in gastrocnemius muscle of db / db controls versus db/+ lean mice , whereas , unexpectedly , dag and ceramide levels were similar between the two groups ( fig . h ) . myriocin treatment of db / db mice had no effect on tag , long - chain acyl - coa , or dag levels in gastrocnemius muscle versus db / db control mice , but did lead to a dramatic reduction in ceramide levels ( fig . insulin - stimulated akt and gsk3 phosphorylation were also depressed in db / db control versus db/+ lean mice , but showed an improvement in db / db mice treated with myriocin ( fig . prevention of insulin resistance in db / db mice via myriocin treatment . a : pretreatment glucose tolerance test ( gtt ) in db / db mice at 6 weeks of age . c : respective areas under the curve for the post - treatment gtt in db / db mice . d : insulin tolerance test ( itt ) in db / db mice treated with vehicle control or myriocin . f : fed and fasted plasma glucose levels in db / db mice treated with vehicle control or myriocin . differences were determined using either a two - tailed student t test , or a one - way or two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the db / db control mice . in vivo metabolic parameters , intramyocellular lipid metabolite profile , and insulin signaling in db / db mice treated with myriocin . rer ( a ) , whole - body oxygen consumption ( b ) , heat production ( c ) , and ambulatory activity ( d ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin . gastrocnemius triacylglycerol ( e ) , long - chain acyl - coa ( f ) , diacylglycerol ( g ) , and ceramide levels ( h ) in db/+ heterozygous mice , and db / db mice treated with vehicle control or myriocin . i : insulin stimulated akt phosphorylation at serine 473 , and ( j ) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of db/+ heterozygous mice and db / db mice treated with vehicle control or myriocin . differences were determined using either a one - way or two - way anova followed by bonferroni post hoc analysis . * p < 0.05 , significantly different from the db / db control mice . our results show that inhibition of spt1 reduces de novo ceramide synthesis in muscle , which has novel effects on whole - body energy metabolism and is associated with a profound reversal of glucose intolerance and insulin resistance induced by chronic high - fat feeding . furthermore , we show that these improvements are dissociated from the other lipid metabolites believed to play a role in the development of insulin resistance . interestingly , obesity - induced insulin resistance in mice is associated with a detriment in aerobic exercise capacity and whole - body oxygen consumption rates , both of which are partially reversed via spt1 inhibition . previous studies have postulated that skeletal muscle insulin resistance is caused by the intramyocellular cytosolic accumulation of lipid metabolites ( tag , long - chain acyl - coa , dag , ceramide , etc . ) in particular , long - chain acyl - coa and dag have received considerable attention because of their ability to activate the classic / novel protein kinase c signaling cascade , which can phosphorylate insulin receptor substrate proteins on serine residues , preventing their activation via the insulin receptor ( 4,5,8,2123 ) . it is important to note , however , that most ( 95% ) acyl - coa esters are located inside the mitochondria ( 24,25 ) , suggesting that if long - chain acyl - coa accumulation does play a role toward insulin resistance development , it is possible that mitochondrial , as opposed to cytosolic long - chain acyl - coa , is the primary contributor . although tag has been shown in numerous studies to be elevated in muscle in association with the development of insulin resistance , recent studies have shown that tag may actually serve as a buffer , protecting the muscle against the accumulation of the more reactive lipid metabolite species ( 10,11 ) . in regards to ceramide , data are mixed with its role in insulin resistance development , because in some studies , ceramide accumulation is not evident in muscle ( 5,26 ) , and in other studies where accumulation does occur , the relative increase in the ceramide pool is not that large ( 12,27 ) . ( 12 ) has shed some light on this issue , as they demonstrated that ceramide accumulation in muscle is dependent on the type of diet fed to the animals . in particular , saturated fatty acids drive de novo ceramide synthesis in muscle via spt1 , whereas unsaturated fatty acids cause insulin resistance via other mechanisms ( 12 ) . such findings may potentially explain why ceramide accumulation is not observed in studies of insulin resistance where the model employed is a lipid infusion that consists primarily of unsaturated fatty acids ( 22 ) . furthermore , holland et al . ( 12 ) showed in their study that preventing de novo synthesis of ceramide via spt1 inhibition with myriocin prevented the development of glucose intolerance in obese zucker rats , and prevented the palmitate - induced inhibition of insulin - stimulated 2-deoxyglucose uptake in isolated soleus muscle . ( 28 ) also reported positive findings with myriocin treatment in leptin - deficient and dio mice , providing further support that ceramide plays a key role in the development of insulin resistance . interestingly , these authors also observed a weight loss effect due to myriocin treatment that we did not observe in our studies . however , the authors in this study used a much longer treatment than ours ( 8 vs. 4 weeks ) , and noted that they did not observe a weight loss effect until later in the treatment period . furthermore , 3 weeks of myriocin treatment in dio mice improved hyperglycemia and whole - body oxygen consumption rates in their mice , despite no change in body weight compared with control - treated dio mice , which is consistent with our results in dio mice treated with myriocin for 2 weeks . yang et al . also observed a dramatic reduction in hepatic steatosis that is consistent with our observations in regards to hepatic tag content . our study adds further support to the studies examining the role of ceramide in mediating insulin resistance ( 12,28 ) by illustrating the potential for targeting spt1 as a treatment against insulin resistance . . moreover , by examining other lipid metabolites such as tag , dag , long - chain acyl - coa , and acyl carnitine content in skeletal muscle , we are able to discern important differences with regard to the relative importance of each metabolite toward the development of skeletal mucle insulin resistance . importantly , reductions in skeletal muscle ceramide accumulation may represent a potential explanation for the exercise paradox observed in humans . dube et al . ( 15 ) showed that obese , insulin - resistant men placed on an aerobic exercise training regime have elevated intramyocellular lipid and tag stores . however , marked reductions in muscle ceramide levels are observed , which may explain the enhanced insulin sensitivity of these men . moreover , bruce et al . ( 14 ) showed that the improved insulin sensitivity observed with exercise training in humans is associated with a drop in muscle ceramide levels , and in particular , the saturated species . ( 29 ) showed that exercise training of rats leads to a dramatic drop in the saturated species of ceramide in muscle , which is associated with an enhanced 2-deoxyglucose uptake . in addition , mice overexpressing diacylglycerol acyl transferase in muscle are protected from high - fat - diet induced insulin resistance and palmitate inhibition of 2-deoxyglucose uptake in isolated muscle , both of which are associated with an elevation of muscle tag and drop in ceramide levels ( 10 ) . our results support these studies , as we show that obese , insulin - resistant mice treated with myriocin had significant increases in intramyocellular tag , long - chain acyl - coa , and dag , but a dramatic drop in ceramide content . moreover , we observed a positive correlation with ceramide content and glucose intolerance , but not with any of the other lipid metabolites . we believe that with this finding , in the setting of obesity , that ceramide may be more vital to the development of skeletal muscle insulin resistance than the other lipid metabolites . support for this statement is also evident in culture models of ceramide accumulation , whereby inhibition of spt1 was able to prevent palmitate - induced insulin resistance in both human and rat l6 myotubes , despite elevated tag and dag levels ( 11,13 ) . furthermore , a recent study in humans demonstrated that insulin resistant muscle is associated with elevated ceramide content , but no change in dag content ( 30 ) . nonetheless , it is also important to note that our measurement of dag assessed total cellular levels of dag , and it is possible that differences in plasma membrane dag were significantly reduced via myriocin treatment . because dag at the membrane is believed to be the specific dag pool responsible for mediating skeletal muscle insulin resistance ( 3 ) , it will be important for future studies to investigate this in more detail . one of the most surprising findings of this study was that chronic high - fat feeding resulted in a dramatic decline in whole - body oxygen consumption rates . the majority of studies that have examined the effect of high - fat feeding on whole - body oxygen consumption rates via use of the clams apparatus have reported elevations in oxygen consumption rates ( 16,17 ) . although the differences between these studies and ours could be due to the duration or composition of the diet , we propose two possible explanations for this observation of ours . first , it has been reported that obesity - induced insulin resistance causes mitochondrial dysfunction that results from an impairment of fatty acid oxidative capacity ( 58 ) . although it may be possible that our model of insulin resistance is inducing mitochondrial dysfunction , it is highly unlikely due to impairments in muscle fatty acid oxidative capacity , as the rer values in obese mice reported in this study are very close to 0.7 , indicating that these animals have no trouble utilizing fat as an energy source . nonetheless , other factors , such as mitochondrial content , protein expression of electron transport chain ( etc ) complexes , or activity of these complexes , may account for potential mitochondrial dysfunction and the subsequent impairment of oxygen consumption rates observed in obese mice ( 31,32 ) . however , we did not observe differences in protein expression of cytochrome c of the etc in any group ( data not shown ) . second , and just as relevant to the findings of this study , is that obesity - induced insulin resistance has been associated with elevated rates of incomplete fatty acid oxidation , which can arise when rates of fatty acid oxidation are disconnected from tca cycle activity ( 19,33,34 ) . this disconnect arises due to the sedentary nature of obese individuals and animals , thus there is no demand for the tca cycle to upregulate its activity to deal with the increased fatty acid supply that is being utilized as an energy source ( 19,33,34 ) . if the tca cycle is unable to accommodate the increasing acetyl coa coming from fatty acid oxidation , reducing equivalents such as nadh and fadh2 would not donate their electrons to the complexes of the etc , accounting for the reduction in oxygen consumption rates . our observation of increased accumulation of long - chain acyl carnitine esters in the muscle of dio mice is thus consistent with elevated rates of incomplete fatty acid oxidation . in contrast , there was an even greater accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice , which at first glance would suggest even greater rates of incomplete fatty acid oxidation in these animals . however , myriocin - treated dio mice actually had a significant reduction in the content of a number of short - chain acyl carnitine esters , and this , in combination with the rise of long - chain acyl carnitine esters , is suggestive of long - chain acyl - coa dehydrogenase and subsequent long - chain fatty acid oxidation inhibition ( 35 ) . another piece of indirect support for fatty acid oxidation inhibition with myriocin treatment in dio mice is the observation that tag accumulated in the muscle of these animals versus their lean counterparts , but not in control - treated dio mice versus their lean counterparts . a reduction in fatty acid oxidation - derived nadh would decrease nadh / nadph oxidase activity and subsequent superoxide production in myriocin - treated dio mice , which would contribute toward their improved mitochondrial function . this improvement in mitochondrial function , coupled together with improvements in glucose metabolism and glucose - derived acetyl coa production for the tca cycle , may contribute to the greater oxygen consumption rates in these animals . obesity - induced decrements in pgc1 protein expression might also explain impairments in mitochondrial function ( 34,36,37 ) , and although not significant , we observed a trend toward a reduction in gastrocnemius pgc1 protein expression in control - treated dio mice ( p = 0.077 ) that was not evident in myriocin - treated dio mice . interestingly , citrulline levels were increased in myriocin - treated dio mice versus their control counterparts ( supplementary fig . a previous study in humans showed that supplementation of citrulline enhances aerobic oxidative metabolism ( 38 ) , supporting our findings of increased whole - body oxygen consumption rates and greater exercise time in myriocin - treated dio mice . how myriocin and subsequent spt1 inhibition would influence skeletal muscle citrulline levels is currently unknown , but is undoubtedly an intriguing avenue for future investigation . in addition , we have previously shown that mcd/ mice ( a genetic model of fatty acid oxidation deficiency ) are protected from obesity - induced insulin resistance . interestingly , we show in this study that these exact same animals do not accumulate ceramide in their muscle after 12 weeks of high - fat feeding , leading to the very intriguing possibility that intramyocellular ceramide accumulation is linked to the mitochondrial dysfunction and enhanced skeletal muscle fatty acid oxidation rates observed in insulin resistance . a limitation with our interpretation of whole - body oxygen consumption rates is that , unlike human studies , we were unable to normalize our oxygen consumption rates to lean body mass . it is entirely possible that whole - body oxygen consumption rates were simply lower in dio mice because of a significant increase in overall adiposity , due to fat mass having a lower metabolic rate than lean body mass . however , the fact that adiposity and body weight were similar between myriocin- and control - treated dio mice suggests that this would not be a contributing factor to the higher oxygen consumption rates observed in the myriocin - treated dio mice . although we believe that the changes accounting for the greater oxygen consumption rates in myriocin - treated dio mice primarily reflect the muscle , we can not ignore possible contributions from changes in other peripheral tissues , such as brown adipose tissue and uncoupling protein activity . the beneficial effects mediated by inhibition of spt1 and prevention of de novo ceramide synthesis could also arise from liver effects in our animals . regardless , we did not observe increases in hepatic ceramide content after diet - induced obesity , and myriocin treatment had no effect on insulin - stimulated akt and gsk3 phosphorylation in obese mice versus their respective controls ( supplementary fig . , recent studies have also shown that high - fat feeding does not increase ceramide content in the liver ( 39 ) , and increases in hepatic ceramide content via genetic overexpression of either dgat1 or dgat2 does not result in any type of insulin resistance or inflammation ( 40 ) . moreover , we reported no difference during a pyruvate challenge of fasted , obese , control- or myriocin - treated mice ( supplementary fig . 8) , suggesting that gluconeogenic capacity was not different between the two groups and that the liver likely does not play a key role with the improved insulin sensitivity observed in myriocin - treated mice . regardless , we can not entirely rule out the possibility that the liver plays a role with the benefit observed during myriocin treatment , as the dio - associated rise in hepatic tag content was reversed via myriocin treatment , and thus it will be important for future studies to delineate the role of hepatic spt1 in greater depth . finally , chronic low - grade inflammation has been shown in a number of studies to play a role in causing obesity - induced insulin resistance ( 4143 ) . inflammatory and stress kinases , such as p38 mapk and jnk , have been proposed to be downstream mediators of this inflammatory effect , as inhibitors of both kinases are able to prevent high - fat diet induced insulin resistance ( 9,4446 ) . unexpectedly , the phosphorylation status of both p38 mapk and jnk was not altered by dio , nor was it altered by myriocin treatment ( supplementary fig . 9 ) , suggesting that inflammation may not play as vital a role in our model of insulin resistance . it may also be possible that inflammation in our model is mediated by some other kinase , such as ikk ( 47,48 ) . with regard to the findings in db / db mice , we report very similar findings to what we observed in the obesity - induced insulin - resistant mice , and that treatment with myriocin also yielded a very similar beneficial profile . interestingly , gastrocnemius ceramide levels , although reduced in myriocin - treated db / db mice , did not differ between db/+ lean and db / db control mice . this suggests , at least in this model , that perhaps ceramide metabolites , such as glucosylceramide , are more important in mediating skeletal muscle insulin resistance than ceramide itself ( 49 ) . furthermore , the ceramide pool is under a dynamic process of synthesis and degradation ( 9 ) , and although de novo synthesis of ceramide may be increased in these animals , a simultaneous increase in ceramide degradation would mask out any noticeable change . in summary , we show that ceramide accumulation in skeletal muscle plays a key role during obesity - induced insulin resistance , whereas the other lipid metabolites , such as tag , long - chain acyl - coa , and dag , may not be as vital . importantly , inhibition of de novo ceramide synthesis has novel effects on whole - body energy metabolism and is sufficient to reverse obesity - induced whole - body glucose intolerance and insulin resistance . furthermore , whole - body oxygen consumption rates and exercise capacity in obese mice are improved via inhibition of de novo ceramide synthesis . last , our finding that muscle ceramide levels are not elevated in db / db mice , but that inhibition of de novo ceramide synthesis still prevents their development of insulin resistance , suggests the possibility that ceramide metabolites may also play a role in the progression of this disease . antibodies against amp kinase ( ampk ) , phosporylated ampk ( pampk ) , acetyl coa carboxylase ( acc ) , phosporylated acc , akt , phosporylated akt , and tubulin were from cell signaling ; anti - oxphos was from mitosciences ; anti cd36-[hrp ] was purchased from novus biologicals , and human recombinant insulin ( novolin ) from novo nordisk canada . this study was performed with the approval of the university of alberta animal policy and welfare committee . experiments were carried out on male wild - type ( c57bl6 ) and cd36 knockout ( ko ) mice ( 19 ) maintained in a temperature - controlled room with a reversed 12-h light/12-h dark cycle . mice were left relatively undisturbed for either 1214 or 5258 weeks of age with free access to water and standard rodent diet ( category no . 5001 ; labdiet ) . at 3234 weeks of age , a subset of mice was randomly divided into a low - fat diet group ( category no . d12450b ; research diets ) and a high - fat diet group ( category no . d12492 ; research diets ) for a period of 12 weeks . indirect calorimetry was performed using the comprehensive lab animal monitoring system ( oxymax / clams ; columbus instruments , colombus , oh ) . following an initial 24-h acclimatization period , mice were monitored every 13 min for 24 h to complete a 12-h dark ( active)/12-h light ( inactive ) cycle . the respiratory exchange ratio ( rer = vco2/vo2 ) was used to estimate the percent contribution of fat and carbohydrate to whole - body energy metabolism in mice in vivo . total activity was calculated by adding z counts ( rearing or jumping ) to total counts associated with ambulatory movement and stereotypical behavior ( grooming and scratching ) . upon phospholipid digestion with phospholipase c ( 2 h at 30c ) and lipid extraction , levels of triglycerides were determined in gastrocnemius muscle lysates by gas - liquid chromatography as previously described ( 20 ) . identification and quantification of the major long - chain acyl - coa molecular species ( c16:0 , c18:0 , and c18:1 ) and c18-ceramides were performed by high - performance liquid chromatography as previously described ( 21 ) . overnight - fasted mice were anesthetized with sodium pentobarbital , and gastrocnemius muscle was rapidly removed and freeze - clamped in liquid nitrogen and stored at 80c . acylcarnitine measurements were made using flow - injection tandem mass spectrometry as previously described ( 14 ) , and organic acids were quantified as previously described ( 22 ) . comparisons between groups were performed using unpaired student 's two - tailed t test or anova with a bonferroni post hoc test of pairwise comparisons where appropriate . a probability value of < 0.05 is considered significant . for further descriptions of the materials and methods , refer to the supplementary material available in an online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1142/dc1 . antibodies against amp kinase ( ampk ) , phosporylated ampk ( pampk ) , acetyl coa carboxylase ( acc ) , phosporylated acc , akt , phosporylated akt , and tubulin were from cell signaling ; anti - oxphos was from mitosciences ; anti cd36-[hrp ] was purchased from novus biologicals , and human recombinant insulin ( novolin ) from novo nordisk canada . this study was performed with the approval of the university of alberta animal policy and welfare committee . experiments were carried out on male wild - type ( c57bl6 ) and cd36 knockout ( ko ) mice ( 19 ) maintained in a temperature - controlled room with a reversed 12-h light/12-h dark cycle . mice were left relatively undisturbed for either 1214 or 5258 weeks of age with free access to water and standard rodent diet ( category no . 5001 ; labdiet ) . at 3234 weeks of age , a subset of mice was randomly divided into a low - fat diet group ( category no . d12450b ; research diets ) and a high - fat diet group ( category no . indirect calorimetry was performed using the comprehensive lab animal monitoring system ( oxymax / clams ; columbus instruments , colombus , oh ) . following an initial 24-h acclimatization period , mice were monitored every 13 min for 24 h to complete a 12-h dark ( active)/12-h light ( inactive ) cycle . the respiratory exchange ratio ( rer = vco2/vo2 ) was used to estimate the percent contribution of fat and carbohydrate to whole - body energy metabolism in mice in vivo . total activity was calculated by adding z counts ( rearing or jumping ) to total counts associated with ambulatory movement and stereotypical behavior ( grooming and scratching ) . upon phospholipid digestion with phospholipase c ( 2 h at 30c ) and lipid extraction , levels of triglycerides were determined in gastrocnemius muscle lysates by gas - liquid chromatography as previously described ( 20 ) . identification and quantification of the major long - chain acyl - coa molecular species ( c16:0 , c18:0 , and c18:1 ) and c18-ceramides were performed by high - performance liquid chromatography as previously described ( 21 ) . overnight - fasted mice were anesthetized with sodium pentobarbital , and gastrocnemius muscle was rapidly removed and freeze - clamped in liquid nitrogen and stored at 80c . acylcarnitine measurements were made using flow - injection tandem mass spectrometry as previously described ( 14 ) , and organic acids were quantified as previously described ( 22 ) . comparisons between groups were performed using unpaired student 's two - tailed t test or anova with a bonferroni post hoc test of pairwise comparisons where appropriate . a probability value of < 0.05 is considered significant . for further descriptions of the materials and methods , refer to the supplementary material available in an online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1142/dc1 . to determine whether an age - related decline in resting metabolic rate and energy expenditure might contribute to the development of insulin resistance , c57bl6 mice of 1214 ( young ) or 5258 ( middle - aged ) weeks of age were analyzed using indirect calorimetry . as mice aged and body weight was increased ( fig . 1a ) , substrate use was altered slightly , with reductions in rer indicating that middle - aged mice used more fatty acid throughout the day compared with young mice ( fig . in addition , significant reductions in oxygen consumption ( vo2 ) ( fig . 1c ) and carbon dioxide production ( vco2 ) ( fig . 1d ) were observed during both the dark ( active ) and light ( inactive ) phases in middle - aged mice compared with young mice . 1e ) was decreased in middle - aged mice compared with young mice , whereas activity measurements were not significantly different between age - groups ( fig . our data indicate that middle - aged mice have a lower metabolic rate than young mice and that this might increase susceptibility to weight gain , obesity , and metabolic disease . body weight of young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice fed a standard laboratory diet ( a ) . indirect calorimetry was performed to measure respiratory exchange ratio ( rer ) ( b ) , vo2 ( c ) , vco2 ( d ) , and heat production adjusted for bodyweight ( e ) ; total activity ( f ) was measured in both the dark ( active ) and light ( inactive ) phases . p < 0.05 indicates comparisons performed between young and middle - aged mice in either dark or light phases ( mann - whitney u test ) . two - way anova was performed for rer ( b ) and indicated the main effect for age ( p < 0.01 ) . given that skeletal muscle metabolism contributes to whole - body basal metabolic rate , we addressed whether skeletal muscle metabolism was depressed in middle - aged mice by assessing the activities of two enzymes involved in regulating mitochondrial metabolism . 2a ) was elevated in the middle - aged compared with the young mice , and citrate synthase ( fig . 2b ) followed a similar upward trend , suggesting that -oxidation and tca cycle activity were not directly compromised in the middle - aged mice . since mitochondrial number ( fig . 2c ) or function did not appear to be altered at middle age , we next assessed whether peroxisome proliferator activated receptor ( ppar) responsive genes or molecular signaling cascades known to regulate skeletal muscle fatty acid flux and fatty acid entry into the mitochondria were associated with this overall reduction in whole - body basal metabolic rate observed in middle age . although a more comprehensive assessment of the multiple mediators of fatty acid utilization may reveal additional mechanisms , we did not observe any changes in protein levels of known ppar responsive genes involved in lipid metabolism , including malonyl - coa decarboxylase and acyl - coa synthetase 1 ( data not shown ) . as a result , we also examined the energy - sensing kinase , ampk , which is known for its ability to govern energy metabolism ( 23 ) . in agreement with previous reports using older rodents ( 24,25 ) , levels of phosphorylated ampk were significantly reduced in skeletal muscle of middle - aged mice compared with young mice ( fig . moreover , the phosphorylation status of acetyl coa carboxylase ( acc ) , the downstream target of ampk that indirectly regulates fatty acid entry into the mitochondria and ultimately -oxidation , was significantly decreased in skeletal muscle from middle - aged mice ( fig . although reduced ampk phosphorylation could be the result of impaired activity of upstream ampk kinases or increased ampk phosphatase activity , it is currently unknown which of these contributes to reduced ampk phosphorylation in our model . however , consistent with decreased energy expenditure , increased adiposity , and reduced ampk activity in skeletal muscle from middle - aged mice , the levels of skeletal muscle triglycerides were significantly elevated in middle - aged mice compared with young mice ( fig . 2 g ) . given that lipid accumulation in skeletal muscle has been proposed in rodents ( 26 ) and humans ( 17,27,28 ) to be one of the primary causes of skeletal muscle insulin resistance , we next investigated whether glucose tolerance was impaired in middle - aged mice . despite elevated intramuscular triglycerides ( fig . 2 g ) and impaired basal and insulin - stimulated akt phosphorylation ( fig . 2h and i , respectively ) in skeletal muscle of middle - aged mice , whole - body glucose tolerance ( fig . 2k ) were not different in middle - aged compared with young mice , suggesting that age - induced alterations in skeletal muscle fatty acid handling and increased triglyceride storage precede the development of insulin resistance and metabolic disease . alterations in fatty acid handling and reduced insulin signaling in skeletal muscle of middle - aged mice do not result in impaired whole - body glucose tolerance . activity of two mitochondrial enzymes -had ( a ) and citrate synthase ( cs ) ( b ) was determined in gastrocnemius muscle from overnight fasted young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice . immunoblot analysis using a total oxphos complex antibody cocktail was performed in gastrocnemius muscle lysates , and immunoblots were normalized against tubulin as a control for protein loading ( c ) . phosphorylation status of ampk at threonine 172 ( d ) and acc at serine 79 ( f ) was detected using immunoblot analysis with phospho - specific antibodies . immunoblots were quantified by densitometry and normalized against total protein levels of ampk ( e ) and acc ( f ) . triglyceride ( tg ) levels ( g ) and phosphorylation status of akt ( h ) were determined in gastrocnemius muscle from overnight - fasted young and middle - aged mice . gastrocnemius muscle was collected from a separate group of overnight - fasted young and middle - aged mice following intraperitoneal injection with either saline or human recombinant insulin ( 10 units / kg ) , and immunoblots were performed to detect phosphorylation status of akt at serine 473 in gastrocnemius muscle lysates ( i ) . glucose tolerance test ( j ) was performed in young ( ) and middle - aged ( ) mice following a 6-h fast . serum insulin was detected in young and middle - aged mice after an overnight fast ( k ) . p < 0.05 indicates comparisons performed between young and middle - aged mice ( mann - whitney u test ) . since we speculated that middle - aged mice are more susceptible than young mice to the development of insulin resistance , young and middle - aged mice were subjected to high - fat feeding for 12 weeks . although young mice fed a high - fat diet displayed weight gain ( fig . 3a ) and signs of glucose intolerance compared with young mice fed a low - fat diet ( fig . in contrast , middle - aged mice fed a high - fat diet showed significant weight gain ( body weight : low fat , 30.18 0.70 g , high fat , 55.14 1.56 g ; p < 0.05 ) and displayed dramatically elevated insulin levels ( fig . in addition , whole - body glucose tolerance in the middle - aged mice fed a high - fat diet was significantly impaired compared with that in middle - aged mice fed a low - fat diet ( fig . although activation of insulin signaling , as determined by the phosphorylation status of akt is not impaired in skeletal muscle of young ( fig . 3j ) mice fed a high - fat diet compared with that in mice fed a low - fat diet , this is likely due to elevated levels of circulating insulin ( fig . 3e and f ) observed in the respective high - fat groups . in support of the glucose tolerance data , homeostasis model assessment of insulin resistance values were significantly higher in the high - fat fed middle - aged mice ( fig . 3k ) , suggesting that high - fat feeding induces more dramatic insulin resistance in middle - aged mice than in young mice . interestingly , young mice on a high - fat diet were of the same weight as middle - aged mice on a low - fat diet ; yet , only the young mice on a high - fat diet had impaired glucose disposal ( data not shown ) . although we can not discriminate between the effects of aging and increased adiposity because these variables coassociate , our findings do suggest that the increased weight gain associated with aging is not sufficient to alter whole - body glucose disposal and that other factors such as high - fat diet are likely involved . aging increases the susceptibility to the development of glucose intolerance and insulin resistance in mice fed a high - fat diet . body weights ( a ) , fed and fasted blood glucose ( b ) , glucose tolerance test ( c ) , and phosphorylation status of akt ( d ) were measured in gastrocnemius muscle from young ( 1214 weeks of age ) mice following 12 weeks of high fat ( hf ) feeding . serum insulin levels in the fasted ( e ) and fed ( f ) states obtained from young ( 1214 weeks of age ) and middle - aged ( 4852 weeks of age ) mice fed a low - fat ( lf ) and high - fat diet for 12 weeks . fed and fasted blood glucose levels ( g ) , glucose tolerance test ( h ) , area under the curve ( auc ) for the glucose tolerance test ( i ) , and phosphorylation status of akt in gastrocnemius muscle ( j ) in middle - aged mice following 12 weeks of high - fat feeding . homeostasis model assessment of insulin resistance ( homa - ir ) as a surrogate marker of insulin resistance in young and middle - aged mice fed a high - fat diet ( k ) . immunoblot analysis using anti - cd36 antibody was performed in gastrocnemius muscle lysates from middle - aged mice fed a low- and high - fat diet , and immunoblots were quantified by densitometry and normalized against tubulin as a control for protein loading ( l ) . triglyceride ( tg ) ( m ) , long - chain acyl - coa ( lccoa ) ( n ) , and c18 ceramide ( o ) levels in gastrocnemius muscle from overnight - fasted middle - aged mice fed a low- or high - fat diet . two - way anova was performed to detect main effects of age , diet , and age diet interactions on insulin levels ( e and f ) and homa - ir ( k ) . significant effect of age , diet , and age diet interaction ( p < 0.05 ) was observed for e , f , and k. * p < 0.05 indicates comparisons performed between young and middle - aged mice or between low- and high - fat fed mice in fed or fasted state ( mann - whitney u test ) . because high - fat diet induced insulin resistance in young rodents is associated with an increased efficiency of fatty acid uptake into skeletal muscle ( 26 ) , protein expression of cd36 , a protein that facilitates fatty acid transport , was determined in skeletal muscle of mice fed a high - fat diet . consistent with previous publications in young mice ( 26,29,30 ) , we observed a modest increase in cd36 protein expression in the muscle of young mice fed a high - fat diet , as well as an increase in intramuscular triglyceride levels ( data not shown ) . consistent with our hypothesis , cd36 expression was significantly elevated in skeletal muscle of middle - aged mice fed a high - fat diet compared with that in mice fed a low - fat diet ( fig . increased cd36 protein expression , skeletal muscle triglyceride levels were significantly elevated in high - fat fed middle - aged mice compared with those in low - fat fed mice ( fig . 3 m ) , as were long - chain acyl - coa esters ( fig . , these data suggest that increased cd36-mediated fatty acid transport may contribute to lipid accumulation and impaired insulin sensitivity in skeletal muscle of middle - aged mice fed a high - fat diet . to investigate whether inhibition of fatty acid transport into the skeletal muscle could alter the observed responses of a middle - aged mouse to a high - fat diet , we utilized the cd36 ko mouse , which has skeletal muscle fatty acid uptake rates 4070% of those in wild - type mice ( 31,32 ) . interestingly , there was a striking difference in weight gain between middle - aged wild - type and cd36 ko mice following 12 weeks of high - fat feeding ( fig . 4a ) , with middle - aged cd36 ko mice accumulating 51% less weight than the wild - type mice over the same period of time ( fig . 4d ) between groups could not account for this dramatic change in weight gain , indirect calorimetry indicated that energy expenditure ( fig . 4 g ) were significantly increased in middle - aged cd36 ko mice fed a high - fat diet compared with those in high - fat fed middle - aged wild - type mice . although this increased activity in the middle - aged cd36 ko mouse fed a high - fat diet could be attributed to the absence of obesity , heat production was also increased in middle - aged cd36 ko mice fed a low - fat diet compared with that in low - fat fed middle - aged wild - type mice ( fig . 4h ) and in high - fat fed middle - aged ko mice when normalized for body weight ( fig . 4i ) . protection of diet - induced obesity in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks . representative image of middle - aged ( 4852 weeks of age ) wild - type ( ) and cd36 ko ( ) mice fed a high - fat diet for 12 weeks ( a ) . weight gain ( b ) and food intake adjusted for body weight ( bw ) ( c ) in wild - type and ko mice fed a high - fat diet . respiratory exchange ratio ( rer ) ( d ) , oxygen consumption ( vo2 ) ( e ) , and carbon dioxide production ( vco2 ) ( f ) in both the dark ( active ) and light ( inactive ) phase following 12 weeks of high - fat feeding in wild - type and ko mice . total activity for a complete dark / light cycle ( g ) , heat production ( h ) , and heat production adjusted for body weight ( i ) in wild - type and ko mice following 12 weeks of high - fat feeding . values are the means sem of n = 610 mice in each group . * p < 0.05 indicates comparisons performed between the low - fat ( lf)-fed mice and between high - fat fed mice ( mann - whitney u test [ b and g ] or anova with bonferroni post hoc test for pairwise comparison ) . ( a high - quality digital representation of this figure is available in the online issue . ) to gain a more comprehensive metabolic assessment of muscle metabolism in middle - aged wild - type and cd36 ko mice fed a low - fat or high - fat diet , we used mass spectrometry to measure a broad range of intermediary metabolites , including acylcarnitines of various chain lengths , organic acids , and amino acids . acylcarnitines are by - products of fuel catabolism that respond to changes in substrate availability or flux limitations at specific mitochondrial enzymes ( 14,33,34 ) . middle - aged cd36 ko mice fed a low - fat diet had elevated levels of acetyl - carnitine ( c2 ) , and -hydroxybutyryl - carnitine ( c4oh ) compared with their wild - type counterparts ( supplemental fig . 1a and supplemental table 1 ) . whereas several short - chain acylcarnitine species , including c2 and c4oh , as well as propionyl - carnitine ( c3 ) and succinyl - carnitine ( c4dc ) , tended to increase in response to a high - fat diet , these same metabolites trended downward in cd36 ko mice fed a high - fat diet ( supplemental fig . 1a and supplemental table 1 ) . in addition , several long - chain acylcarnitine species were reduced in muscle from wild - type mice fed a high - fat diet while at the same time levels of hydroxylated long - chain acylcarnitine ( lcoh ) species were increased , resulting in a robust increase in the long - chain to lcoh acylcarnitine ratio ( fig . c ) . given that long - chain acylcarnitines accumulate when their production by mitochondrial carnitine palmitoyl transferase ( cpt)1 exceeds flux through -oxidation enzymes , such as long - chain acyl - coa dehydrogenase ( lcad ) and -had ( 35 ) , this pattern is consistent with a diet - induced shift in flux limitation from lcad to -had . notably , levels of many long - chain and lcoh acylcarnitines were lower in the cd36 ko mice fed a high - fat diet than in the wild - type mice fed a high - fat diet ( fig . 5a ; supplemental table 1 ) . the organic acids were less responsive to both diet and genotype , although subtle changes were detected in succinate , fumarate , and citrate levels ( supplemental fig . muscle levels of amino acids were higher in wild - type mice fed a low - fat diet but were dramatically decreased following high - fat feeding . by comparison , amino acid levels remained unchanged in cd36 ko mice in response to a high - fat diet ( supplemental fig . although these metabolite measurements do not fully characterize mitochondrial substrate flux , together the data suggest that ablation of cd36 not only alters baseline mitochondrial and intermediary metabolism but also significantly impacts the muscle response to lipid exposure . altered skeletal muscle lipid handling prevents development of insulin resistance in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks . acylcarnitine levels were measured in gastrocnemius muscle from overnight - fasted middle - aged ( 4852 weeks of age ) wild - type ( wt ) and cd36 ko mice fed a low- ( lf ) or high - fat diet for 12 weeks . levels of individual long chain ( lc ) and lcoh species ( a ) , the sum total of all long - chain or lcoh species ( b ) , and the ratio of total lcoh to long - chain species ( c ) . phosphorylation status of ampk ( thr172 ) ( d ) and acc ( ser 79 ) ( e ) was detected in gastrocnemius muscle using immunoblot analysis . immunoblots were quantified by densitometry and normalized against total ampk ( d ) and acc ( e ) . serum levels of free fatty acids ( ffas ) from high - fat fed wild - type and cd36 ko mice were determined after 12 weeks of diet ( f ) . intramuscular levels of triglyceride ( tg ) ( g ) , lccoa ( h ) , and c18 ceramide ( i ) were determined in gastrocnemius muscle obtained from wild - type and ko mice fed a high - fat diet for 12 weeks . glucose tolerance testing ( j ) was performed in high - fat fed wild - type and ko mice fasted for 6 h. fasted blood glucose ( k ) and serum insulin ( l ) levels obtained from middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks . insulin tolerance test with blood glucose levels expressed as percent change of blood glucose at time zero in middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks ( m ) . immunoblots were performed on gastrocnemius muscle isolated from middle - aged wild - type and ko mice following high - fat diet and phosphorylation status of akt measured and normalized to total akt levels ( n ) . main effects of genotype , diet , and genotype diet interactions on acylcarnitine levels ( a c ) were detected by two - way anova . for simplicity , symbols indicate metabolites that were affected by genotype , diet , or a genotype - diet interaction . detailed results of the statistical analysis for all acylcarnitine species are presented in supplemental table 1 . * p < 0.05 indicates comparisons performed between low - fat fed wild - type and low - fat fed ko mice and between high - fat fed wild - type and high - fat fed ko mice ( mann - whitney u test or anova with bonferroni post hoc test [ j and m ] ) . interestingly , despite the changes in muscle acylcarnitine levels , the activity of -had was not altered ( data not shown ) and middle - aged cd36 ko mice fed a high - fat diet were not protected from decreased levels of ampk or acc phosphorylation ( fig . 5d and e ) compared with young wild - type mice ( fig . 2d and f ) , suggesting that alternate mechanisms are responsible for the metabolic phenotype observed in cd36 ko mice . similarly , absolute expression of ampk and acc in skeletal muscle from aged cd36 mice is not different between groups ( data not shown ) . although serum free fatty acid levels were elevated in high - fat fed cd36 ko mice ( fig . 5f ) , cd36 ablation resulted in a significant reduction in skeletal muscle triglycerides ( fig . by contrast , ceramide levels remained similar between high - fat fed groups ( fig . 5i ) , suggesting that accumulation of lipid - derived intermediates other than ceramides , may contribute to impaired insulin sensitivity . indeed , reduced intramuscular lipid accumulation in middle - aged cd36 ko mice was associated with both improved whole - body glucose tolerance ( fig . 5k ) compared to high - fat fed middle - aged wild - type mice . moreover , fasted insulin levels were significantly reduced ( fig . 5 m ) in high - fat fed middle - aged cd36 ko mice compared to high - fat fed middle - aged wild - type mice , suggesting that insulin sensitivity is restored by preventing lipid accumulation in skeletal muscle . interestingly , despite improved glucose utilization and reduced plasma insulin levels in these mice , the phosphorylation status of akt was similar in skeletal muscle from middle - aged wild - type and cd36 ko mice on high - fat diet ( fig . furthermore , we found no difference in glycogen content in livers from cd36 ko mice fed a low - fat or high - fat diet ( data not shown ) , suggesting that the improved glucose tolerance observed in these mice is the result of increased glucose uptake and/or glucose oxidation in skeletal muscle from cd36 ko mice and not alterations in hepatic glucose metabolism . to determine whether an age - related decline in resting metabolic rate and energy expenditure might contribute to the development of insulin resistance , c57bl6 mice of 1214 ( young ) or 5258 ( middle - aged ) weeks of age were analyzed using indirect calorimetry . as mice aged and body weight was increased ( fig . 1a ) , substrate use was altered slightly , with reductions in rer indicating that middle - aged mice used more fatty acid throughout the day compared with young mice ( fig . in addition , significant reductions in oxygen consumption ( vo2 ) ( fig . 1c ) and carbon dioxide production ( vco2 ) ( fig . 1d ) were observed during both the dark ( active ) and light ( inactive ) phases in middle - aged mice compared with young mice . 1e ) was decreased in middle - aged mice compared with young mice , whereas activity measurements were not significantly different between age - groups ( fig . our data indicate that middle - aged mice have a lower metabolic rate than young mice and that this might increase susceptibility to weight gain , obesity , and metabolic disease . body weight of young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice fed a standard laboratory diet ( a ) . indirect calorimetry was performed to measure respiratory exchange ratio ( rer ) ( b ) , vo2 ( c ) , vco2 ( d ) , and heat production adjusted for bodyweight ( e ) ; total activity ( f ) was measured in both the dark ( active ) and light ( inactive ) phases . p < 0.05 indicates comparisons performed between young and middle - aged mice in either dark or light phases ( mann - whitney u test ) . two - way anova was performed for rer ( b ) and indicated the main effect for age ( p < 0.01 ) . given that skeletal muscle metabolism contributes to whole - body basal metabolic rate , we addressed whether skeletal muscle metabolism was depressed in middle - aged mice by assessing the activities of two enzymes involved in regulating mitochondrial metabolism . 2a ) was elevated in the middle - aged compared with the young mice , and citrate synthase ( fig . 2b ) followed a similar upward trend , suggesting that -oxidation and tca cycle activity were not directly compromised in the middle - aged mice . since mitochondrial number ( fig . 2c ) or function did not appear to be altered at middle age , we next assessed whether peroxisome proliferator activated receptor ( ppar) responsive genes or molecular signaling cascades known to regulate skeletal muscle fatty acid flux and fatty acid entry into the mitochondria were associated with this overall reduction in whole - body basal metabolic rate observed in middle age . although a more comprehensive assessment of the multiple mediators of fatty acid utilization may reveal additional mechanisms , we did not observe any changes in protein levels of known ppar responsive genes involved in lipid metabolism , including malonyl - coa decarboxylase and acyl - coa synthetase 1 ( data not shown ) . as a result , we also examined the energy - sensing kinase , ampk , which is known for its ability to govern energy metabolism ( 23 ) . in agreement with previous reports using older rodents ( 24,25 ) , levels of phosphorylated ampk were significantly reduced in skeletal muscle of middle - aged mice compared with young mice ( fig . moreover , the phosphorylation status of acetyl coa carboxylase ( acc ) , the downstream target of ampk that indirectly regulates fatty acid entry into the mitochondria and ultimately -oxidation , was significantly decreased in skeletal muscle from middle - aged mice ( fig . although reduced ampk phosphorylation could be the result of impaired activity of upstream ampk kinases or increased ampk phosphatase activity , it is currently unknown which of these contributes to reduced ampk phosphorylation in our model . however , consistent with decreased energy expenditure , increased adiposity , and reduced ampk activity in skeletal muscle from middle - aged mice , the levels of skeletal muscle triglycerides were significantly elevated in middle - aged mice compared with young mice ( fig . 2 g ) . given that lipid accumulation in skeletal muscle has been proposed in rodents ( 26 ) and humans ( 17,27,28 ) to be one of the primary causes of skeletal muscle insulin resistance , we next investigated whether glucose tolerance was impaired in middle - aged mice . despite elevated intramuscular triglycerides ( fig . 2 g ) and impaired basal and insulin - stimulated akt phosphorylation ( fig . 2h and i , respectively ) in skeletal muscle of middle - aged mice , whole - body glucose tolerance ( fig . 2k ) were not different in middle - aged compared with young mice , suggesting that age - induced alterations in skeletal muscle fatty acid handling and increased triglyceride storage precede the development of insulin resistance and metabolic disease . alterations in fatty acid handling and reduced insulin signaling in skeletal muscle of middle - aged mice do not result in impaired whole - body glucose tolerance . activity of two mitochondrial enzymes -had ( a ) and citrate synthase ( cs ) ( b ) was determined in gastrocnemius muscle from overnight fasted young ( 1214 weeks of age ) and middle - aged ( 5258 weeks of age ) mice . immunoblot analysis using a total oxphos complex antibody cocktail was performed in gastrocnemius muscle lysates , and immunoblots were normalized against tubulin as a control for protein loading ( c ) . phosphorylation status of ampk at threonine 172 ( d ) and acc at serine 79 ( f ) was detected using immunoblot analysis with phospho - specific antibodies . immunoblots were quantified by densitometry and normalized against total protein levels of ampk ( e ) and acc ( f ) . triglyceride ( tg ) levels ( g ) and phosphorylation status of akt ( h ) were determined in gastrocnemius muscle from overnight - fasted young and middle - aged mice . gastrocnemius muscle was collected from a separate group of overnight - fasted young and middle - aged mice following intraperitoneal injection with either saline or human recombinant insulin ( 10 units / kg ) , and immunoblots were performed to detect phosphorylation status of akt at serine 473 in gastrocnemius muscle lysates ( i ) . glucose tolerance test ( j ) was performed in young ( ) and middle - aged ( ) mice following a 6-h fast . serum insulin was detected in young and middle - aged mice after an overnight fast ( k ) . p < 0.05 indicates comparisons performed between young and middle - aged mice ( mann - whitney u test ) . since we speculated that middle - aged mice are more susceptible than young mice to the development of insulin resistance , young and middle - aged mice were subjected to high - fat feeding for 12 weeks . 3a ) and signs of glucose intolerance compared with young mice fed a low - fat diet ( fig . in contrast , middle - aged mice fed a high - fat diet showed significant weight gain ( body weight : low fat , 30.18 0.70 g , high fat , 55.14 1.56 g ; p < 0.05 ) and displayed dramatically elevated insulin levels ( fig . in addition , whole - body glucose tolerance in the middle - aged mice fed a high - fat diet was significantly impaired compared with that in middle - aged mice fed a low - fat diet ( fig . although activation of insulin signaling , as determined by the phosphorylation status of akt is not impaired in skeletal muscle of young ( fig . 3j ) mice fed a high - fat diet compared with that in mice fed a low - fat diet , this is likely due to elevated levels of circulating insulin ( fig . 3e and f ) observed in the respective high - fat groups . in support of the glucose tolerance data , homeostasis model assessment of insulin resistance values were significantly higher in the high - fat fed middle - aged mice ( fig . 3k ) , suggesting that high - fat feeding induces more dramatic insulin resistance in middle - aged mice than in young mice . interestingly , young mice on a high - fat diet were of the same weight as middle - aged mice on a low - fat diet ; yet , only the young mice on a high - fat diet had impaired glucose disposal ( data not shown ) . although we can not discriminate between the effects of aging and increased adiposity because these variables coassociate , our findings do suggest that the increased weight gain associated with aging is not sufficient to alter whole - body glucose disposal and that other factors such as high - fat diet are likely involved . aging increases the susceptibility to the development of glucose intolerance and insulin resistance in mice fed a high - fat diet . body weights ( a ) , fed and fasted blood glucose ( b ) , glucose tolerance test ( c ) , and phosphorylation status of akt ( d ) were measured in gastrocnemius muscle from young ( 1214 weeks of age ) mice following 12 weeks of high fat ( hf ) feeding . serum insulin levels in the fasted ( e ) and fed ( f ) states obtained from young ( 1214 weeks of age ) and middle - aged ( 4852 weeks of age ) mice fed a low - fat ( lf ) and high - fat diet for 12 weeks . fed and fasted blood glucose levels ( g ) , glucose tolerance test ( h ) , area under the curve ( auc ) for the glucose tolerance test ( i ) , and phosphorylation status of akt in gastrocnemius muscle ( j ) in middle - aged mice following 12 weeks of high - fat feeding . homeostasis model assessment of insulin resistance ( homa - ir ) as a surrogate marker of insulin resistance in young and middle - aged mice fed a high - fat diet ( k ) . immunoblot analysis using anti - cd36 antibody was performed in gastrocnemius muscle lysates from middle - aged mice fed a low- and high - fat diet , and immunoblots were quantified by densitometry and normalized against tubulin as a control for protein loading ( l ) . triglyceride ( tg ) ( m ) , long - chain acyl - coa ( lccoa ) ( n ) , and c18 ceramide ( o ) levels in gastrocnemius muscle from overnight - fasted middle - aged mice fed a low- or high - fat diet . two - way anova was performed to detect main effects of age , diet , and age diet interactions on insulin levels ( e and f ) and homa - ir ( k ) . significant effect of age , diet , and age diet interaction ( p < 0.05 ) was observed for e , f , and k. * p < 0.05 indicates comparisons performed between young and middle - aged mice or between low- and high - fat fed mice in fed or fasted state ( mann - whitney u test ) . because high - fat diet induced insulin resistance in young rodents is associated with an increased efficiency of fatty acid uptake into skeletal muscle ( 26 ) , protein expression of cd36 , a protein that facilitates fatty acid transport , was determined in skeletal muscle of mice fed a high - fat diet . consistent with previous publications in young mice ( 26,29,30 ) , we observed a modest increase in cd36 protein expression in the muscle of young mice fed a high - fat diet , as well as an increase in intramuscular triglyceride levels ( data not shown ) . consistent with our hypothesis , cd36 expression was significantly elevated in skeletal muscle of middle - aged mice fed a high - fat diet compared with that in mice fed a low - fat diet ( fig . 3l ) . in accordance with increased cd36 protein expression , skeletal muscle triglyceride levels were significantly elevated in high - fat fed middle - aged mice compared with those in low - fat fed mice ( fig . 3 m ) , as were long - chain acyl - coa esters ( fig . together , these data suggest that increased cd36-mediated fatty acid transport may contribute to lipid accumulation and impaired insulin sensitivity in skeletal muscle of middle - aged mice fed a high - fat diet . to investigate whether inhibition of fatty acid transport into the skeletal muscle could alter the observed responses of a middle - aged mouse to a high - fat diet , we utilized the cd36 ko mouse , which has skeletal muscle fatty acid uptake rates 4070% of those in wild - type mice ( 31,32 ) . interestingly , there was a striking difference in weight gain between middle - aged wild - type and cd36 ko mice following 12 weeks of high - fat feeding ( fig . 4a ) , with middle - aged cd36 ko mice accumulating 51% less weight than the wild - type mice over the same period of time ( fig . 4d ) between groups could not account for this dramatic change in weight gain , indirect calorimetry indicated that energy expenditure ( fig . 4 g ) were significantly increased in middle - aged cd36 ko mice fed a high - fat diet compared with those in high - fat fed middle - aged wild - type mice . although this increased activity in the middle - aged cd36 ko mouse fed a high - fat diet could be attributed to the absence of obesity , heat production was also increased in middle - aged cd36 ko mice fed a low - fat diet compared with that in low - fat fed middle - aged wild - type mice ( fig . 4h ) and in high - fat fed middle - aged ko mice when normalized for body weight ( fig . 4i ) . protection of diet - induced obesity in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks . representative image of middle - aged ( 4852 weeks of age ) wild - type ( ) and cd36 ko ( ) mice fed a high - fat diet for 12 weeks ( a ) . weight gain ( b ) and food intake adjusted for body weight ( bw ) ( c ) in wild - type and ko mice fed a high - fat diet . respiratory exchange ratio ( rer ) ( d ) , oxygen consumption ( vo2 ) ( e ) , and carbon dioxide production ( vco2 ) ( f ) in both the dark ( active ) and light ( inactive ) phase following 12 weeks of high - fat feeding in wild - type and ko mice . total activity for a complete dark / light cycle ( g ) , heat production ( h ) , and heat production adjusted for body weight ( i ) in wild - type and ko mice following 12 weeks of high - fat feeding . * p < 0.05 indicates comparisons performed between the low - fat ( lf)-fed mice and between high - fat fed mice ( mann - whitney u test [ b and g ] or anova with bonferroni post hoc test for pairwise comparison ) . ( a high - quality digital representation of this figure is available in the online issue . ) to gain a more comprehensive metabolic assessment of muscle metabolism in middle - aged wild - type and cd36 ko mice fed a low - fat or high - fat diet , we used mass spectrometry to measure a broad range of intermediary metabolites , including acylcarnitines of various chain lengths , organic acids , and amino acids . acylcarnitines are by - products of fuel catabolism that respond to changes in substrate availability or flux limitations at specific mitochondrial enzymes ( 14,33,34 ) . middle - aged cd36 ko mice fed a low - fat diet had elevated levels of acetyl - carnitine ( c2 ) , and -hydroxybutyryl - carnitine ( c4oh ) compared with their wild - type counterparts ( supplemental fig . 1a and supplemental table 1 ) . whereas several short - chain acylcarnitine species , including c2 and c4oh , as well as propionyl - carnitine ( c3 ) and succinyl - carnitine ( c4dc ) , tended to increase in response to a high - fat diet , these same metabolites trended downward in cd36 ko mice fed a high - fat diet ( supplemental fig . 1a and supplemental table 1 ) . in addition , several long - chain acylcarnitine species were reduced in muscle from wild - type mice fed a high - fat diet while at the same time levels of hydroxylated long - chain acylcarnitine ( lcoh ) species were increased , resulting in a robust increase in the long - chain to lcoh acylcarnitine ratio ( fig . 5a c ) . given that long - chain acylcarnitines accumulate when their production by mitochondrial carnitine palmitoyl transferase ( cpt)1 exceeds flux through -oxidation enzymes , such as long - chain acyl - coa dehydrogenase ( lcad ) and -had ( 35 ) , this pattern is consistent with a diet - induced shift in flux limitation from lcad to -had . notably , levels of many long - chain and lcoh acylcarnitines were lower in the cd36 ko mice fed a high - fat diet than in the wild - type mice fed a high - fat diet ( fig . the organic acids were less responsive to both diet and genotype , although subtle changes were detected in succinate , fumarate , and citrate levels ( supplemental fig . muscle levels of amino acids were higher in wild - type mice fed a low - fat diet but were dramatically decreased following high - fat feeding . by comparison , amino acid levels remained unchanged in cd36 ko mice in response to a high - fat diet ( supplemental fig . although these metabolite measurements do not fully characterize mitochondrial substrate flux , together the data suggest that ablation of cd36 not only alters baseline mitochondrial and intermediary metabolism but also significantly impacts the muscle response to lipid exposure . altered skeletal muscle lipid handling prevents development of insulin resistance in middle - aged cd36 ko mice fed a high - fat ( hf ) diet for 12 weeks . acylcarnitine levels were measured in gastrocnemius muscle from overnight - fasted middle - aged ( 4852 weeks of age ) wild - type ( wt ) and cd36 ko mice fed a low- ( lf ) or high - fat diet for 12 weeks . levels of individual long chain ( lc ) and lcoh species ( a ) , the sum total of all long - chain or lcoh species ( b ) , and the ratio of total lcoh to long - chain species ( c ) . phosphorylation status of ampk ( thr172 ) ( d ) and acc ( ser 79 ) ( e ) was detected in gastrocnemius muscle using immunoblot analysis . immunoblots were quantified by densitometry and normalized against total ampk ( d ) and acc ( e ) . serum levels of free fatty acids ( ffas ) from high - fat fed wild - type and cd36 ko mice were determined after 12 weeks of diet ( f ) . intramuscular levels of triglyceride ( tg ) ( g ) , lccoa ( h ) , and c18 ceramide ( i ) were determined in gastrocnemius muscle obtained from wild - type and ko mice fed a high - fat diet for 12 weeks . glucose tolerance testing ( j ) was performed in high - fat fed wild - type and ko mice fasted for 6 h. fasted blood glucose ( k ) and serum insulin ( l ) levels obtained from middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks . insulin tolerance test with blood glucose levels expressed as percent change of blood glucose at time zero in middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks ( m ) . immunoblots were performed on gastrocnemius muscle isolated from middle - aged wild - type and ko mice following high - fat diet and phosphorylation status of akt measured and normalized to total akt levels ( n ) . main effects of genotype , diet , and genotype diet interactions on acylcarnitine levels ( a c ) were detected by two - way anova . for simplicity , symbols indicate metabolites that were affected by genotype , diet , or a genotype - diet interaction . detailed results of the statistical analysis for all acylcarnitine species are presented in supplemental table 1 . * p < 0.05 indicates comparisons performed between low - fat fed wild - type and low - fat fed ko mice and between high - fat fed wild - type and high - fat fed ko mice ( mann - whitney u test or anova with bonferroni post hoc test [ j and m ] ) . interestingly , despite the changes in muscle acylcarnitine levels , the activity of -had was not altered ( data not shown ) and middle - aged cd36 ko mice fed a high - fat diet were not protected from decreased levels of ampk or acc phosphorylation ( fig . 5d and e ) compared with young wild - type mice ( fig . 2d and f ) , suggesting that alternate mechanisms are responsible for the metabolic phenotype observed in cd36 ko mice . similarly , absolute expression of ampk and acc in skeletal muscle from aged cd36 mice is not different between groups ( data not shown ) . although serum free fatty acid levels were elevated in high - fat fed cd36 ko mice ( fig . 5f ) , cd36 ablation resulted in a significant reduction in skeletal muscle triglycerides ( fig . by contrast , ceramide levels remained similar between high - fat fed groups ( fig . 5i ) , suggesting that accumulation of lipid - derived intermediates other than ceramides , may contribute to impaired insulin sensitivity . indeed , reduced intramuscular lipid accumulation in middle - aged cd36 ko mice was associated with both improved whole - body glucose tolerance ( fig . 5k ) compared to high - fat fed middle - aged wild - type mice . 5 m ) in high - fat fed middle - aged cd36 ko mice compared to high - fat fed middle - aged wild - type mice , suggesting that insulin sensitivity is restored by preventing lipid accumulation in skeletal muscle . interestingly , despite improved glucose utilization and reduced plasma insulin levels in these mice , the phosphorylation status of akt was similar in skeletal muscle from middle - aged wild - type and cd36 ko mice on high - fat diet ( fig . furthermore , we found no difference in glycogen content in livers from cd36 ko mice fed a low - fat or high - fat diet ( data not shown ) , suggesting that the improved glucose tolerance observed in these mice is the result of increased glucose uptake and/or glucose oxidation in skeletal muscle from cd36 ko mice and not alterations in hepatic glucose metabolism . consistent with previous reports ( 17 ) , our data show a significant decline in metabolic rate in middle - aged mice when compared to their young counterparts ( fig . interestingly , although mitochondrial function was not directly assessed in this study , the decline in overall metabolic rate did not appear to stem from compromised mitochondrial function in skeletal muscle of middle - aged mice compared to young mice . indeed , whole body rer was modestly decreased with aging and muscle activity of -had increased , suggesting there is a shift in substrate selection from carbohydrates toward fatty acids . however , the age - associated reduction in overall metabolic rate did not appear to correlate with changes in maximal activities of mitochondrial enzymes in muscle from young and middle - aged mice ( fig . 2a and 2b ) , suggesting other factors may influence substrate oxidation in muscle , as well as overall metabolic rate . consistent with this , the ampk / acc signaling axis was significantly reduced in skeletal muscle from middle - aged mice ( fig . 2d and 2f ) . still unclear is whether reduced ampk phosphorylation in the older mice reflects a cause or consequence of reduced metabolic rate . although results of a recent study suggest that acc - mediated shifts in fat oxidation per se do not impact whole body energy expenditure or susceptibility to diet - induced obesity ( 36 ) , ampk acts on a broad range of enzymatic and transcriptional targets that could affect energy balance via mechanisms other than substrate selection ( 25,37 ) . nonetheless , cd36 deficiency raised metabolic rate without activating ampk , indicating that other mechanisms are operative in this model ( see below ) . although akt phosphorylation is reduced in skeletal muscle from middle - aged mice compared to young mice ( fig . 2h and 2i ) whole - body glucose tolerance and plasma insulin levels remained normal ( fig . 2j and 2k ) , suggesting that impaired activation of insulin - signaling parameters in skeletal muscle precede overt changes in whole body glucose disposal and may increase the susceptibility of aged mice to diet - induced insulin resistance . to determine whether middle - aged mice indeed are more susceptible to developing insulin resistance in response to a high - fat diet compared to their younger counterparts , we subjected middle - aged mice to 12 weeks of a low - fat or high - fat diet . as expected , middle - aged mice gained significantly more weight than young mice when fed a high - fat diet ( weight gain : middle - aged , 22.8 1.9 g versus young , 12.0 1.1 g ; p < 0.01 ) . 3c , 3h and 3i ) , high - fat diet - induced hyperinsulinemia was only observed in middle - aged mice ( fig . 3e and 3f ) , indicating that increased -cell insulin secretion was sufficient to offset overt hyperglycemia in this age - group ( fig . 3 g ) . given that prolonged hypersecretion of insulin by the -cells to compensate for peripheral insulin resistance can contribute to -cell failure ( 38 ) and potentially type 2 diabetes ( 39 ) , our data suggest that middle - aged mice have a heightened susceptibility to the development of insulin resistance . however , this susceptibility might not be due to aging per se , since adiposity was also increased in the older mice . as our study design does not permit to discriminate between the effects of aging and adiposity on skeletal muscle metabolism and the development of insulin resistance , future experiments should be directed at conducting weight loss ( food restriction ) studies or exercise studies to determine if these nevertheless , as aging and increased adiposity co - associate our findings likely reflect the majority of middle - aged humans in the western world who are at risk of developing insulin resistance . although intramuscular lipid accumulation associated with aging did not appear to result from mitochondrial dysfunction , we do show a significant twofold increase in cd36 protein expression in skeletal muscle of high - fat fed middle - aged mice compared to low - fat fed middle - aged mice ( fig . 3l ) , suggesting fatty acid transport into muscle exceeded the capacity for their oxidation . although it is unknown what caused increased cd36 expression in our study , it may have resulted from high levels of plasma glucose levels that have been shown to regulate cd36 expression through both transcriptional and/or translational mechanisms in rodents and humans ( 40,41 ) . to determine whether reduced fatty acid transport and metabolism could rescue the high - fat diet - induced phenotype consistent with our prediction , cd36 deficiency prevented the decline in metabolic rate and energy expenditure in middle - aged mice fed a high - fat diet as compared to age - matched wild - type mice ( fig . 4e and 4f ) . since food intake was similar between groups ( fig . 4c ) , we propose that increased energy expenditure in high - fat fed middle - aged cd36 ko mice contributes to their protection against diet - induced obesity ( fig . 4b ) . the blunted decline in metabolic rate in the middle - aged cd36 ko mice fed a high - fat diet ( fig . 4e and 4f ) was accompanied by alterations in muscle concentrations of several metabolic intermediates ( fig . 1 ) , but no improvement in ampk and acc phosphorylation ( fig . 5d and 5e , respectively ) . the impact of the diet on muscle metabolites in this study differed to some extent as compared to a previous report ( 14 ) in which tissue specimens were harvested from younger animals in the fed state . herein , tissues were collected after an overnight fast because we sought to evaluate a state of heightened fatty acid oxidation . under these conditions , the drop in long - chain acylcarnitines could reflect decreased fatty acid availability , lower cpt1 activity or increased long - chain acyl coa flux through lcad . first , the high - fat diet increases rather than decreases lipid delivery to muscle . second , of the two major products of cpt1 ( palmitoylcarnitine ( c16 ) and oleylcarnitine ( c18:1 ) , only the unsaturated species was reduced by the diet ( fig . 5a ) , suggesting upregulation of the isomerase enzyme that catalyzes conversion of the double bond ( 42 ) . 5c ) in response to chronic lipid exposure suggest a shift in flux limitation from the earlier to later steps in -oxidation . lastly , the diet resulted in a robust drop in whole body rer , indicative of a systemic increase in fat oxidation . notably , cd36 deficiency altered baseline levels of several muscle metabolites , and in general , tended to mitigate diet - induced changes in several acylcarnitine and amino acid species . this apparent resistance to diet - induced metabolic perturbations in the cd36 ko mice might be directly related to a reduction in fat delivery and/or secondary to enhanced energy expenditure and insulin sensitivity . although further work is necessary to fully understand the implications of these results , it is clear that loss of cd36 has a global impact on muscle fuel metabolism . in addition , it is possible that other organs such as adipose tissue , liver and brain ( 43,44 ) are also involved in maintaining a high level of energy expenditure in the middle - aged cd36 ko mice . overall , ablation of cd36 was associated with an improvement in whole - body glucose utilization and insulin sensitivity ( fig . although the mechanisms responsible for this are not known , excessive intramuscular lipid accumulation induced by high - fat feeding was prevented in skeletal muscle of middle - aged cd36 ko mice ( fig . 5 g and 5h ) . whereas elevated intramuscular triglyceride levels were not associated with insulin resistance in young mice ( fig . 2j2k ) , preventing the more dramatic age- and diet - induced accumulation of intramuscular triglyceride and long - chain acyl coa levels in middle - aged cd36 ko mice correlated with improved whole - body insulin sensitivity . although there is ample evidence indicating that cd36 ablation significantly reduces skeletal muscle fatty acid uptake ( 31,32 ) , it is also possible that the effects that we report using the cd36 ko mice are secondary to changes in fatty acid metabolism . notwithstanding this later possibility , our data demonstrate that limiting cd36-mediated skeletal muscle fatty acid transport guards against whole - body and muscle insulin resistance in middle - aged mice fed a high - fat diet . this finding suggests a potential therapeutic strategy for combating metabolic disease in the face of age - related abnormalities .

objectiveit has been proposed that skeletal muscle insulin resistance arises from the accumulation of intramyocellular lipid metabolites that impede insulin signaling , including diacylglycerol and ceramide . we determined the role of de novo ceramide synthesis in mediating muscle insulin resistance.research design and methodsmice were subjected to 12 weeks of diet - induced obesity ( dio ) , and then treated for 4 weeks with myriocin , an inhibitor of serine palmitoyl transferase-1 ( spt1 ) , the rate - limiting enzyme of de novo ceramide synthesis.resultsafter 12 weeks of dio , c57bl/6 mice demonstrated a doubling in gastrocnemius ceramide content , which was completely reversed ( 141.5 15.8 vs. 94.6 10.2 nmol / g dry wt ) via treatment with myriocin , whereas hepatic ceramide content was unaffected by dio . interestingly , myriocin treatment did not alter the dio - associated increase in gastrocnemius diacyglycerol content , and the only correlation observed between lipid metabolite accumulation and glucose intolerance occurred with ceramide ( r = 0.61 ) . dio mice treated with myriocin showed a complete reversal of glucose intolerance and insulin resistance which was associated with enhanced insulin - stimulated akt and glycogen synthase kinase 3 phosphorylation . furthermore , myriocin treatment also decreased intramyocellular ceramide content and prevented insulin resistance development in db / db mice . finally , myriocin - treated dio mice displayed enhanced oxygen consumption rates ( 3,041 124 vs. 2,407 124 ml / kg / h ) versus their control counterparts.conclusionsour results demonstrate that the intramyocellular accumulation of ceramide correlates strongly with the development of insulin resistance , and suggests that inhibition of spt1 is a potentially promising target for the treatment of insulin resistance . objectivealthough advanced age is a risk factor for type 2 diabetes , a clear understanding of the changes that occur during middle age that contribute to the development of skeletal muscle insulin resistance is currently lacking . therefore , we sought to investigate how middle age impacts skeletal muscle fatty acid handling and to determine how this contributes to the development of diet - induced insulin resistance.research design and methodswhole - body and skeletal muscle insulin resistance were studied in young and middle - aged wild - type and cd36 knockout ( ko ) mice fed either a standard or a high - fat diet for 12 weeks . molecular signaling pathways , intramuscular triglycerides accumulation , and targeted metabolomics of in vivo mitochondrial substrate flux were also analyzed in the skeletal muscle of mice of all ages.resultsmiddle-aged mice fed a standard diet demonstrated an increase in intramuscular triglycerides without a concomitant increase in insulin resistance . however , middle - aged mice fed a high - fat diet were more susceptible to the development of insulin resistance a condition that could be prevented by limiting skeletal muscle fatty acid transport and excessive lipid accumulation in middle - aged cd36 ko mice.conclusionour data provide insight into the mechanisms by which aging becomes a risk factor for the development of insulin resistance . our data also demonstrate that limiting skeletal muscle fatty acid transport is an effective approach for delaying the development of age - associated insulin resistance and metabolic disease during exposure to a high - fat diet .

sarcopenia is present in approximately 513% of persons over the age of 60 years and defined as the loss of skeletal muscle mass and strength with progressive decline in mobility and function . the pathogenesis of sarcopenia is associated with many intrinsic and extrinsic factors , including proinflammatory cytokine accumulation , oxidative stress , mitochondrial dysfunction , insulin resistance , and aging - related loss of anabolic hormones and motor neuron end plates . the loss of skeletal muscle results from an imbalance of protein metabolism , which is the dynamic balance between protein degradation and protein synthesis . the protein degradation systems in skeletal muscle are modulated by a coordinated network of signaling pathways activated or suppressed by hormones and cytokines ; therefore , catabolism is stimulated by a variety of proinflammatory cytokines , glucocorticoids , and reactive oxygen species ( ros ) [ 5 , 6 ] . it is important to note that impaired cellular immune function combined with low - grade inflammation represents a continuous impact in aging process . although aging is associated with prolonged inflammatory activity that is mainly attributed to progressively worsening muscle weakness , it is unclear whether these processes are cross - talked . molecular signals and pathways connecting inflammatory system and muscle degeneration may be the key to reveal the interactions responsible for the progression of sarcopenia . the interplay seems to exist between the mass loss of skeletal muscle and elevated systemic inflammation ( figure 1 ) . sarcopenia is a complex process with a subclinical state of inflammation driven by proinflammatory cytokines and oxidative stress , which increases the infiltration of immune cells into injured muscles . in turn , inflammation aggravates muscle loss and fat accumulation in the aging skeletal muscle and further decreases muscle function and physical activity . the increase in chronic inflammation response associated with high - level proinflammatory mediators as the extension of age has been considered as one of the diagnostic hallmarks and a significant contributor to aging - related atrophy of skeletal muscle . the transcription factor , nuclear factor-b ( nf-b ) , has been considered as an important mediator underlying the relationship between inflammation and aging [ 10 , 11 ] . the correlation between inflammation and sarcopenia exists as a possible linkage describing the effect of inflammation on the balance between protein anabolism and catabolism , with the presence of cd68 + macrophage infiltration . it is worth noting that substantial evidence also demonstrates the connection between obesity and sarcopenia , namely , sarcopenic obesity . in fact , obesity always plays an important role in sarcopenia , by the way of adding inflammatory burden . during obesity , adipose tissue is characterized by a chronic inflammatory state , through the release of numerous proinflammatory cytokines including tumor necrosis factor - alpha ( tnf- ) , interleukin-6 ( il-6 ) , and interleukin-1 beta ( il-1 ) as the factors largely responsible for insulin resistance in obese adipose tissue combined with aging - related skeletal muscle loss . moreover , impaired mitochondria can be both the reason and the consequence of inflammation during aging . increasing evidence shows that mitochondria may contribute to inflammation via ros production , nf-b activation , calcium homeostasis , impaired autophagy , and atp deficiency [ 17 , 18 ] . dysfunctional mitochondria are able to modulate aging - related inflammatory processes through direct activation of nlrp3 inflammasome , which can correspondingly result in the activation of caspase-1 or redox - sensitive inflammatory signaling pathways , thus leading to the production of il-1 and il-18 [ 19 , 20 ] . it should be noted that increasing ros could stimulate the activation of nf-b via nf-b - inducing kinase ( nik ) and ib kinase and ( ikk/ ) [ 21 , 22 ] . since the increased redox activation in the presence of transcription factor nf-b , excessive ros generation plays an important role in impaired mitochondrial function and oxidative capacity and accelerates the aging process of skeletal muscle . as described above , sarcopenia is a common feature in the elderly and mainly related to the release of inflammatory mediators from damaged tissue . these responses are controlled by a combination of various cytokines responsible for inflammatory pathways [ 10 , 24 ] . given that the inflammatory response is a complex system , cytokines are important not only as the indicators for mediating chronic inflammatory state through increasing protein degradation and reducing protein synthesis , but also as the mediators for controlling muscle wasting by directly targeting muscle tissue . in particular , proinflammatory cytokines are well known to impinge on protein metabolism in skeletal muscle and result in the activation of catabolism signals or upregulate inflammatory pathways such as nf-b and stat3 , thus finally leading to the increased activation of ubiquitin - proteasome and autophagy system [ 26 , 27 ] . the chronic inflammatory aging process depends not only on increased expression of proinflammatory factors , but also on reduced levels of anti - inflammatory factors such as il-10 , one of the anti - inflammatory cytokines . since the presence and function of cytokines have been demonstrated in the pathogenesis of sarcopenia , their origins and types must be identified . in fact , cytokines can be secreted by various types of cells like inflammatory and stromal cells , as well as skeletal muscle cells . in skeletal muscle , the constitutive expression of cytokines is generally stronger in differentiated myotubes compared with myoblasts , which usually results in inconspicuous change in cytokine release to stimuli [ 29 , 30 ] . accumulating evidences over the past decade have demonstrated that those proinflammatory cytokines such as tnf- , il-6 , and c - reactive protein ( crp ) cause a significant increase in aging skeletal muscle cells and play a key role in the complex network of inflammatory signals in charge of muscle homeostasis connected with aging - related disability and mortality [ 31 , 32 ] . has originally been classified as a prototypical proinflammatory cytokine to exhibit marked pleiotropy , and its anti - inflammatory property has also been identified later [ 34 , 35 ] . il-6 plays an important role in the pathogenesis of several chronic diseases including sarcopenia by regulating inflammatory and metabolic functions . il-6 signaling involves the binding to the membrane - bound il-6 receptor in skeletal muscle and the activation of downstream signaling pathways including stat3 , mapk / erk , p38 , myostatin , and foxo3 pathways [ 3740 ] . additionally , il-6 has confirmed to have the function of activating ampk and/or phosphatidylinositol-3-kinase ( pi3k ) and regulating the metabolism in skeletal muscle [ 41 , 42 ] . the overexpression of il-6 can result in reduced body mass and impaired insulin - stimulated glucose uptake in mouse skeletal muscle . furthermore , the infusion of il-6 in skeletal muscle can reduce the phosphorylation of s6k1 , which is activated by akt / mtor and associated with the inhibition of anabolic process . a comparative analysis of cytokine levels has confirmed the upregulation of proinflammatory il-6 and crp in the elderly , along with increased risk for the loss of skeletal muscle mass and strength . according to a recent study , the serum high - sensitivity crp ( hs - crp ) levels in the obesity only and in the sarcopenic obesity groups are significantly higher than that in the normal group after multivariate adjustments , which provides the evidence that obesity and sarcopenic obesity are associated with increased levels of serum hs - crp among males . tnf- is a cytokine implicated in the metabolic disturbance of chronic inflammation , with the formation of il-1 , which has been identified as a circulatory factor to increase gluconeogenesis , lipolysis , and proteolysis , accompanied with the decrease in protein , lipid , and glycogen synthesis in skeletal muscle . during the stages of muscle regeneration , tnf- and il-1 are observed in injured muscle with an accumulation of macrophages [ 48 , 49 ] . also , tnf- and il-1 have been confirmed to promote il-6 secretion through activating nf-b in cultured skeletal muscle cells . several previous studies have confirmed that tnf- at the elevated level can increase catabolism in skeletal muscle by suppressing akt / mtor pathway . tnf- and its soluble receptors have been described as the important contributors or biomarkers for the loss of mass and strength in aged skeletal muscle . there is a strongly negative correlation between protein breakdown and tnf- concentration in the elderly . injecting tnf- into mice has revealed the activation of ubiquitin - proteasome system and the decrease of skeletal muscle function . in vivo , the synthesis rate of myosin heavy chain protein is correlated negatively with the expression of tnf- in skeletal muscle . tnf- induces skeletal muscle loss through increased myofibrillar protein degradation and cell apoptosis , thus resulting in muscle atrophy and the inhibition of muscle regeneration following injury [ 55 , 56 ] . additionally , it seems that tnf- may antagonize the anabolic effect of insulin growth factor-1 ( igf-1 ) , due to the development of growth hormone resistance , which decreases both circulating and muscular igf-1 [ 57 , 58 ] . recent studies have shown that g / a-308 tnf- polymorphism as a marker of sarcopenia in normal weight obese syndrome , suggesting the importance of tnf- in the diagnosis of sarcopenia . it is important to note that an increase in tnf- alone is not sufficient to cause muscle atrophy . the upregulation of nf-b can cause muscle atrophy in rodents and contribute to the progressive muscle loss of advancing age . other mediators such as interferon- ( ifn- ) are produced in the microenvironment of skeletal muscle and play a critical role during myogenesis . moreover , il-15 is usually mentioned among paracrine effectors , while cytokines such as irisin and myonectin are able to induce anti - inflammatory cytokines ( il-1 receptor antagonist and il-10 ) , especially during contraction and aging conditions . the levels of tlr4 protein and il6 , il10 , and il15 mrna expression are increased after short - time bed rest in healthy older adults , while the levels of ifn- and macrophage inflammatory protein-1-beta ( mip-1 ) are elevated in aging skeletal muscle . furthermore , peroxisome proliferator - activated receptor gamma coactivator-1-alpha ( pgc-1 ) is regarded to have an anti - inflammatory function by inhibiting the function of foxo3 , which could promote inflammatory cytokine expression and downregulate antioxidant enzyme expression in aging muscle . the reduced expression of pgc-1 results in a low level and a systemic inflammatory response to exhibit negative impacts on skeletal muscle . inversely , the upregulated pgc-1 can reduce the activity of nf-b , which contributes to the inhibition of proinflammatory cytokines to be benefit for the prevention of mass and strength loss of skeletal muscle and functional decline of other organs , as well as the ultimate impact on homeostasis in human body [ 68 , 69 ] . proinflammatory cytokines have a wide variety of roles in inflammation systems and may be the important factor predisposing to muscle catabolism response in the elderly . however , the roles of these cytokines in aging skeletal muscle are still not fully understood . furthermore , the tissue - specific inflammatory signaling pathways in response to cytokines along with the elevation of systemic cytokines are important elements to be considered . mirnas are short noncoding rnas with approximately 22 nucleotides in length and are involved in the complex posttranscriptional regulatory networks and the maintenance of healthy cellular functions such as growth , development , and metabolism . skeletal muscle is the most abundant tissue in human body , comprising 4050% of body mass . it is estimated that approximately 60% of human genes are regulated by mirnas , suggesting that highly enriched mirnas in skeletal muscle play important roles in biological processes by gene silencing , including aging process . the functions of mirnas can be achieved either by suppressing the translation of target messenger rnas ( mrnas ) or by promoting the degradation of mrnas , thereby providing a powerful and sensitive regulator to tune gene expression and cell functions during the aging process of skeletal muscle . mirnas , from noncoding rna genes or within protein - coding genes , are transcribed into primicrornas by rna polymerase ii or polymerase iii in some cases and subsequently embedded into premicrorna hairpins like rna duplex by drosha [ 73 , 74 ] . premicrorna hairpins are exported from nucleus by exportin-5 and processed into double - stranded mature mirnas by dicer in combination with its rna - binding cofactor . after dicer - mediated maturation , the mirnas orient the risc complex with the removal or the preservation of one strand as the guide strand and preferentially load on the risc complex in position at regulatory sequences in target genes . although the precise mechanisms for mirna targeting and activity still remain to be fully explored , mirna activity appears to be largely dependent on its binding capacity to the target mrna molecule [ 7779 ] . generally , mrnas contain a predicted binding site in the 3 untranslated region ( utr ) , less commonly , in the 5 utr , and many mrnas contain multiple potential binding sites . according to the binding complementarity of the seed sequence , argonaute proteins such as ago-2 can directly cleave messenger rna and normally repress gene expression by targeting the mrna for degradation ( complete match , risc binds to mrna with perfect match ) or by mediating translation inhibition or mrna deadenylation leading to mrna destabilization at the condition with mismatches between mrna sequence and the risc ( incomplete match , risc binds to mrna with some mismatches ) . however , there are still unsettled questions regarding mirna - binding rules , thereby resulting in a lack of consensus in previous studies . establishing direct cause - and - effect links between mirnas and mrna targets is a key to understand underlying molecular mechanisms behind health and diseases and to develop effective targeted therapeutic strategies . now that mirnas have multiple gene targets , each target may be regulated by a suite of mirnas . the roles of mirnas in inflammation and sarcopenia have only been initially explored and future investigations will unravel their roles in immunity and metabolism . according to the analysis of mirna expression profiling , mirnas are critical regulators for both proinflammatory cytokines and skeletal muscle function [ 82 , 83 ] . in order to elucidate which mirna is important in the production of proinflammatory cytokines in aging - related muscle wasting , mrna targets and specific roles in regeneration and protein synthesis in skeletal muscle need to be established . several tissue - specific mirnas are known to be associated with the aging of skeletal muscle , which are named as myomirnas and consistently identified , including mir-1 , mir-133 , mir-206 , mir-208 , mir-486 , mir-431 , and mir-499 [ 8486 ] . these myomirnas can induce significant effects on development and myogenesis of skeletal muscle by targeting myogenic factors such as srf , mef2 , and myostatin . local injection of mir-206 can accelerate muscle regeneration , and mir-133 can promote the proliferation of myoblasts , while mir-1 can suppress the proliferation of myoblasts . although there is no obvious difference in the expression of mature mir-1 , mir-133 , or mir-206 in skeletal muscle from younger adults , an increased expression of these primary mirnas can be observed during aging , and the effect of anabolic stimulus on the levels of these mirnas can be perturbed in the elderly . on the other hand , many studies have implicated the regulation of inflammatory response through inflammatory mirnas such as mir-155 and mir-146a , suggesting their roles in the immune system . since inflammation is rather a broad concept , there are some overlaps between mirnas involved in inflammation and aging . therefore , cytokine - associated mirnas appear to have central roles in both inflammation and sarcopenia . a recent rna sequencing study has demonstrated the differential expression of mirnas in skeletal muscle from old and young rhesus monkeys . mir-181a , with its role in tuning the threshold of t - cell receptor ( tcr ) signaling originally described , not only acts as a myomirna , but also impacts inflammatory system . it can downregulate sirtuin 1 ( sirt1 ) gene expression as a regulator so that the expression of mir-181a and its target gene is disrupted in aged skeletal muscle . moreover , based on earlier reports showing proinflammatory cytokines such as tnf- , il-6 , il-1 , and il-8 as the proposed targets of mir-181a , the reduction of mir-181a can be responsible for an increase in the abovementioned proinflammatory cytokines in skeletal muscle during aging process . besides , tnf- and il-1 are significantly negatively correlated with decreased expression of myomirs and can suppress the differentiation of c2c12 myoblasts to myocytes / myotubes through nf-b , jak / stat , mapk p38 , or other key pathways . a newly discovered proinflammatory cytokine , tnf - like weak inducer of apoptosis ( tweak ) , belongs to tnf family and has revealed the function for causing muscle atrophy . one of the mechanisms proposed for the induction of skeletal muscle wasting by tweak is regulating differential expression of several growth - related mirnas , including mir-1 , mir-23 , mir-133a , mir-133b , and mir-206 in c2c12 myotubes ; however , it can reduce mir-1 , mir-133a , and mir-133b only in mouse skeletal muscle . while the treatment with tweak regulates several mirnas involved in the growth of skeletal muscle , it is not known whether their regulation is a cause of muscle wasting or a compensatory response to prevent further muscle wasting . let-7 mirna , the first known human mirna , has been reported to be critical for promoting differentiation and inhibiting cellular proliferation . the elevation of let-7 mirna may be responsible for the damage - repairing capability through the activation and proliferation of satellite cells in skeletal muscle from the elderly , therefore contributing to the attenuated regenerative capacity of skeletal muscle in the elderly . moreover , let-7 mirna can inhibit the secretion of inflammatory cytokine il-13 in human myotubes . recent study has also found that the overexpression of mirna let-7c can inhibit lps - induced production of tnf- , il-6 , and il-1 by inhibiting the phosphorylation of stat3 . compared to younger individuals , skeletal muscle from older individuals shows a significant elevation in let-7b and let-7e under resting conditions , suggesting the involvement of these mirnas in regulating cell cycle based on let-7 target genes . mir-146a , which negatively regulates the expression of il-1 and il-6 , is highly expressed in aged mice as a consequence of an aberrant nf-b binding to mir-146a promoter . as a result , the negative feedback regulation loop inducing the downregulation of inflammatory factors can be interrupted in aged mice . in macrophages isolated from aged mice , both dna methyl - transferase inhibitor and histone deacetylase inhibitor are able to significantly upregulate mir-146a through transcriptional activation by altering dna binding activity of nf-b . reduced mir-146a in oxldl - activated macrophages is linked to an increase of its target , toll - like receptor 4 ( tlr4 ) , involved in lipid uptake and inflammatory cytokine secretion . due to the upregulation in inflammatory condition in various primary disorders of skeletal muscle , mir-155 is identified as an important regulator of mef2a expression and exhibits its function in myoblast differentiation , and it also plays a critical role in the regulation of inflammation that affects both innate and adaptive immunity . the upregulation of mir-155 is the most characteristic feature of the mirna expression signature in lps - stimulated macrophages through the binding to nf-b . moreover , the inflammatory response mediated by mir-155 is induced by tlr ligands and can enhance the translation of tnf- during the pathogenesis of metabolic syndromes . in addition , mir-23a negatively regulates pgc-1 as a key activator of mitochondrial biogenesis and function . in another study , the level of mir-696 is upregulated in skeletal muscle of mice subjected to hind limb immobilization while the level of pgc-1 as the target of mir-696 exhibits an obvious decrease . consistent with this observation , the overexpression of mir-696 in myocytes shows a decrease in pgc-1 , suggesting its involvement in mitochondrial function and metabolism , and its importance in controlling the metabolism , adaptation , and atrophy of skeletal muscle . since a large number of mirnas have been identified in skeletal muscle , the investigation of mirnas is a promising but relatively unexplored area for understanding the regulatory mechanisms of wasting process associated with cytokine expression and secretion in aging skeletal muscle . clearly targeting these mirnas could provide an efficient and noninvasive approach for the diagnosis , prevention , or treatment of sarcopenia through the regulation of proinflammatory and anti - inflammatory factors . potential interventions for sarcopenia include nutritional supplements , physical activity / resistance exercise , caloric restriction , anabolic hormones , anti - inflammatory agents , and antioxidants . a key question is whether sarcopenia is a truly distinct syndrome or a milder form of a cachexia continuum . it is difficult to estimate the prevalence of sarcopenia , mostly because of practical difficulties in assessing skeletal muscle mass . sarcopenia - associated difference in the production of proinflammatory cytokines in vivo shows a prolonged inflammation activity . there remains a lack of understanding of individual contributions of various cytokines during the aging process of skeletal muscle , because of their wide - ranging effects on cell proliferation , differentiation , migration , survival , and apoptosis . as reviewed here , mirnas play major roles in the inflammatory regulation as the pivotal regulators for modulating cell functions or the critical factors for affecting the therapeutic outcome of sarcopenia . although the central role of inflammation in sarcopenia and proinflammatory cytokines including tnf- and il-6 as the central mediators of skeletal muscle atrophy has been documented , the roles of inflammatory mirnas in mass maintenance and functional development of skeletal muscle still need to be further explored and confirmed . whether mirnas can be used for the diagnosis of inflammatory involvement in the aging process of skeletal muscle proinflammatory cytokine - associated mirnas such as mir-146a , mir-181 , and mir-21 are frequently detected at a high level in skeletal muscle ; however , their functions are still not fully clear . therefore , further exploration of their targets , regulatory networks , and functions is highly desired . in conclusion , the discovery of mirnas with regulatory capacity of inflammatory response during aging process of skeletal muscle will open a novel avenue for the diagnosis , prevention , and therapy of sarcopenia through effectively modulating inflammatory signal pathways . temporary henna tattoos or pseudotattoo have become increasingly widespread among children and adolescent , as a safe and economic alternative to permanent tattoos . it is well - known that allergic skin reactions to natural henna are rare , due to its extremely low rate of sensitization . in india , north of africa , china , and egypt , it is used in weddings and religious ceremonies ; in occident , it is used to dye hair and cosmetics . paraphenylenediamine ( ppd ) , a powerful allergen , is added to the henna tattoo mixtures ( black henna tattoo ) to decrease application time and intensify the color . we describe the case of a 7-year - old boy who reported erythematous papular bulls - eye shaped lesions and consolidated edema primarily in the upper and lower extremities [ figure 1 ] . he also showed an erythematous - eczematous lesion on his leg , shaped like a dolphin [ figure 2 ] , and lesions compatible with erythema multiforme - like reaction . erythematous papular lesions contact eczema in the tattoo area dolphin shaped patch tests were performed , and we observed a high sensitivity after 48 h and moderate after 96 h. we reported a positive reaction to ppd . henna has been used to paint the skin for adornment and religious reasons for 9000 years and in over 60 countries . christians , jews , muslims , hindus , and buddhists have used henna as part of their religious customs . the henna is a flowering plant native to northern africa , western and southern asia in semi - arid zones , used since antiquity to dye skin . it has a great affinity for keratinocytes , and it is used to create temporary tattoos , without it being necessary to puncture the skin . black henna contains an ingredient in addition to pure henna to achieve its ebony color . in most cases , this added ingredient is ppd , a powerful sensitizer that should not be directly applied to the skin as it may cause mild contact dermatitis . one of the most dangerous applications of this chemical is when it is added to henna because the dye is applied while the ppd is in its oxidation process , and its potential as allergen is increased . when added to henna , the concentration of ppd is often much higher than what is approved for use in hair dyes . the cause of the sensitivity to ppd is unknown ; it is believed that the mechanism involved in the pathogenesis may be a reaction mediated by type iii immune complexes and associated with type iv retarded hypersensitivity . various topicals allergens cause erythema multiforme , including topical drugs such as corticosteroids , nonsteroidal anti - inflammatory drugs , iodine povidone , imiquimod ; rubber gloves ; nickel and herbicides . three possible causes of the residual hypopigmentation have been described : a reduction in melanin synthesis , selective destruction of the melanocytes , or photoleukomelanodermitis due to pigment blocking . as henna tattoos are becoming increasingly popular , prevention requires the provision of information to consumers , especially young people and their parents . it is important for the population to be aware of this circumstance and the risk entailed by sensitization to ppd . to conclude , we believe that temporary black henna tattooing should be controlled by health authority legislation to minimize the appearance of new cases of reaction to ppd and the serious and permanent consequences we have presented . it is important for the population to be aware of the risk entailed by sensitization to ppd due to popular henna tattoos .

sarcopenia has been defined as the aging - related disease with the declined mass , strength , and function of skeletal muscle , which is the major cause of frailty and falls in elders . the activation of inflammatory signal pathways due to diseases and aging is suggested to reveal the critical impact on sarcopenia . several proinflammatory cytokines , especially interleukin-6 ( il-6 ) and tumor necrosis factor - alpha ( tnf- ) , play crucial roles in modulation of inflammatory signaling pathway during the aging - related loss of skeletal muscle . micrornas ( mirnas ) have emerged as the important regulators for the mass and functional maintenance of skeletal muscle through regulating gene expression of proinflammatory cytokines . in this paper , we have systematically discussed regulatory mechanisms of mirnas for the expression and secretion of inflammatory cytokines during sarcopenia , which will provide some novel targets and therapeutic strategies for controlling aging - related atrophy of skeletal muscle and corresponding chronic inflammatory diseases . temporary henna tattoos or pseudotattoos have become increasingly widespread among children and adolescent . a generalized skin reaction , type erythema multiforme - like reaction is unusual , and rarely reported . we describe the case of a 7-year - old boy who reported erythematous papular bulls - eye shaped lesions and consolidated edema primarily in the upper and lower extremities . these lesions were compatibles with erythema multiforme - like reaction . he also showed an erythematous - eczematous lesion on his leg , shaped like a dolphin . in this area , a temporary henna tattoo was painted 1-month earlier . patch test was positive for paraphenylenediamine ( ppd ) . skin reactions due to henna are rare . most of the reactions are due to additives , especially ppd , an aniline derivative , which is added to speed up the process of skin dyeing and to give a darker brown to black color ( black henna ) . as henna tattoos are becoming increasingly popular , prevention requires the annual provision of information to consumers , especially young people and their parents .

the romaleidae family accounts for more than 200 species of grasshoppers comprising three subfamilies : romaleinae , aucacrinae and trybliophorinae . it is the second most diverse family of the superfamily acridoidea occurring from semiarid regions to tropical rainforests ( carbonell , 1977 ; roberts and carbonell , 1982 ; carbonell , 1984 , 1986 , 2002 ) . the genus radacridium is dispersed across the northeast region of brazil , where it seems to be well - adapted to the severely arid conditions ( carbonell , 1984 ) . two species are found in the state of pernambuco : radacridium mariajoseae , which is typical of the agreste region , and radacridium nordestinum , typical of the caatinga biome ( carbonell , 1984 , 1996 ) . cytogenetic studies involving representatives of romaleidae have revealed a great karyotypic conservation ( mesa et al . , 1982 , 2004 ; souza and kido , 1995 ; rocha et al . , 1997 ; pereira and souza , 2000 ) . most of analyzed species , including r. mariajoseae and r. nordestinum , presented karyotypes with 2n = 23 male , 24 female , an x0/xx sex chromosome system and exclusively acrocentric chromosomes . the constitutive heterochromatin ( ch ) in romaleidae is predominantly located in the pericentromeric regions of all chromosomes , as observed in xyleus angulatus , brasilacris gigas , chromacris nuptialis , radacridium nordestinum e phaeoparia megacephala ( souza and kido , 1995 ; rocha et al . , analyses with dapi and cma3 in romaleids showed a predominance of gc - rich ( cma3 ) regions . in xyleus angulatus and xestotrachelus robustus in contrast , few gc - rich ch regions were observed by loreto et al . ( 2005 ) in two chromacris species . c. nuptialis had only one bivalent ( m6 ) with a pericentromeric cma3 block , whereas c. speciosa presented cma3 blocks in two autosomal bivalents , a proximal one in m6 and a telomeric one in l2 . a large part of the eukaryotic genome is formed by repetitive dna , including tandem sequences mainly comprising satellite dna and multigene families . ribosomal dna and histone gene families include a variable number of copies with various genome locations ( charlesworth et al . fluorescence in situ hybridization ( fish ) with rdna and histone genes probes has proven useful for mapping and their location and in clarifying the genome organization of several organisms . among invertebrates , rdna gene mapping has been used in several groups , including worms ( vitturi et al . , 2002 ) , mollusks ( colomba et al . , 2002 ) , insects ( cabrero and camacho , 2008 ; cabral - de - mello et al . , 2011a ; panzera et al . , 2012 ) , the studies involving histone genes mapping in grasshoppers included some species of the acrididae family ( cabrero et al . , 2009 ; oliveira et al . , 2011 ) and four species of the proscopiidae family ( cabral - de - mello et al . , 2011b ) were mapped . this study aimed at understanding the pattern of organization of multigene families in two species of radacridium . our results showed a great variability in the number and location of rdna clusters in both species , whereas the histone h4 genes were highly conserved in number . these results are compared with data from other grasshopper species and are discussed based on the possible mechanisms involved in repetitive dna diversification . we analyzed ten individuals of r. mariajoseae from gravat ( 081204 s ; 353353 w ) and bezerros ( 081400 s ; 354749 w ) and ten adult males of radacridium nordestinum from surubim ( 074959 s ; 354517 w ) , all located in the agreste region of the state of pernambuco , brazilian northeast . specimens were processed and their testes were fixed in carnoy solution ( 3:1 ethanol : acetic acid ) . chromosome preparations were obtained by the testicular follicles squashing technique , in one drop of 45% acetic acid . the slides were stained with cma3 ( 0.5 mg / ml ) for one hour , washed with distilled water and stained with da ( distamicine a , 0.1 mg / ml ) for 45 min . the slides were rewashed and stained with dapi ( 2 g / ml ) for 20 min and mounted in glycerol / mcilvaine buffer / mgcl2 . probes were obtained by pcr performed according to ayres ( 2002 ) , with modifications , using genomic dna from both species and the primers : 18s dnar - sca18s1f ( f 5 - ccc cgt aat cgg aat gag ta - 3 ) ; sca18s1r ( r 5- gag gtt tcc cgt gtt gag tc -3 ) , 5srdna f(5- aac gac cat acc acg ctg aa -3 ) ; r ( 5- aag cgg tcc ccc atc taa gt - 3 ) , histone h4 f-1 ( 5 - tsc gig aya aca tyc agg gia tca c - 3 ) and r-1 ( 5 - cky tti agi gcr tai acc acr tcc at - 3 ) . the pcr products analyzed after electrophoresis in a 1% agarose gel had the expected sizes . the 18s rdna and histone h4 probes were labeled with biotin-16-dutp and the 5s rdna probe , with digoxigenin-11-dutp . the chromosome preparations were submitted to an alcohol series pretreatment and then treated with rnase and pepsin . the hybridization mix contained 1 l of probe and the denaturation was performed in a humid chamber at 75 c , followed renaturation overnight at 37 c . immunodetection was performed with mouse anti - biotin ( m743 , dako ) and rabbit anti - mouse - tritc ( r270 , dako ) for biotin and sheep anti - digoxigenin ( roche 1 207 741 ) and sheep anti - rabbit - fitc ( dako - f0135 ) for digoxigenin . chromosomes were counterstained with 4,6 diamidine-2-phenyl indole ( dapi ) and the slides were mounted with vectashield ( vector ) . images were obtained in a leica epifluorescence microscope , captured with the cw4000 program ( leica ) and adjusted in adobe photoshop cs5 . radacridium mariajoseae and r. nordestinum presented 2n = 23(male ) , a sex determination mechanism of the x0(male ) type and exclusively acrocentric chromosomes . chromosomes of both species were grouped in three large ( l1-l3 ) , five medium ( m4-m8 ) and three small ( s9-s11 ) pairs . cma3/da / dapi sequential staining showed gc - rich ( cma3 positive ) constitutive heterochromatin ( ch ) blocks in the interstitial region of only one medium - sized bivalent ( m5 ) and in the pericentromeric region of the x chromosome in r. mariajoseae ( figure 1a ) . in r. nordestinum , cma3 blocks were present in the interstitial region of the l2 bivalent and in the pericentromeric region of all chromosomes , except the l1 bivalent ( figure 1c ) . after fish , the 18s rdna probe labeled a single site in the pericentromeric region of the x chromosome in r. mariajoseae ( figure 2a ) and three pericentromeric sites , in the bivalents l2 , s9 and s10 of r. nordestinum ( figure 2b ) . the 5s rdna genes were located in the pericentromeric region of the two largest bivalents ( l1 and l2 ) and in the x chromosome of r. mariajoseae , ( figure 2c ) and in the bivalents l2 , l3 m4 and m5 of r. nordestinumm ( figure 2d ) . the histone h4 probe was mapped in a proximal location in a medium - sized autosome bivalent ( m5 ) in both species ( figure 2e and f ) . the karyotypic similarities detected after conventional analysis between the two species of radacridium did not extend to their cma3 and dapi staining patterns . our results showed gc - richness ch heterogeneity . in r. mariajoseae only one autosome pair and the x chromosome presented cma3 blocks , which resembled the scarcity in gc - rich regions of other romaleidae , as two species of chromacris with a single cma3 block ( loreto et al . , 2005 ) . the presence of a large number of cma3 blocks is more frequent in romaleidae , as observed in xyleus angulatus , phaeoparia megacephala and xestotrachelus robustus , in which all ch was shown to be gc - rich ( souza et al . , 1998 ; pereira and souza , 2000 ; souza et al . , 2003 ) . a great difference in the 18s and 5s rdna distribution r. mariajoseae presented a single 18s rdna site in the x chromosome , confirming the finding of only one active nucleolus organizer region ( nor ) after silver nitrate impregnation ( agno3 ) by rocha et al . ( 1997 ) . although three autosome bivalents showed 18s hybridization signals in r. nordestinum , only the l2 pair had a corresponding active nor identified by rocha et al . these data indicate a possible dispersion of the 18s rdna sequences , which may have been caused by structural chromosome rearrangements , ectopic recombination and transpositions ( cabrero and camacho , 2008 ) . these kind of events have been observed in other insects ( nguyen et al . , 2010 ; cabral - de - mello et al . , the ancestral rdna site could have been present in the m9 bivalent in a common ancestor . two points support this hypothesis : the presence of a 18s rdna site in this pair in r. nordestinum , which was considered as a megameric chromosome by rocha et al . ( 1997 ) and the preferential localization of nors is in this type of chromosome in several species of grasshoppers ( rufas et al . , 1985 ) . the 18s rdna probe location in the x chromosome of r. mariajoseae could have resulted from a chromosome rearrangement , such as a translocation or transposition , that moved it from m9 to the x , as a meiotic association between this chromosome and the megameric one has been described ( loreto et al . , 2008a ) . 18s rdna sites restricted to autosomes , as observed in r. nordestinum , were also detected in xestotrachelus robustus , chromacris nuptialis and c. speciosa ( souza et al . on the other hand , 18s rdna sites located both in autosomes and in the sex chromosome have been described , for example , in xyleus discoideus angulatus ( souza et al . 18s rdna sites have been frequently observed associated with gc - rich regions in romaleidae ( pereira and souza , 2000 ; loreto et al . , 2005 ) , 2004 ) , proscopiidae ( souza and moura , 2000 ) and ommexechidae ( carvalho et al . , 2011 ) , a pattern also observed in the two species analyzed herein . the histone h4 genes were located in a single chromosome pair ( m5 ) in both species . a single chromosome pair bearing histone genes was also described in other species of grasshoppers . ( 2009 ) analyzed the location of the histone h3 and h4 genes in 35 species of grasshoppers from the acrididae family . they observed that the great majority of species analyzed showed only one histone site , located in an autosome pair . in the same work , double fish performed in 11 randomly chosen species revealed that , in all cases , both genes were present in the same chromosome site , indicating a great conservation of histone gene location in acrididae . cabral - de - mello et al . ( 2011b ) , using a histone h3 probe in four species of proscopiidae ( tetanorhynchus silvai , scleratoscopia protopeirae , s. spinosa and stiphra robusta ) , observed a single site in the m4 bivalent in all the species . oliveira et al . ( 2011 ) observed multiples sites of histone h3 in rhammatocerous brasiliensis ( acrididae ) and concluded that this would be a derived condition and the presence of a single histone site , as observed in both species studied herein , would be the ancestral form . the 5s and the 18s genes co - localized in the l2 chromosome of r. nordestinum and in the x chromosome of r. mariajoseae . similarly to what we observed in both radacridium species , an extensive variation in 5s rdna distribution has been found in others grasshoppers , in which species presenting single sites and extending to all chromosome pairs have been described ( loreto et al . , 2008b ; cabral - de - mello et al . , 2011a ) . the results obtained in this study indicate a great level of karyotypic differentiation between r. mariajoseae and r. nordestinum . these data reinforce the fact that the high conservation observed at the chromosome level , including chromosome number and morphology , in radacridium and other romaleids , is not reflected at the genomic level . our results also contribute to the understanding of chromosome evolution patterns in the family romaleidae . the relation between chronic subclinical low - grade inflammation and insulin resistance ( ir ) has long been known [ 1 , 2 ] . ir is the major contributor and mediating factor in the development of type 2 dm ( t2 dm ) along with concomitant hypertension ( ht ) and cardiovascular disease ( cvd ) [ 3 , 4 ] . the relationship between the development of dm and some markers of inflammation such as c - reactive protein ( crp ) , il-6 , fibrinogen , and pai-1 has been described previously . serum concentration of crp increases in both impaired glucose tolerance ( igt ) and overt t2 dm [ 3 , 510 ] . on the other hand , some studies reported that elevation of crp is an indicator of development of t2 dm . compared with the conventional ogtt ( 2-hpg ) as recommended by who as gold standard , fasting plasma glucose ( fpg ) and hba1c are more convenient , simpler , and cost - effective diagnostic methods that are currently in use for the diagnosis of t2 dm [ 5 , 1217 ] . however , each test recognizes people with different metabolic features and groups who may be diagnosed by different tests but do not overlap substantially . while high postchallenge plasma glucose is a strong predictor of cvd , fasting glucose is not an independent predictor of cvd . to the best of our knowledge , there is no previous report specifically comparing the role of hs - crp in people with newly diagnosed dm with the criteria based on the 2-hpg , fpg , and hba1c . therefore , the aim of this study was to identify the optimal cut - off points of hs - crp in new - onset ( previously undiagnosed ) people with dm diagnosed based on the current 2-hpg , fpg , and hba1c diagnostic criteria . in this study , hs - crp results obtained from a nationally representative population - based survey are being reported . data derived from the turkish epidemiology survey of diabetes , hypertension , obesity and endocrine diseases ( turdep - ii ) , a population - based study , which was included randomly assigned 26,499 adult people from 270 urban and 270 rural centers . the field survey was performed between january and june 2010 , with a participation rate of 85% . the study was approved by the local ethical board ( istanbul medical faculty ethical committee , 16.4.2008/699 ) . people with known dm or other systemic diseases who had hs - crp levels of 10 mg / l ( 95.2 nmol / l ) or above were excluded from this study due to a possible infection . all biochemical tests including glucose , insulin , and lipid profile were measured in fasting blood samples using roche diagnostics modular autoanalyzer system ( roche diagnostics , germany ) in the central biochemistry laboratory of istanbul medical faculty . concentration of hs - crp was analyzed by immunoturbidimetric assay ( roche / hitachi 912 , modular p analyzers : acn 210 ; crpl3 tina - quant c - reactive protein gen . 3 ) and hba1c by turbidimetric inhibition immunoassay ; both the system and the laboratory have been regularly certified ( roche diagnostics tq hba1c gen . 3 ; ngsp certificate of traceability ; september 2010 - 2011 ) . a detailed medical history of each participant was obtained , and measurements of anthropometry ( height , weight , waist , and hip circumference ) and systolic and diastolic blood pressure ( sbp , dbp ) were done . body mass index ( bmi ) , homa - ir (= fasting glucose fasting insulin/405 ) , and non - hdl - cholesterol (= total cholesterol hdl - cholesterol ) were calculated accordingly . glomerular filtration rate ( egfr ) was estimated using chronic kidney disease epidemiology collaboration ckd - epi equation . risk factors for dm were evaluated using chi - square test ; mean values by sex were compared using nonparametric mann - whitney u test . homogeneity of variance and normal distribution of variables were tested by kolmogorov - smirnov test . pearson correlation coefficients ( r values ) were calculated to assess the association between hs - crp and other laboratory parameters . mean levels of hs - crp in each of the new dm groups were further compared using univariate analysis after being adjusted for age , bmi , smoking and alcohol drinking , and sbp and dbp . an optimum cut - off point of hs - crp was estimated using the receiver operating characteristic ( roc ) curve and area under the curve ( auc ) with 95% cis was calculated for each of the new dm groups that were diagnosed with fpg , 126 mg / dl ( 7.0 mmol / l ) , 2-hpg : 200 mg / dl ( 11.1 mmol / l ) , or hba1c : 6.5% ( 48 mmol / mol ) [ 13 , 17 ] . optimum cut - off points of hs - crp were defined for men and women , separately . as the raw data not normally distributed when log transformed , we obtained a square root transformation ( sqrt ) of the hs - crp levels , and after defining the cut - off points we recalculated the squares . data were analyzed using spss for windows ( version 21.0 ; spss / ibm , chicago , il ) . demographic characteristics and laboratory findings of women and men in turdep - ii study are presented in table 1 . in brief , men were significantly older and had higher mean weight , waist circumference , sbp , dbp , triglycerides ( tg ) , hdl - cholesterol , and non - hdl - cholesterol values than women . women had significantly higher bmi , hip circumference , heart rate , fpg , hba1c , 1-hpg , 2-hpg , hdl - cholesterol , and egfr values than men . median ( interquartile range , iqr ) concentration of hs - crp in women was significantly higher than in men ( women , 1.85 [ 3.09 ] total serum cholesterol , fasting serum insulin , and homa - ir values did not differ between men and women ( table 1 ) . there was a positive correlation between hs - crp levels and age , bmi , waist , hip , sbp , dbp , pulse , fpg , hba1c , 1-hpg , 2-hpg , tg , non - hdl - cholesterol , and homa - ir ; and there was a negative correlation with hdl - cholesterol and egfr . when we repeated the analysis after controlling for ht , age , sex , smoking and alcohol use , bmi , and waist circumference , the positive correlations between hs - crp levels and hba1c , 1-hpg , 2-hpg , tg , non - hdl - cholesterol , egfr , and homa - ir and the negative correlation with hdl - cholesterol and creatinine remained significant ( table 2 ) . among people with new dm , the highest hs - crp level was in the group detected by hba1c criterion ( hs - crp median [ iqr ] ; hba1c : 3.45 [ 3.82 ] mg / dl , 32.9 [ 36.4 ] nmol / l ; 2-hpg : 2.7 [ 3.14 ] mg / dl , 25.4 [ 29.9 ] nmol / l ; and fpg : 2.4 [ 3.0 ] mg / dl , 22.4 [ 28.5 ] nmol / l , data not shown ) . high sensitive crp level was significantly higher in women than men with newly diagnosed dm groups based on 2-hpg , fpg , and hba1c criteria . among the newly diagnosed dm groups , the median [ iqr ] level of hs - crp was highest in those detected with hba1c in both genders ( hba1c - group : women : 4.0 [ 4.1 ] mg / dl , 38.4 [ 39.1 ] nmol / l ; men : 2.7 [ 3.1 ] mg / dl , 25.9 [ 29.1 ] nmol / l ; fpg group : women : 3.3 [ 4.1 ] mg / dl , 31.1 [ 39.3 ] nmol / l ; men : 2.4 [ 3.0 ] mg / dl , 22.4 [ 28.5 ] nmol / l ; and 2-hpg group : women : 2.8 [ 3.3 ] mg / dl , 26.5 [ 31.3 ] nmol / l ; men : 2.4 [ 2.8 ] mg / dl , 23.3 [ 26.9 ] sex differences in hs - crp did not change after the data adjusted with respect to age , bmi , waist circumference , and ht ; the mean hs - crp was highest in newly diagnosed patients based on hba1c criterion in both sexes . again , the average hs - crp levels of women were higher than men ( table 3 ) . in women with newly diagnosed dm based on fpg , mean hs - crp level was comparable to those diagnosed with 2-hpg but lower than the group diagnosed with hba1c ( p = 0.000032 ) . women with newly detected dm based on 2-hpg had also lower mean hs - crp than those detected with hba1c ( p < 0.000001 ) . in contrast , men with newly detected dm based on fpg criterion had lower mean hs - crp than those diagnosed with 2-hpg ( p = 0.017 ) and hba1c ( p = 0.003 ) but mean hs - crp levels were comparable in 2-hpg and hba1c based new dm groups ( table 3 ) . the specificity and sensitivity of the optimal cut - off points for hs - crp to detect dm in women were for the fpg group , 60% and 57% for 2.5 mg / l ( 23.6 nmol / l ) , 2-hpg group , 60% and 54% for 2.1 mg / l ( 19.7 nmol / l ) , and 65% and 64% for hba1c group , 2.9 mg / l ( 27.5 nmol / l ) . in men the corresponding specificity and sensitivity values were as follows : fpg group : 60% and 57% for 1.8 mg / l ( 16.9 nmol / l ) ; 2-hpg group : 60% and 57% for 1.8 mg / l ( 16.9 nmol / l ) ; and hba1c group : 65% and 60% for 2.0 mg / l ( 19.0 nmol / l ) ( roc curves figure 1 and table 4 ) . the largest auc value for hs - crp to detect dm was found in women and men when using hba1c ( women : 0.700 ; men : 0.656 ) . the positive and negative predictive values ( ppv and npv ) corresponding to the above - mentioned cut - points of hs - crp in women were calculated as 58% and 59% for fpg , 57% and 58% for 2-hpg , and 64% and 65% for hba1c and in men were 58% and 59% for fpg , 58% and 59% for 2-hpg , and 61% and 61% for hba1c . our current population - based study identified 1,727 people with newly diagnosed dm based on at least one of the three methods . however , the people identified to have dm were substantially different for each of the three methods . in other words , the concordance rate for dm among the different methods of glycaemia testing was low . characteristics of the people with dm and their risk factors may vary by the method used for the detection of dm . several studies have suggested that inflammation is associated with ir that takes part in the pathogenesis of t2 dm and atherosclerotic disease [ 14 , 6 ] . environmental factors such as infections , overnutrition , and lack of physical activity are believed to contribute serum crp levels although the mechanism is not properly understood . on the other hand , hyperglycaemia per se may induce inflammation and this may enhance the development of dm [ 21 , 22 ] . demonstrated that people who developed dm ( detected by an ogtt ) had higher baseline serum crp levels than those who did not develop dm . there was a linear increasing trend in the incidence of dm as the baseline crp quartile increased . in pizarra prospective study , people with baseline hs - crp 3 mg / l ( 28.6 nmol / l ) developed dm . in our previous report , we demonstrated a linear increasing trend for hs - crp levels from normal glucose tolerance through impaired fasting glucose ( ifg ) , igt , and new dm . a recent meta - analysis including 18 prospective studies demonstrated that high baseline crp levels associated with future t2 dm diagnosed based on fpg and/or 2-hpg criteria . all these findings support the chronic low - grade inflammation hypothesis in the development of dm . in our study , we found a positive correlation between hs - crp levels and all glycaemia and ir parameters . however , after adjustment for age , sex , smoking , bmi , waist , and ht , positive correlations were maintained with hba1c , 1-hpg and 2-hpg , fasting insulin , and homa - ir but not with fpg . in their later report festa et al . stated that postchallenge glucose but not fpg was strongly correlated with baseline crp . other studies have also shown an association between crp and dm , which remained significant after adjusting for bmi or other covariates . our findings and others suggested that adiposity is not sufficient to explain the relationship between high levels of inflammatory markers and increased dm risk . the predictive value of hs - crp did not seem to be fully independent of obesity in several [ 3 , 23 , 24 , 27 , 28 ] but not all studies [ 7 , 9 , 10 , 22 ] . our results showed that among people with new dm the highest hs - crp levels were obtained in those identified with the hba1c criterion . hba1c elevation at diagnosis is an indication of overt dm , but in a more advanced stage compared with new - onset dm detected with the fpg or 2-hpg criteria . as we did show a positive correlation between hs - crp and hba1c , it has been reported that people with dm with poorer glycaemic control had higher crp levels . some commonly used medications like aspirin and statin may synergistically reduce serum crp concentrations [ 29 , 30 ] . in the present study , we excluded people with known systemic disease and those who self - reported regularly using such medications ; however , we may not ascertain all people using aspirin and/or statin . aronson et al . reported that crp levels among middle - aged people were higher in those with dm and ifg when compared with the healthy subjects . similar to our findings showing women had higher hs - crp levels than men regardless of having dm or not , the adopt investigators have reported that hs - crp levels in women were higher than in men in both with and without the metabolic syndrome ( ms ) . they reported a positive correlation between hs - crp and hba1c , bmi and homa - ir and the number of ms components in people with new dm . in women 's health study , the incidence of dm was four times higher in women with hs - crp levels in the upper quartile in comparison with those with hs - crp levels in the lowest quartile . wu and coworkers reported that high levels of hs - crp were correlated with high levels of hba1c and fpg in men and with only fpg in women . other studies reported strong correlations between hs - crp and fasting insulin , homa - ir , and fpg [ 21 , 34 ] and an inverse correlation with crp and hdl - cholesterol [ 3 , 21 ] ; all were confirmed in our study . we estimated the optimal cut - off hs - crp and auc - crp with 95% cis for dm for each of the three diagnostic methods separately . to the best of our knowledge , this is the first study aiming at determining hs - crp cut - off points indicating new dm compared with each of the three methods assessing hyperglycaemia . the highest cut - off point for hs - crp was obtained with hba1c based new dm detection compared to fpg and 2-hpg methods . in fact we previously showed that new dm group detected with hba1c has a more advanced metabolic disorder ( higher bmi , waist , blood pressure , non - hdl - cholesterol , triglycerides , and insulin but lower hdl - cholesterol ) than other new dm groups detected with fpg or 2-hpg . den engelsen et al . attempted to find a cut - off point for hs - crp that would indicate the presence of the ms . if hs - crp cut - off point was set at 3 mg / l ( 28.6 nmol / l ) , the sensitivity and specificity were 72% and 37% ; at this point ppv and npv were 42% and 67% . in another study performed in a japanese population , a cut - off point of hs - crp of 0.65 mg / l ( 6.2 nmol / l ) for fpg 100 mg / dl ( 5.6 mmol / l ) or higher was capable of defining the ms with a 100% sensitivity and 77% specificity in women and with a 65% sensitivity and 63% specificity in men . in our study the optimum cut - point of hs - crp of 2.5 mg / l ( 23.6 nmol / l ) in women and 1.8 mg / l ( 16.9 nmol / l ) in men for fpg 126 mg / dl ( 7.0 mmol / l ) or higher was capable of defining the new dm with a 60% sensitivity and 57% specificity in both genders . the optimum cut - point for hba1c 6.5% and over was 2.9 mg / l ( 27.5 nmol / l ) in women with a 65% sensitivity and 64% specificity and 2.0 mg / l ( 19.0 nmol / l ) in men with a 60% sensitivity and 62% specificity , and the largest auc value for hs - crp to detect new dm was found in women and men when using hba1c ( women : 0.700 ; men : 0.656 ) . these high cut - off points of crp may be related with the more advanced diabetic state as compared to fpg or 2-hpg based detected cases . one of the greatest strengths of the present study is its national representative sampling with a large sample size and wide age range . in addition , this is the first study where all three currently proposed methods ( 2-hpg , fpg , and hba1c ) were used to define dm and each of them was compared with the inflammation marker , hs - crp . the major limitations are the cross - sectional design and somewhat higher participation rate in women , controlled in the data analyses . in brief , an hs - crp level 1.8 mg / l ( 16.9 nmol / l ) generally detects more than half of the people with new dm . external validation of our findings needs to be carried out in additional studies in other populations with reasonably large sample size before these findings can be generalized . our results revealed that hs - crp may not further strengthen the diagnosis of new - onset dm . however , the highest hs - crp among people with new dm was found in those identified with the hba1c criterion . this suggests that high hba1c may recognize new dm cases at a more advanced stage than fpg or 2-hpg in an ogtt . . it would be important to find out if people with newly detected dm with high hs - crp require a more intensive therapy than those with low hs - crp .

in this study , two species of romaleidae grasshoppers , radacridium mariajoseae and r.nordestinum , were analyzed after cma3/da / dapi sequential staining and fluorescence in situ hybridization ( fish ) to determine the location of the 18s and 5s rdna and histone h4 genes . both species presented karyotypes composed of 2n = 23 , x0 with exclusively acrocentric chromosomes . cma3 + blocks were detected after cma3/da / dapi staining in only one medium size autosome bivalent and in the x chromosome in r. mariajoseae . on the other hand , all chromosomes , except the l1 bivalent , of r. nordestinum presented cma3 + blocks . fish analysis showed that the 18s genes are restricted to the x chromosome in r. mariajoseae , whereas these genes were located in the l2 , s9 and s10 autosomes in r. nordestinum . in r. mariajoseae , the 5s rdna sites were localized in the in l1 and l2 bivalents and in the x chromosome . in r. nordestinum , the 5s genes were located in the l2 , l3 , m4 and m5 pairs . in both species the histone h4 genes were present in a medium size bivalent . together , these data evidence a great variability of chromosome markers and show that the 18s and 5s ribosomal genes are dispersed in the radacridium genome without a significant correlation . fasting plasma glucose ( fpg ) and hemoglobin a1c ( hba1c ) have been used to diagnose new - onset diabetes mellitus ( dm ) in order to simplify the diagnostic tests compared with the 2-hour oral glucose tolerance test ( ogtt ; 2-hpg ) . we aimed to identify optimal cut - off points of high sensitive c - reactive protein ( hs - crp ) in new - onset dm people based on fpg , 2-hpg , or hba1c methods . data derived from recent population - based survey in turkey ( turdep - ii ) . the study included 26,499 adult people ( 63% women , response rate 85% ) . the mean serum concentration of hs - crp in women was higher than in men ( p < 0.001 ) . the people with new - onset dm based on hba1c had higher mean hs - crp level than fpg based and 2-hpg based dm cases . in hba1c , 2-hpg , and fpg based new - onset dm people , cut - off levels of hs - crp in women were 2.9 , 2.1 , and 2.5 mg / l [ 27.5 , 19.7 , and 23.5 nmol / l ] and corresponding values in men were 2.0 , 1.8 , and 1.8 mg / l ( 19.0 , 16.9 , and 16.9 nmol / l ) , respectively ( sensitivity 6065% and specificity 5464% ) . our results revealed that hs - crp may not further strengthen the diagnosis of new - onset dm . nevertheless , the highest hs - crp level observed in new - onset dm people diagnosed with hba1c criterion supports the general assumption that this method might recognize people in more advanced diabetic stage compared with other diagnostic methods .

. they can be caused by mutations in nuclear genes either relating to mitochondrial components or mitochondrial dna ( mtdna ) . the prevalence of mitochondrial diseases due to mutations in mtdna and related nuclear genes is estimated to be 1 in 5000 . mutations in mtdna are much higher than those in the related nuclear genes , probably because that mitochondrial genome has been exposed in reactive oxygen species(ros ) and lacked protection structure . in 1990 , the adenine to guanine transition at the mtdna position of 3243 encoding trna was found to be the molecular basis for mitochondrial encephalomyopathy with lactic acidosis and stroke - like episodes ( melas ) . epidemiological studies showed that m.3243a > g mutation is the most frequent pathogenic mutation in mtdna . the prevalence of m.3243a > g mutation in mtdna is 3.5/100,000 adults in the north east of england and approximately 1/6000 in an adult population in finland . seizures , encephalopathy , and stroke - like episodes were found in about 80% patients with melas , and short stature , deafness , cognitive impairment , exercise intolerance , migraines , depression , cardiomyopathy , cardiac conduction defects , and diabetes mellitus were also found in some melas cases . here , we summarized the clinical spectrum of m.3243a > g mutation in chinese pediatric patients , to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease . clinical data of 100 chinese pediatric patients who were first diagnosed as mitochondrial diseases through gene test for m.3243a > g mutation in peking university first hospital from 2007 to 2013 were retrospectively reviewed . this study was approved by the medical ethics committee of peking university first hospital ( no . a total of ten clinical characteristics including vision impairment , hearing loss , encephalopathy , myopathy , and gastrointestinal disturbances were collected from these patients . short stature was defined as the body height less than two standard deviations below the mean height of normal children . the peripheral blood samples were collected from these patients when they were first screened for the gene mutation . detection of m.3243a > g mutation ratio was performed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism method . the pcr product was digested with restriction enzyme apa i , and then separated in 2% agarose gel . the patients were divided into three groups based on mutation ratio ( low level : 030% ; middle level : 3160% ; and high level : 61100% ) . age and mutation ratio inconsistent with gaussian distribution were presented as median ( q1 , q3 ) . correlation between m.3243a > g mutation ratio and age was evaluated with spearman 's rank correlation method . the differences in clinical symptom frequency of patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test . clinical data of 100 chinese pediatric patients who were first diagnosed as mitochondrial diseases through gene test for m.3243a > g mutation in peking university first hospital from 2007 to 2013 were retrospectively reviewed . this study was approved by the medical ethics committee of peking university first hospital ( no . a total of ten clinical characteristics including vision impairment , hearing loss , encephalopathy , myopathy , and gastrointestinal disturbances were collected from these patients . short stature was defined as the body height less than two standard deviations below the mean height of normal children . the peripheral blood samples were collected from these patients when they were first screened for the gene mutation . detection of m.3243a > g mutation ratio was performed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism method . the pcr product was digested with restriction enzyme apa i , and then separated in 2% agarose gel . the patients were divided into three groups based on mutation ratio ( low level : 030% ; middle level : 3160% ; and high level : 61100% ) . age and mutation ratio inconsistent with gaussian distribution were presented as median ( q1 , q3 ) . correlation between m.3243a > g mutation ratio and age was evaluated with spearman 's rank correlation method . the differences in clinical symptom frequency of patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test . the age of the diagnosis of mitochondrial disease ranged from 2 months to 18 years , with the median age of 9 years ( 5.8 years , 12.0 years ) . the m.3243a > g mutation ratio varied from 5% to 93% , with a median level of 44% ( 36% , 54% ) . there was no significant difference in m.3243a > g mutation ratio between males and females ( t = 0.691 , p = 0.491 ) . patients at the onset had one or more symptoms , including seizures ( 54% ) , muscle weakness ( 29% ) , headache complicated with vomiting ( 25% ) , decreased vision ( 18% ) , hearing loss ( 10% ) , impaired verbal communication ( 6% ) , and heart preexcitation syndrome ( 5% ) . the most prevalent symptom of these patients was seizures ( 76% ) , followed by short stature ( 73% ) , elevated plasma lactate ( 70% ) , abnormal magnetic resonance imaging / computed tomography ( mri / ct ) changes ( 68% ) , vomiting ( 55% ) , decreased vision ( 52% ) , headache ( 50% ) , and muscle weakness ( 48% ) . most of the patients were multisymptomatic , only two patients had one symptom , and five patients manifested two symptoms . seizures were present in 76 patients ( 76% ) , of which 13 patients had stroke during or shortly after seizures . recurrent headaches were found in 50 patients ( 50% ) , of which most were complicated with vomiting and/or vision loss . sixty - eight patients ( 68% ) were found to have mri / ct abnormalities , including abnormal asymmetric signals in occipital area ( 51/68 , 75% ) , temporal area ( 21/68 , 31% ) , parietal area ( 20/68 , 29% ) , bilateral basal ganglia calcification ( 16/68 , 24% ) , cerebral atrophy ( 10/68 , 15% ) , and thalamus and brainstem lesions ( 5/68 , 7% ) . slurred speech was present in 16 patients , of whom 3 had progressively worsening speech , and 2 had delayed speech development ( at 1.5 and 3.0 years of age ) . seventy - three patients ( 73% ) had a short stature and 69 patients ( 69% ) had a weight loss . fifty - five patients ( 55% ) had experienced recurrent vomiting and 38 patients ( 38% ) had diarrhea or / and constipation . plasma lactate ranged from 1.4 to 19.0 mmol / l ( normal range : 0.72.0 mmol / l ) . elevated plasma lactate was detected in 70 patients ( 70% ) , of which 90% had a plasma lactate > 5 mmol / l . vision impairment was found in 52 patients ( 52% ) , of which 33 experienced blurred vision and 16 had decreased visual acuity . reduced muscle strength was reported in 48 patients ( 48% ) , of which 31 had minimal muscle weakness in upper limbs and shoulders and 17 manifested muscle weakness in lower limbs , and 2 had walk difficulties . thirty - seven patients ( 37% ) complained of difficulties in maintaining stability , of them 18 experienced frequent tumbling , and 38 patients ( 38% ) had compromised exercise tolerance , which appeared during running or going upstairs in most cases and during walk on flat places in only two cases . heart diseases were detected in 35 patients ( 35% ) , of which 17 were found to have abnormal electrocardiogram such as st - t changes and arrhythmias . left ventricular hypertrophy was found in eight patients , right ventricular hypertrophy in five patients , and preexcitation syndrome in five patients . twenty - one patients ( 21% ) had hearing impairment , presenting tinnitus or hearing loss ( mild deafness in 14 patients , moderate deafness in 5 patients , and severe deafness in 2 patients ) . the m.3243a > g mutation ratio in peripheral leukocytes was determined in all the patients , and 32% of them had a mutation ratio above 50% . the relationship between m.3243a > g mutation ratio and onset age was analyzed by spearman 's rank correlation method , which showed that m.3243a > g mutation ratio was correlated negatively with onset age [ r = 0.470 , p < 0.001 ; figure 1 ] . correlation between m.3243a > g mutation ratio in peripheral leukocytes and onset age ( r=0.470 , n=100 ) . the differences in clinical symptom frequency among patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test [ table 1 ] . there were significant differences in the frequencies of hearing loss , decreased vision , myopathy , and gastrointestinal disturbance among three groups . however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer from hearing loss , decreased vision , and gastrointestinal disturbance than high level group . analysis of clinical symptom frequency in different distributions of 3243a > g mutation ratio , n low level : a mutation ratio range 030% ; middle level : a mutation ratio range 3160% ; high level : a mutation ratio range 61100% ; mri : magnetic resonance imaging ; ct : computed tomography . patients at the onset had one or more symptoms , including seizures ( 54% ) , muscle weakness ( 29% ) , headache complicated with vomiting ( 25% ) , decreased vision ( 18% ) , hearing loss ( 10% ) , impaired verbal communication ( 6% ) , and heart preexcitation syndrome ( 5% ) . the most prevalent symptom of these patients was seizures ( 76% ) , followed by short stature ( 73% ) , elevated plasma lactate ( 70% ) , abnormal magnetic resonance imaging / computed tomography ( mri / ct ) changes ( 68% ) , vomiting ( 55% ) , decreased vision ( 52% ) , headache ( 50% ) , and muscle weakness ( 48% ) . most of the patients were multisymptomatic , only two patients had one symptom , and five patients manifested two symptoms . seizures were present in 76 patients ( 76% ) , of which 13 patients had stroke during or shortly after seizures . recurrent headaches were found in 50 patients ( 50% ) , of which most were complicated with vomiting and/or vision loss . sixty - eight patients ( 68% ) were found to have mri / ct abnormalities , including abnormal asymmetric signals in occipital area ( 51/68 , 75% ) , temporal area ( 21/68 , 31% ) , parietal area ( 20/68 , 29% ) , bilateral basal ganglia calcification ( 16/68 , 24% ) , cerebral atrophy ( 10/68 , 15% ) , and thalamus and brainstem lesions ( 5/68 , 7% ) . slurred speech was present in 16 patients , of whom 3 had progressively worsening speech , and 2 had delayed speech development ( at 1.5 and 3.0 years of age ) . seventy - three patients ( 73% ) had a short stature and 69 patients ( 69% ) had a weight loss . fifty - five patients ( 55% ) had experienced recurrent vomiting and 38 patients ( 38% ) had diarrhea or / and constipation . plasma lactate ranged from 1.4 to 19.0 mmol / l ( normal range : 0.72.0 mmol / l ) . elevated plasma lactate was detected in 70 patients ( 70% ) , of which 90% had a plasma lactate > 5 mmol / l . vision impairment was found in 52 patients ( 52% ) , of which 33 experienced blurred vision and 16 had decreased visual acuity . reduced muscle strength was reported in 48 patients ( 48% ) , of which 31 had minimal muscle weakness in upper limbs and shoulders and 17 manifested muscle weakness in lower limbs , and 2 had walk difficulties . thirty - seven patients ( 37% ) complained of difficulties in maintaining stability , of them 18 experienced frequent tumbling , and 38 patients ( 38% ) had compromised exercise tolerance , which appeared during running or going upstairs in most cases and during walk on flat places in only two cases . heart diseases were detected in 35 patients ( 35% ) , of which 17 were found to have abnormal electrocardiogram such as st - t changes and arrhythmias . left ventricular hypertrophy was found in eight patients , right ventricular hypertrophy in five patients , and preexcitation syndrome in five patients . twenty - one patients ( 21% ) had hearing impairment , presenting tinnitus or hearing loss ( mild deafness in 14 patients , moderate deafness in 5 patients , and severe deafness in 2 patients ) . the m.3243a > g mutation ratio in peripheral leukocytes was determined in all the patients , and 32% of them had a mutation ratio above 50% . the relationship between m.3243a > g mutation ratio and onset age was analyzed by spearman 's rank correlation method , which showed that m.3243a > g mutation ratio was correlated negatively with onset age [ r = 0.470 , p < 0.001 ; figure 1 ] . correlation between m.3243a > g mutation ratio in peripheral leukocytes and onset age ( r=0.470 , n=100 ) . the differences in clinical symptom frequency among patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test [ table 1 ] . there were significant differences in the frequencies of hearing loss , decreased vision , myopathy , and gastrointestinal disturbance among three groups . however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer from hearing loss , decreased vision , and gastrointestinal disturbance than high level group . analysis of clinical symptom frequency in different distributions of 3243a > g mutation ratio , n low level : a mutation ratio range 030% ; middle level : a mutation ratio range 3160% ; high level : a mutation ratio range 61100% ; mri : magnetic resonance imaging ; ct : computed tomography . the m.3243a > g mutation has been shown to lead to reduced levels of the trna , decrease in aminoacylation , and absence of the normal modification with 5-taurinomethyl group at the wobble base . mitochondrial disease caused by this mutation may result from the reduction of mtdna - encoded proteins and oxidative phosphorylation activity in mitochondrial translation . several clinical syndromes including melas , myoclonic epilepsy with ragged - red fibers , chronic progressive external ophthalmoplegia , and leigh 's syndrome may associate with m.3243a > g mutation . patients with m.3243a > g mutation but without clinical symptoms are not uncommon . to define the spectrum of clinical manifestations due to m.3243a > g mutation , we retrospectively reviewed clinical data of 100 chinese pediatric patients with m.3243a > g mutation regardless of their clinical presentations . central nervous system ( cns ) is frequently involved in mitochondrial diseases because of its higher energy demand . seizures may result from neuronal energy depletion , oxidative stress , impaired calcium signaling , immune disturbances , and deficiencies of vitamins , cofactors , and amino acids . in this study , the prevalence of seizures was as high as 76% , and was nearly 100% in our previous study , similar to the prevalence of 70.5% in a report of pediatric mitochondrial disease from taiwan , china . cns symptoms are , therefore , more common and severe in children than adults . the prevalence of seizures ranged from 9% to 20% in adults . in melas patients , recurrent stroke - like episodes were more frequent in those with m.3243a > g mutation than those without the mutation . we found stroke - like episodes in 22% patients , higher than the prevalence from patients with mitochondrial mutations other than m.3243a > g mutation . most of our patients showed abnormal brain image findings were probably not due to vascular injuries . bilateral basal ganglia were the most vulnerable site in this disease , followed by temporal , parietal , and occipital area , similar to the previous report . chae et al . also observed the tendency that basal ganglia were frequently involved in patients without m.3243a > g mutation . vomiting was more frequent than constipation or / and diarrhea , and 68% vomiting cases were complicated with headaches . a higher prevalence ( 42% ) of these symptoms therefore , mitochondrial diseases should be considered clinically in children with unexplained vomiting and headaches . the prevalence of cardiac involvement was highly variable in patients with mtdna mutations , ranging from 17% to 40% . in this study , 35% patients exhibited cardiac involvement with the main manifestations of abnormal electrocardiogram ( 17% ) and left ventricular hypertrophy ( 8% ) . the prevalence of left ventricular hypertrophy was less in our patients than the patients with m.3243a > g mutation ( 56% ) reported by majamaa - voltti et al . pediatric patients may be the reason of that lower prevalence of cardiac involvement was present in this study . for patients suspected of mitochondrial disease , routine electrocardiogram , and ultrasonocardiography the m.3243a > g mutation ratio in peripheral leukocytes was negatively correlated with patients onset age . myopathy was frequently seen in patients with high level of m.3243a > g mutation ratio . however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer from hearing loss , decreased vision , and gastrointestinal disturbance . in conclusion , this study summarized the common symptoms and onset symptoms in a large cohort of chinese pediatric patients with m.3243a > g mutation . in view of the fact that the diagnosis of 66% of patients was delayed an average of 2 years , we suggested that examination of m.3243a > g mutation in mtdna should be considered in children with one or more of the symptoms including seizures , short stature , muscle weakness , headache complicated with vomiting , decreased vision , and hearing loss . although the mutation ratio in blood sample is an available test for diagnosis of mitochondrial disease , the m.3243a > g mutation ratio is usually higher in muscle and urine sample . therefore , the mutation ratio in other tissues , such as muscle and urine cell , should be included in the future study . this study was supported by grants from national natural science foundation of china ( no . this study was supported by grants from national natural science foundation of china ( no . 81271256 and no . prolonged exposure to microgravity has significant effects on physiological conditions . many studies have investigated the effects of microgravity on the neuromuscular system . most of these studies have focused on skeletal muscle changes under microgravity . however , to the best of our knowledge , there is limited research regarding the effects of microgravity on the peripheral nervous system . one study examined the conduction velocity ( cv ) in branching axon terminals following a space mission or bed rest and identified a decreased cv ; however , it did not measure the velocity in the main trunk of nerve fibers . for example , one study investigated the manifestation of somatosensory - evoked potentials under hypokinesia , which included both the peripheral and central sensory nervous systems . however , the conditions under hypokinesia / bed rest are different compared with microgravity ; therefore , the effect of microgravity on the peripheral nervous system should be further evaluated . ground - based models are a suitable alternative to space flights and induce similar modifications in the neuromuscular system . the hindlimb unloading ( hu ) model is characterized by the absence of weightbearing and reduced motor activity , and it is typically applied in rats . moreover , the 6 head - down tilt ( hdt ) is another experimental model used to simulate the space flight environment of humans . to determine changes in nerve conduction under simulated microgravity , the current study aimed to investigate the nerve conduction characteristics of rhesus monkeys before and after prolonged exposure to hdt . all experimental procedures conducted with rhesus monkeys were approved by the ethical committee of the china astronaut research and training center , which is in accordance with the principles of the association for assessment and accreditation of laboratory animal care international ( aaalac ) , approved by institutional animal care and use committee ( iacuc ) . the hdt model in rhesus monkeys was selected as the model because the peripheral nervous system of primates is the most similar to humans compared with other species . six rhesus monkeys at the age of 34 years ( three males and three females ) were laid on beds , which were tilted backward 6 from the horizontal . the head - down animals wore a special cloth , which enabled them to be fixed to the bed ; however , their arms and legs were free to move , and they were provided access to food and water . the hdt model functions to reduce mechanical loading to the hindlimbs , which provides a similar condition as microgravity . the rhesus monkeys were provided with a regular dietary program , which included primate biscuits , fresh fruits , and vitamin syrup . the animals were housed one per cage or bed in rooms with air temperature maintained at 23 2c and a standard 12:12 h dark light cycle ( lights were turned on at 8:00 a.m. and off at 8:00 p.m. ) . the general health condition of the animals was carefully monitored . the monkeys were provided with toys ( for example , the drum - shaped rattle , a chinese traditional toy ) during housing throughout the duration of the study , and the toys were available all the time except during experimental procedures . nerve conduction studies ( ncss ) were performed using surface electrodes on the median , ulnar , tibial , and fibular nerves on the right side to assess motor function in the upper and lower limbs using the key point electromyography system ( 31a06 , medoc ltd . , the motor conduction velocities , the proximal and distal amplitudes of the compound muscle action potentials ( cmaps ) , and the proximal and distal latencies of the median , ulnar , tibial , and fibular nerves were recorded ( representative images of the ncs of the tibial nerve are shown in figure 1 ) . the ncs was performed in the order of the median , ulnar , tibial , and fibular nerves before hdt and on 7 , 21 , and 42 days of hdt . during the procedure , toys were provided , and the researchers accompanied the monkeys to reduce pain and suffering . all animal housing and experiments occurred in the animal facility at china astronaut research and training center . ncs : nerve conduction studies ; hdt : head - down tilt . statistical analysis was performed using spss software version 19.0 ( ibm , chicago , il , usa ) . analysis of variance ( anova ) using a randomized block design was conducted to compare the differences in the ncs before hdt and at 7 , 21 , and 42 days of hdt . post hoc analyses , including least significant difference and student newman keuls , were performed if significant differences were identified in the anova . one rhesus monkey died on the day 21 of hdt because of inadaptation to the simulated microgravity , and one monkey injured its upper limbs as a result of rigorous movement . thus , the anova with a randomized block design and the anova of the median and ulnar nerve parameters were performed using data collected from the remaining four rhesus monkeys . data from the tibial and fibular nerves were obtained from the five remaining rhesus monkeys . all experimental procedures conducted with rhesus monkeys were approved by the ethical committee of the china astronaut research and training center , which is in accordance with the principles of the association for assessment and accreditation of laboratory animal care international ( aaalac ) , approved by institutional animal care and use committee ( iacuc ) . the hdt model in rhesus monkeys was selected as the model because the peripheral nervous system of primates is the most similar to humans compared with other species . six rhesus monkeys at the age of 34 years ( three males and three females ) were laid on beds , which were tilted backward 6 from the horizontal . the head - down animals wore a special cloth , which enabled them to be fixed to the bed ; however , their arms and legs were free to move , and they were provided access to food and water . the hdt model functions to reduce mechanical loading to the hindlimbs , which provides a similar condition as microgravity . the rhesus monkeys were provided with a regular dietary program , which included primate biscuits , fresh fruits , and vitamin syrup . the animals were housed one per cage or bed in rooms with air temperature maintained at 23 2c and a standard 12:12 h dark light cycle ( lights were turned on at 8:00 a.m. and off at 8:00 p.m. ) . the monkeys were provided with toys ( for example , the drum - shaped rattle , a chinese traditional toy ) during housing throughout the duration of the study , and the toys were available all the time except during experimental procedures . nerve conduction studies ( ncss ) were performed using surface electrodes on the median , ulnar , tibial , and fibular nerves on the right side to assess motor function in the upper and lower limbs using the key point electromyography system ( 31a06 , medoc ltd . , the motor conduction velocities , the proximal and distal amplitudes of the compound muscle action potentials ( cmaps ) , and the proximal and distal latencies of the median , ulnar , tibial , and fibular nerves were recorded ( representative images of the ncs of the tibial nerve are shown in figure 1 ) . the ncs was performed in the order of the median , ulnar , tibial , and fibular nerves before hdt and on 7 , 21 , and 42 days of hdt . during the procedure , toys were provided , and the researchers accompanied the monkeys to reduce pain and suffering . all animal housing and experiments occurred in the animal facility at china astronaut research and training center . ncs of tibial nerve before hdt and 42 days after hdt in rhesus monkeys . ( a ) before hdt . statistical analysis was performed using spss software version 19.0 ( ibm , chicago , il , usa ) . analysis of variance ( anova ) using a randomized block design was conducted to compare the differences in the ncs before hdt and at 7 , 21 , and 42 days of hdt . post hoc analyses , including least significant difference and student newman keuls , were performed if significant differences were identified in the anova . one rhesus monkey died on the day 21 of hdt because of inadaptation to the simulated microgravity , and one monkey injured its upper limbs as a result of rigorous movement . thus , the anova with a randomized block design and the anova of the median and ulnar nerve parameters were performed using data collected from the remaining four rhesus monkeys . data from the tibial and fibular nerves were obtained from the five remaining rhesus monkeys . the parameters of ncs before hdt ( under normal conditions ) and 7 , 21 , and 42 days of hdt are presented in table 1 . the proximal amplitude of the cmap of the median nerve was significantly decreased at 21 and 42 days of hdt compared with the amplitude before hdt ( 4.38 2.83 vs. 8.40 2.66 mv , f = 4.85 , p = 0.013 and 3.30 2.70 vs. 8.40 2.66 mv , f = 5.93 , p = 0.004 , respectively ) . the proximal amplitude of the cmap of the median nerve at 42 days of hdt was significantly decreased compared with that at 7 days of hdt ( 3.30 2.70 vs. 7.70 1.50 mv , f = 4.40 , p = 0.022 ) . ncs before and at 7 , 21 , and 42 days of hdt in rhesus monkeys ( n = 6 ) data represent means sds . * for the median nerve , statistically different versus the proximal cmap before hdt ; for the median nerve , statistically different versus distal cmap before hdt ; for the tibial nerve , statistically different versus the proximal cmap before hdt ; for the median nerve , statistically different versus the proximal cmap 7 days of hdt ; for the median nerves , statistically different versus distal cmap 7 days of hdt ; for the tibial nerve , statistically different versus the proximal cmap 7 days of hdt . ncs : nerve conduction study ; hdt : head - down tilt ; mcv : motor conduction velocity ; cmap : compound muscle action potential ; sds : standard deviations . the distal amplitude of the cmap of the median nerve was significantly decreased at 7 , 21 , and 42 days of hdt compared with the amplitude before hdt ( 7.28 1.27 vs. 10.25 3.40 mv , f = 4.03 , p = 0.039 ; 5.05 2.01 vs. 10.25 3.40 mv , f = 6.25 , p = 0.04 ; and 3.95 2.79 vs. 10.25 3.40 mv , f = 7.35 , p = 0.01 , respectively ) . the distal amplitude of the cmap of the median nerve at 42 days of hdt was significantly decreased compared with that at 7 days of hdt ( 3.95 2.79 vs. 7.28 1.27 mv , f = 3.33 , p = 0.08 ) . the proximal amplitude of the cmap of the tibial nerve was significantly decreased at 42 days of hdt compared with the amplitude before hdt ( 6.14 1.94 vs. 11.87 3.19 mv , f = 5.02 , p = 0.039 ) . the proximal amplitude of the cmap of the tibial nerve at 42 days of hdt was significantly decreased compared with that at 7 days of hdt ( 6.14 1.94 vs. 12.98 4.99 mv , f = 6.84 , p = 0.008 ) . the proximal and distal latencies of the nerves and the nerve cvs were prolonged and decreased slightly compared with the parameters before hdt but were not significantly different . previous studies regarding the effects of microgravity on the neuromuscular system have mainly focused on muscle atrophy induced by muscle disuse . few studies have investigated the electrophysiological characteristics of the peripheral nervous system after prolonged microgravity . the current investigation focused on the electrophysiological characteristics of the peripheral nervous system and identified a significant decrease in the proximal and distal amplitudes of the cmap of the median and tibial nerves as early as day 7 of hdt . previous research identified a longer duration and a slower falling rate of the action potentials of muscle fibers , thereby reflecting a change in the motor fiber impulses of its nerve , which may contribute to the decreased amplitude . furthermore , atypical combinations of myosin heavy chain mrna isoforms have been demonstrated to be transiently increased in the soleus fibers of rats that experienced 7 days of hu , which suggests a disruption of transcriptional and translational activity . these changes were present at a very early stage and may explain the cmap amplitude decrease in the ncs identified in the current study . furthermore , as the recording surface electrodes were attached at the muscle belly , a decrease in muscle power and muscle atrophy may contribute to the cmap amplitude decrease of the median and tibial nerves . however , there were no significant differences in the proximal and distal amplitudes of the cmap of the ulnar and fibular nerves at 42 days of hdt . the reason for this finding may be that 42 days of hdt is not sufficient to produce dramatic differences . studies of rats under microgravity have typically lasted for 14 days or less , and significant changes in multiple parameters such as the nerve cv and axon diameter have been identified . in humans , primates most closely resemble humans ; however , 42 days of hdt was likely not sufficient to induce similar changes . moreover , one study demonstrated that the equilibrium state in rats shifted to a different level at 14 days of hu . the transitional period between these two equilibrium states may have been marked by a transitory disorganization . we infer that the electrophysiological characteristics of peripheral nerves were under a transitory disorganization on day 42 of hdt . thus , the amplitudes of the cmap of the ulnar and peroneal nerves did not exhibit significant decreases . . a significant decrease in the cv of muscle fibers and a reduction in the cv in branching axon terminals were previously identified . in this study , the latencies of the nerves and the nerve cvs were not significantly different after hdt . the mechanisms that modulate the cv in nerve fibers are not clear , and changes in the membrane properties at the nodes of ranvier may represent one factor that plays a role in this modulation . changes in the membrane properties may be induced through modifications in the synthesis of voltage - gated membrane channels in excitable muscle membranes . wittwer et al . demonstrated gene expression alterations following prolonged unloading of rat soleus muscles . there was an increase in the mrnas that coded for ion channels , which may elucidate the increased shortening velocity of the soleus muscle following spaceflight . in our study , the most likely explanations for the lack of change in the latencies of the nerves and nerve cvs following hdt were that the 42 days of exposure to hdt was not sufficient to generate significant changes , and the electrophysiological characteristics of the peripheral nerve fibers were under a transitory disorganization . furthermore , to the best of our knowledge , the nerve cv depends on the axonal diameter , myelin sheath thickness , and internodal distance . previous research indicates that although myelin was thinner following hu , the mean axonal diameter for the small fibers was slightly decreased in hu rats ; in contrast , it was not significantly different for the large fibers , which were in charge of motor function , and the distances between the nodes of ranvier and the global structure of the nerve fibers were not significantly different . moreover , the current findings are consistent with previous research regarding fatigue , in which the cv along nerve fibers was not substantially reduced . in this study , rhesus monkeys were subjected to hdt to resemble the space flight environment , which is a model of substantial value in the investigation of microgravity effects on astronauts . experiments that involve astronauts , including ground - based experiments , are expensive , and the impact of microgravity on the motor function of astronauts is often minimized through countermeasures . furthermore , the pretest conditions of astronauts are challenging to control ; thus , the potential heterogeneity may impact the reliability of the findings . moreover , although studies have evaluated changes in muscle fibers and axon terminals under microgravity , few studies have investigated these variables in nerve trunks . the current findings provide novel evidence related to the electrophysiological characteristics of nerve trunks under microgravity . despite the importance of the experimental model and findings identified in this study , thus , future studies with a larger sample size and a longer duration of hdt are necessary to confirm these findings . conclusively , this study demonstrates that the cmap amplitudes of nerves are decreased under hdt in rhesus monkeys . moreover , results of the present study suggest that rhesus monkeys exposed to hdt might be used as an experimental model for the study of ncs under microgravity .

background : mitochondrial diseases are a group of energy metabolic disorders with multisystem involvements . variable clinical features present a major challenge in pediatric diagnoses . we summarized the clinical spectrum of m.3243a > g mutation in chinese pediatric patients , to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease.methods:clinical data of one - hundred pediatric patients with symptomatic mitochondrial disease harboring m.3243a > g mutation from 2007 to 2013 were retrospectively reviewed . detection of m.3243a > g mutation ratio was performed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism . correlation between m.3243a > g mutation ratio and age was evaluated . the differences in clinical symptom frequency of patients with low , middle , and high levels of mutation ratio were analyzed by chi - square test.results:sixty-six patients ( 66% ) had suffered a delayed diagnosis for an average of 2 years . the most frequent symptoms were seizures ( 76% ) , short stature ( 73% ) , elevated plasma lactate ( 70% ) , abnormal magnetic resonance imaging / computed tomography ( mri / ct ) changes ( 68% ) , vomiting ( 55% ) , decreased vision ( 52% ) , headache ( 50% ) , and muscle weakness ( 48% ) . the mutation ratio was correlated negatively with onset age ( r = 0.470 , p < 0.001 ) . myopathy was more frequent in patients with a high level of mutation ratio . however , patients with a low or middle level of m.3243a > g mutation ratio were more likely to suffer hearing loss , decreased vision , and gastrointestinal disturbance than patients with a high level of mutation ratio.conclusions:our study showed that half of chinese pediatric patients with m.3243a > g mutation presented seizures , short stature , abnormal mri / ct changes , elevated plasma lactate , vomiting , and headache . pediatric patients with these recurrent symptoms should be considered for screening m.3243a > g mutation . clinical manifestations and laboratory abnormalities should be carefully monitored in patients with this point mutation . background : few studies have focused on peripheral nerve conduction during exposure to microgravity . the 6 head - down tilt ( hdt ) comprises an experimental model used to simulate the space flight environment . this study investigated nerve conduction characteristics of rhesus monkeys before and after prolonged exposure to hdt.methods:six rhesus monkeys ( 34 years old ) were tilted backward 6 from the horizontal . nerve conduction studies ( ncss ) were performed on the median , ulnar , tibial , and fibular motor nerves . analysis of variance with a randomized block design was conducted to compare the differences in the ncs before and 7 , 21 , and 42 days after the 6 hdt.results:the proximal amplitude of the cmap of the median nerve was significantly decreased at 21 and 42 days of hdt compared with the amplitude before hdt ( 4.38 2.83 vs. 8.40 2.66 mv , f = 4.85 , p = 0.013 and 3.30 2.70 vs. 8.40 2.66 mv , f = 5.93 , p = 0.004 , respectively ) . the distal amplitude of the cmap of the median nerve was significantly decreased at 7 , 21 , and 42 days of hdt compared with the amplitude before hdt ( 7.28 1.27 vs. 10.25 3.40 mv , f = 4.03 , p = 0.039 ; 5.05 2.01 vs. 10.25 3.40 mv , f = 6.25 , p = 0.04 ; and 3.95 2.79 vs. 10.25 3.40 mv , f = 7.35 , p = 0.01 ; respectively ) . the proximal amplitude of the cmap of the tibial nerve was significantly decreased at 42 days of hdt compared with the amplitude before hdt ( 6.14 1.94 vs. 11.87 3.19 mv , f = 5.02 , p = 0.039).conclusions : this study demonstrates that the compound muscle action potential amplitudes of nerves are decreased under simulated microgravity in rhesus monkeys . moreover , rhesus monkeys exposed to hdt might be served as an experimental model for the study of ncs under microgravity .

a 55 year - old man visited our emergency department because of increasing frequency of chest pain . he had undergone off - pump coronary artery bypass grafting ( cabg ) 10 years ago because of unstable angina associated with three vessel coronary artery disease . at the initial operation , the in situ right internal thoracic artery ( ita ) , in situ left ita and in situ right gastroepiploic artery ( rgea ) grafts were used to revascularize the left anterior descending coronary artery , two obtuse marginal coronary branches , and posterior descending coronary artery , respectively . an excess segment of the distal right ita was connected to the side of left ita as a y - composite graft and anastomosed to the first diagonal coronary artery . coronary angiography and myocardial single photon emission computed tomography ( spect ) were performed at 5 years after surgery as a follow - up study . the 5-year angiography showed all patent grafts and the myocardial spect demonstrated no perfusion decrease . exertional chest pain recurred at 7 years after surgery , and a repeated coronary angiography showed patent previous grafts including faint visualization of the in situ rgea graft associated with significant stenosis at the os of the celiac axis . the computed tomographic angiogram also demonstrated a 90% stenosis at the celiac os , which had been without stenosis on abdominal angiography taken before the surgery ( fig . redo off - pump cabg was performed 10 years after the initial surgery because of an increasing frequency of angina and an aggravated finding of the follow - up myocardial spect , which was a newly developed reversible perfusion decrease in the inferior wall ( fig . , the great saphenous vein was harvested from the lower leg and interposed between the middle part of in situ right ita and distal part of in situ rgea grafts used previously , to supply blood flow from the right ita graft to the posterior descending coronary artery . one year after redo surgery , the patient had no symptoms of angina and coronary angiogram was performed and revealed patent grafts , including an interposed saphenous vein graft ( fig . 3a ) . the myocardial spect test was also performed and demonstrated that there was no perfusion decrease including the inferior wall ( fig . reoperations for coronary artery disease have been increased due to the increased number of isolated cabg . the society of thoracic surgeons statistics indicated that nearly 5% of the current cabg procedures done in the us were repeat surgical revascularization . angiographic indications for reoperation are progression of native coronary atherosclerosis , previous graft failure or a combination of both . one previous study demonstrated that 4 out of 400 patients who underwent cabg using the rgea graft needed percutaneous interventions due to the rgea graft failure during postoperative follow - up of 2211 months . one of those 4 patients required an angioplasty for a newly developed stenosis of the celiac trunk . in the present case , an indication for reoperation the patient had been free of angina , and the angiographic and myocardial spect follow - up studies revealed no abnormal findings at postoperative 5 years . when the patient suffered from recurred angina at postoperative 7 years , coronary angiography showed a faint visualization of the in situ rgea graft associated with significant stenosis at the os of the celiac axis . the 10-year follow - up myocardial spect test demonstrated a newly developed reversible perfusion decrease in the inferior wall . the prevalence of celiac axis stenosis was 7.3% in a korean population although it was lower than the previously reported incidence of celiac axis stenosis in western populations ranged from 12.5% to 24% . in the present case , celiac artery stenting could be an alternative option in such a case . however , we performed a redo operation because celiac axis stenting was associated with a high incidence of late restenosis . the aorta or another in situ arterial graft could be chosen as a blood source . alternatively , patent in situ grafts used previously may be re - used as an inflow conduit . with regards to our patient , the 3 in situ arterial grafts had already been used . the saphenous vein graft was interposed between the middle part of right ita and distal part of in situ rgea grafts used previously . to report a case of inadvertent anterior chamber and cornea stromal injection with high dose antibiotics and steroids during cataract operation . during cataract operation on a 78 year - old female patient , high dose gentamicin ( 20 mg/0.5 ml ) and dexamethasone ( 2 mg/0.5 ml ) were inadvertently injected into the anterior chamber and cornea stroma when making cornea edema for sealing of the incision sites . on postoperative day one , extensive cornea edema was noted , and best - corrected visual acuity was 0.2 . postoperatively , corneal edema fully resolved , and best - corrected visual acuity was 0.8 . however , some descemet 's membrane folds still remained , and a decrease in the number of endothelial cells was noted by specular microscope . in this case involving anterior chamber and cornea stromal injection with high dose antibiotics and steroids , immediate anterior chamber irrigation with balanced salt solution seemed an appropriate management , and the patient 's long - term visual acuity appears good . to prevent such mistakes , precise labeling of all solutions and use of different syringe needles should be considered . a 78 year - old woman underwent extracapsular cataract extraction with phacoemulsification via temporal clear corneal and a single superior side port incision . an intraocular lens was inserted in the bag , and viscoelastic materials were removed by irrigation and aspiration , without any complications . at the end of the operation , the nurse inadvertently handed over the wrong syringe and , and high dose antibiotics and steroids were inadvertently injected into the anterior chamber and corneal stroma through a 19 g needle during the stromal hydration procedure for wound closure at the incision sites . the antibiotics and steroids given were gentamicin ( 20 mg/0.5 ml ) and dexamethasone ( 2 mg/0.5 ml ) , originally prepared for subconjunctival injection . about 10 seconds after the injection , we recognized the mistake , and the anterior chamber was irrigated with 100ml of a balanced salt solution ( cytosol ophthalmicstm ) for one minute . on postoperative day one , the patient complained of blurred vision and foreign body sensations . severe corneal edema and descemet 's membrane folds invading the visual axis were seen extending from the 12 to 4 o'clock position ( fig . best - corrected visual acuity was 0.2 , and intraocular pressure was 18 mmhg by noncontact tonometer . postoperatively , cravit ( levofloxacin 0.5% , santen , osaka , japan ) , 1% prednisolone , and 5% nacl were administered every two hours . oral prednisolone ( 20 mg ) and ofloxacin ( 300 mg ) were used as well . a pressure patch with ofloxacin and dexamethasone ointment the corneal edema persisted until postoperative day three , and the best - corrected visual acuity decreased to 0.15 . the vision improved to 0.4 after two weeks , but the corneal edema persisted without improvement . the patient continued with the original and medications and dosages . on the third postoperative week , the best - corrected visual acuity stabilized at 0.4 , and the corneal edema began to resolve . the visual acuity continued to improve to 0.7 , and the corneal edema finally resolved four weeks postoperatively . however , the linear descemet 's membrane folds persisted up to postoperative week 16 ( fig . 2 , 3 ) . at the same site of the descemet 's membrane fold , corresponding endothelial damage was also observed , manifesting as a definite dark acelluar area on specular microscopy ( fig . 4 ) . the preoperative endothelial cell density on specular microscopy was 2717 cells / mm and the mean cell size was 368 m . at postoperative month three , the mean cell size was 424 m and endothelial cell density had decreased to 2358 cells / mm . no other changes were observed after that , and the best - corrected visual acuity improved to 0.8 at postoperative month four . almost every ocular drug can be potentially toxic to the corneal endothelium , and the risk of endothelial toxicity is increased when used in the anterior chamber because of its direct contact to the endothelium . corneal endothelium is vulnerable even to osmotic change or ph change of the irrigating fluid , and is easily damaged by electrolyte imbalance . moreover , permanent endothelial dysfunction , cystoid macular edema , pupil dilation , and glaucoma due to destruction of trabecular meshwork can occur and have been reported in severe cases.8 - 10 therefore , it is important to control the concentration of the solution when antibiotics are added to the irrigating solution in order to prevent postoperative endophthalmitis.1,2,4 however , the exact nontoxic concentrations of the antibiotics and steroids have not been fully investigated . montan et al.11 studied doses and exposure durations of cefuroxime when injected in the anterior chamber , reporting that 1 mg/0.1 ml of cefuroxime is safe and maintains a high concentration for one hour postoperatively . kang et al.12 studied gentamicin toxicity in different concentrations on three types of human corneal cells under culture and suggested that the safe local concentration of gentamicin for endothelial and stromal cell was 1 mg / ml in the human eye . with increasing concentration of gentamicin ( from 2 mg / ml to 4 mg / ml ) , the toxicity of gentamicin to the corneal cells was in direct proportion to its concentration and duration of incubation . petroutsos et al.13 reported that endothelial toxicity occurred when 1 mg / ml of gentamicin was injected into the anterior chamber in rabbit eyes . medin14 used a weight recording system to demonstrate the possible toxic effect of gentamicin on the endothelium of rabbit corneas stored in organ culture . lin et al.15 reported that 15% cell damage was found in tissue cultured bovine endothelial cells when exposed to 1.6 mg / ml of gentamicin . when the concentration of gentamicin was doubled , cell damage reached 40% , and the recovery of cellular morphology was much slower . mester and stein16 studied the effect of gentamicin concentration on rabbit endothelial cells and reported that 0.50% gentamicin was toxic to endothelium , but 0.25% gentamicin was not significantly different as compared to the control . besides irreversible damage to the corneal endothelium and corneal edema , high concentrations of gentamicin solutions can induce retinal hemorrhage , edema , and obliteration of the retinal vasculature when injected in the vitreous , resulting in optic atrophy , retinal pigment degeneration , or neovascular glaucoma.17,18 in our case , the toxicity of the gentamicin to the corneal endothelium was expected to be significant due to the fact that the concentration of the gentamicin injected in anterior chamber and corneal stroma was 40 times more than that of previously mentioned studies . wang et al.19 tested the cytotoxicity of five antibiotics ( amphotericin - b , colistin - m , sulbenicillin , amikacin , cephradine ) and a steroid ( betamethasone ) to cultured porcine corneal endothelial cells and demonstrated that the steroid was less toxic to the endothelium than other antibiotics . in our case , the inadvertent injection of antibiotics and steroids resulted in severe corneal edema from 12 to 4 o'clock position involving the visual axis , along with extensive descemet 's membrane folds . we used a hypertonic solution of about 1720 osm / l ( 5% nacl ) topically to reduce the stromal edema . this concentration is known to be nontoxic,20,21 and the solution was effective in our case . on postoperative week 12 , the descemet 's membrane folds were observed by specular microscopy and persisted 16 weeks after the operation . in our case , corneal edema and the descemet 's membrane fold were probably due to inadvertent corneal stromal injection with high dose gentamicin and dexamethasone rather than injection directly into the anterior chamber . the duration of exposure and the concentration of the drugs were negligible , due to prompt irrigation of the anterior chamber with balanced salt solution . we found the localized endothelial defect on specular microscopy manifesting as a definite dark acelluar area , which corresponded to the area of the descemet 's membrane fold and site of inadvertent stromal injection . also , irrigation with a balanced salt solution might play a role in causing corneal edema . on postoperative week four , the corneal edema resolved , and best corrected visual acuity improved to 0.7 . our case showed relatively good visual prognosis despite the high concentration of the injected antibiotics and steroids . this favorable outcome might be due to the immediate irrigation of the anterior chamber with balanced salt solution . as such , long - term endothelial exposure to high dose antibiotics and steroid can be avoided . the immediate irrigation was possible because the balanced salt solution was prepared before the operation . hansany and basu22 demonstrated that a 0.1 mg / ml concentration of gentamicin was not toxic to the corneal endothelium until seven days postop , but after seven days corneal endothelial toxicity was demonstrated for that concentration . this study suggested that duration of drug exposure ( rather than drug concentration ) is also an important factor for corneal endothelial damage . therefore , considering our case and this study , we can conclude that immediate irrigation of the anterior chamber is important in preventing corneal endothelial damage when high dose antibiotics and steroids are injected in anterior chamber by mistake during cataract or other intraocular surgery . to dilute the intrastromal antibiotics and steroids , we considered stromal injection of a balanced salt solution , but this was not performed because separation of descemet 's membrane from stroma can occur . mcdonald et al.23 insisted that clear communication between the surgeon and nursing staff , in addition to precise labeling of every injectable solution , is necessary to prevent accidents , such as this . in addition , it is wise to draw up injectable antibiotics just before injection , and to use different gauge needles for the different solutions , which could easily be recognized by physician under the microscope . in our case , we reported a favorable visual outcome with immediate irrigation of the anterior chamber with a balanced salt solution after the inadvertent injection of high dose antibiotics and steroids . however , to prevent such an accident during surgery , strict precautions ( mentioned previously ) and clear communication between surgeons and nursing staff , including scrub nurses , are considered to be of great importance .

we report a redo coronary artery bypass grafting ( cabg ) in a 55-year - old man . angina recurred 7 years after the initial surgery . coronary angiography showed all patent grafts except a faint visualization of the in situ right gastroepiploic artery ( rgea ) graft , which was anastomosed to the posterior descending coronary artery , associated with celiac axis stenosis . redo - cabg was performed at postoperative 10 years because of aggravated angina and decreased perfusion of the inferior wall in the myocardial single photon emission computed tomography . the saphenous vein graft was interposed between the 2 in situ grafts used previously ; the right internal thoracic artery and rgea grafts . angina was relieved and myocardial perfusion was improved . purposeto report a case of inadvertent anterior chamber and cornea stromal injection with high dose antibiotics and steroids during cataract operation.methodsduring cataract operation on a 78 year - old female patient , high dose gentamicin ( 20 mg/0.5 ml ) and dexamethasone ( 2 mg/0.5 ml ) were inadvertently injected into the anterior chamber and cornea stroma when making cornea edema for sealing of the incision sites . anterior chamber irrigation with balanced salt solution ( bss ) was immediately administered . on postoperative day one , extensive cornea edema was noted , and best - corrected visual acuity was 0.2 . descemet 's membrane folds were observed around the corneal incision sites . topical 5% nacl and 1% prednisolone were started.resultsfour weeks postoperatively , corneal edema began to reduce significantly . at four months postoperatively , corneal edema fully resolved , and best - corrected visual acuity was 0.8 . however , some descemet 's membrane folds still remained , and a decrease in the number of endothelial cells was noted by specular microscope.conclusionsin this case involving anterior chamber and cornea stromal injection with high dose antibiotics and steroids , immediate anterior chamber irrigation with balanced salt solution seemed an appropriate management , and the patient 's long - term visual acuity appears good . to prevent such mistakes , precise labeling of all solutions and use of different syringe needles should be considered .

autosomal - dominant polycystic kidney disease ( adpkd ) is characterized by cyst formation and occurs primarily in the kidneys . due to the replacement of the normal renal parenchyma , adpkd results in end - stage renal failure in 45% of patients . approximately 711% of patients receiving renal replacement therapy in the western world are doing so due to adpkd . the underlying cause of adpkd is a mutation in the polycystin-1 and -2 plasma proteins , located in the primary cilia . this mutation leads to abnormal function of renal tubular epithelia and inadequate calcium influx followed by cyst formation . cardiovascular disease is the most frequent cause ( 36% ) of mortality in patients with adpkd . patients with adpkd often develop hypertension at an earlier age than the general population before any impairment of kidney function . it is hypothesized that the renin - aldosterone system and endothelial dysfunction caused by impaired nitric oxide release are the important factors in the development of hypertension in adpkd patients . we describe an uncommon case of a middle - aged man with a spontaneous coronary artery dissection ( cad ) and adpkd . a 41-year - old caucasian man presented at an emergency department with acute chest pain . the chest pain began 2 h before presentation . medical history recorded a subdural haematoma , as a result of trauma . according to the family history , our patient was a non - smoker , with no history of hypertension , diabetes mellitus or hypercholesterolaemia . on physical examination , the blood pressure was 155/102 mmhg and the pulse 72 beats / minute ; other vital parameters were normal . electrocardiography showed a sinus rhythm of 65 beats / minute and st elevation in the precordial leads ( figure 1 ) . / l , creatine kinase mb was 17.4 u / l and troponin t was 0.21 ng / ml . the other laboratory test results were normal . echocardiography revealed a hypokinetic septum and a slightly impaired left ventricular function with an ejection fraction of 4560% . electrocardiography on presentation shows a sinus rhythm of 65 beats / minute and st elevation in v3 , ii and iii . based on these results , a presumptive diagnosis of acute septal myocardial infarction was made . coronary artery angiography ( cag ) revealed a transient occlusion of the left anterior descending ( lad ) coronary artery , most probably as a result of myocardial bridging ( figure 2a ) . the cag ( a ) shows the compression of the lad coronary artery during the systole resulting in narrowing and ( b ) performed after the second chest pain attack demonstrating a dissection in the distal left anterior descending coronary artery . however , the chest pain returned 3 days after the presentation . a second electrocardiography showed persistent inverted t waves in the precordial leads without st elevation . it disclosed an open lad with a dissection in the distal part and a double lumen , which was not observed during the first angiography ( figure 2b ) . the definitive diagnosis of non - q - wave anterior infarct as a result of a spontaneous lad dissection was made . based on the cysts in both kidneys combined with a family history of adpkd , the diagnosis of adpkd was made ( ravine s criteria ) . since cerebral aneurysms are one of the extrarenal manifestations of adpkd , a computerized tomography angiography of the brain was performed , with a negative result for vascular anomalies . an exercise stress test , performed 16 days after the onset of chest pain , was normal . the lad was patent without a significant infarction , but still with a double lumen appearance . at 2-year follow - up by a nephrologist , our patient , a relatively young man with a negative cardiovascular profile and a history of subdural haematoma , developed a myocardial infarction secondary to dissection of the lad . the cad was not recognized during the first cag because the relevant coronary artery can obturate the dissection in the acute phase . this condition is found when the segmental coronary artery has an intramyocardial course , being compressed during systole and restored during diastole . therefore , one would require a difference in coronary artery contraction in the systolic and diastolic phase which was not observed on the first cag . four cases were reported on the occurrence of a spontaneous cad in middle - aged adpkd patients . predisposing factors for cad in adpkd patients are still undetermined , especially in the absence of traditional cardiovascular risk factors . furthermore , it is not known if arterial dissection is an extrarenal manifestation of adpkd or if is just secondary to hypertension . the estimated prevalence of spontaneous cad is 0.7% , and it is in 2% of cases , the cause of acute coronary syndrome . the lad is affected in 80% of the patients with cad [ 8 , 9 ] . the majority of patients with a cad often lack classical risk factors for cardiovascular disease and are female . de maio et al . identified three groups of patients with cad : ( i ) patients with atherosclerotic cardiovascular disease , ( ii ) women in the postpartum period and ( iii ) an idiopathic group . several underlying conditions in the idiopathic group are suggested , such as polyarteritis nodosa , lupus erythematosus , marfan s syndrome , ehlers danlos syndrome , intense physical exercise , the use of cocaine , cyclosporin and oral contraceptives . the tunica media , the middle layer of elastic arteries , include smooth muscles with interposing layers of elastic lamellae . the connection between the intracellular contractile filaments and extracellular elastic fibres is provided by dense plaque sites . localization in the dense plaques assigns a significant function to polycystins in maintaining vascular integrity . furthermore , qian et al . suggested that abnormal intracellular calcium concentration in the vascular smooth muscle cells is linked to vascular phenotype in the case of inactivation of polycystin-2 protein . literature also confirms the observation of intracranial aneurysms and myocardial infarction secondary to coronary aneurysms in certain families and the occurrence of vascular rupture and haemorrhage in homozygous polycystic kidney disease ( pkd)-1 knockout mice [ 4 , 11 ] . considering these results , and the high plasma renin activity and impaired nitric oxide release in adpkd patients , we speculate that the vascular abnormalities are most likely a direct result of pkd mutations rather than a secondary cause of hypertension . in conclusion , polycystins seem to play a main role in the stability of the arterial vasculature . therefore , a spontaneous cad should be considered as an extrarenal manifestation of adpkd . the clinician should be aware of cad if adpkd patients present with chest pain or discomfort . cag can be inconclusive in respect to the mechanism of coronary occlusion in the acute phase and should be repeated , especially when the complaints persist . optimal treatment of cancers in children often requires combined - modality therapy , including : chemotherapy , surgery , and/or radiotherapy . chemotherapy is not always sufficient to achieve the cure of solid tumors in children ; either resection or radiation may be needed for local tumor control as well ( 1 ) . children with radiosensitive malignant tumors typically require radiation therapy for a number of sessions over a period of several weeks . although the procedure is painless , young children need to be sedated or anesthetised in order to provide a motionless state during the procedure . a short period of sedation , analgesia or general anaesthesia the patient and anaesthesia equipment are observed continuously by closed - circuit television , and monitors are mirrored to the remote observation site outside the treatment room . different anesthesia methods and anesthetics have been recommended to provide a safe and optimal situation of motionless with a short recovery period in children undergoing general anaesthesia or sedation for external beam irradiation ( 37 ) . this report describes the accidental detection of a missed complication by the anesthetist at the time of radiotherapy , from the previous chemotherapy , which was preliminarily and unduly attributed to anesthesia . a 2.5 year - old , 13-kg boy , asa ( american society of anesthesiologists ) class ii , with acute lymphoblastic leukaemia ( all ) , was scheduled for radiotherapy . anesthesia was planned by the anesthetist , and the physical examination was normal before radiotherapy . routine monitoring was established , and for peripheral pulse monitoring , the distal portion of the right upper limb of the child was rested out of the covers . after about 5 minutes , when the anesthetic effect of ketamine began , we noticed a lack of right radial pulse . anesthesia - related hemodynamic instability was expected , however no other manifestation of hypoxia or hypo - perfusion was detected . furthermore , carotid pulsation was normal , and examination of the left radial pulse and other peripheral pulses showed normal pulsation . the patient showed a scar and swelling on the right antecubital area . a more detailed history taken from the child s parents showed his history of chemotherapy during which extravasation of the chemotherapy drug had resulted in severe inflammation and edema at the site of injection . a colour doppler ultrasound of the antecubital area showed deep edema and chronic compression on the antecubital tissue and confirmed the diagnosis . permission was obtained from the patient s parents to use the patient s reports , however the patient s information was to remain confidential . the incidence range of extravasations of cytostatic drugs in cancer patients has been reported from 0.2 to 1.4% in a five - year study ( 8) . extravasations in cytostatic treatment may cause a wide range of symptoms , from patients discomfort to severe complications such as necrosis and amputation . ( 9 ) eccrine squamous syringometaplasia , while rare , has also occurred in patients who have received chemotherapy treatment ( 10 ) . yeung et al described a case of metastatic ovarian carcinoma with repeated thrombosis of the femoral arteries following intravenous carboplatin - based combination chemotherapy . persistent withdrawal occlusion ( pwo ) is frequently caused by fibrin sheath formation around venous access devices . small doses of thrombolytic drugs ( such as urokinase ) could manage pwo , but could also serious complicate chemotherapy drug extravasation ( 12 ) . keratolytic ointment was applied for old lesions , whereas in new lesions , multiple subcutaneous injections of hydrocortisone solution were used before the application of betamethasone ointment . application of conservative agents in radiotherapy - induced extravasation areas may avoid tissue necrosis and consequently , reconstructive surgery ( 13 ) . management of cytotoxic drug extravasation in humans is based on the experimental evidences and available case reports because of lack of randomized trials . for instance , topical dimethylsulfoxide ( dmso ) and cooling for extravasation of anthracyclines or mitomycin , local injection of hyaluronidase for extravasation of vinca alkaloids , and local injection of sodium thiosulfate ( sodium hyposulfite ) for extravasation of chlormethine ( mechlorethamine ; mustine ) should be empirically recommended . in the case of failed conservative treatment , history and physical examination before injection of anesthetic agents could be useful in preventing patient mismanagement . a weak pulse in a child can be a significant problem for the anesthesiologist and in this case , it was shown to be a critically misleading factor . increased emphasis is on clinical evaluation and pulse checking is necessary , especially in children with a history of chemotherapy . providing thorough pulse evaluation when we work on children and avoiding immediate aggressive intervention before being certain about the cause of weak pulse are additional issues of importance shown by this case report .

little is known about the association between autosomal - dominant polycystic kidney disease ( adpkd ) and coronary artery dissection ( cad ) . we suggest that the genetic disorder in adpkd is the main cause of instable artery vasculature . our case also shows that cad can be missed in the acute phase . therefore , we recommend additional investigation in patients with adpkd presenting with acute chest pain . we report a case of a patient who developed a myocardial infarction due to a spontaneous dissection of the left anterior descending coronary artery . adpkd was diagnosed during the additional investigation . the patient received medical management . treatment of cancer in children often requires a combination of chemotherapy , surgery , and/or radiotherapy . radiotherapy and chemotherapy are not painful processes , but children undergoing these procedures must be made motionless through anesthesia or sedation . there are a few reports of complications during these procedures in relation to the procedures themselves or to the anesthesia given . this report describes an unexpected pulseless radial artery which was preliminarily and unduly attributed to anesthesia . a 2.5 year - old male pediatric patient with an acute lymphoblastic leukaemia was scheduled for radiotherapy . anesthesia with intramuscular ketamine was induced before starting radiotherapy . about 5 minutes after injection of ketamine we found the right radial pulse undetectable . there was no other manifestation of hypoxia or hypo - perfusion . carotid pulsation was normal . examination of the left radial pulse and other peripheral pulses showed normal pulsation . the procedure was continued uneventfully . the next follow - up after radiotherapy , showed a scar and swelling on the right antecubital area , caused by extravasation of chemotherapeutic agent in the prior period of chemotherapy . doppler ultrasonography of the antecubital vein confirmed the diagnosis . this case study therefore demonstrates that proper intravenous cannula establishment before chemotherapy is of great importance . furthermore , accurate history and physical examination before induction of anesthesia or sedation may be useful in preventing mismanagement in pediatric cancer procedures .

annually , more than 2.5 million muslims from about 140 countries go on the hajj pilgrimage , which is the largest mass gathering in the world . the presence of such a vast number of people in the holy places , especially in the cities of mecca and medina , and the massive pilgrim crowds in certain places at certain times , causes infectious diseases to spread more easily . racial , cultural and health differences , as well as the level of access to health , medical and welfare services at the time of stay in these places are important factors that increase the risk of communicable diseases . both , viral or bacterial respiratory tract infections are very common among pilgrims in all age and sex groups , and is regarded as the most common disease among pilgrims in some reports . some studies have also reported the prevalence of different types of these infections up to 85.3% . to prevent these infections , some preventive measures have been recommended for the pilgrims , such as mandatory injection of tetravalent meningococcal meningitis vaccine , recommendation to inject seasonal flu vaccine , and using surgical masks . on the other hand , each country plans and implements some specific measures to prevent and control these diseases among pilgrims . more than 100,000 iranians in about 600 caravans undertook the hajj in 2010 . according to the current policies of the hajj medical center of iran , before traveling any caravan common diseases detected among pilgrims are treated in the caravan itself , or referred to the medical center , if needed . the reports show conflicting results about the effects of preventive measures for respiratory tract infections and in fact , a precise study on the effects of each one of these preventive measures has been rarely done . this research is trying to examine the effects of general preventive measures on respiratory tract infections through a nested case - control study , which is more precise design than ordinary case - control studies . we used nested case - control design , which is based on risk set sampling . the studied cohort consist two caravans ( 338 pilgrims ) , one of which was the first to enter mecca , and the other the first to enter medina . the outcome of this study was all types of respiratory tract infections other than the common cold including tonsillitis , pharyngitis , laryngitis , sinusitis , otitis media , bronchitis , pneumonia and influenza . as soon as any patient in the caravans was identified , a data collection form was completed for that individual . considering that the level of presence in saudi arabia and the amount of contact with all types of viruses and bacteria are regarded as risk , on the day of identifying each patient , two pilgrims in the same caravan were randomly selected as control group . each control pilgrim was not affected by the mentioned outcome at the time of investigation , although these pilgrims may be enrolled as a case in future days . every pilgrim who become ill with studied disease and had consent to participate , was enrolled in the study . all pilgrims who did not get the studied disease at time of selection and had consent to participate , could be enrolled as controls . body mass index ( bmi ) is calculated by dividing the weight in kilograms to height square in meter . if another patient with studied disease existed in the same room then the pilgrim considered as having room contacty . if the pilgrims used face masks in crowding , even occasionally were considered as using face mask . pilgrims considered as vaccinated against influenza if they vaccinated by this vaccine since two years before traveling to saudi arabia . pilgrims , who washed their mouth and throat with salt water at least once a day , were considered as having salt water gargling . pilgrims with a past history of at least one systemic diseases including asthma , diabetes mellitus , hypertension , copd and cardiovascular diseases were considered as having systemic diseases . present or past smoker with any type and amount of smoking were considered as smokers . if the pilgrims used personal prayer carpet in holy places were considered as using personal prayer carpet . the effect of each preventive measure on the occurrence of respiratory tract infections was studied through a univariable model . at the next stage , all variables which were significant in the first stage ( p < 0.2 ) , were entered into the multiple models , and the adjusted odds ratio was obtained for each one of the variables . we used nested case - control design , which is based on risk set sampling . the studied cohort consist two caravans ( 338 pilgrims ) , one of which was the first to enter mecca , and the other the first to enter medina . the outcome of this study was all types of respiratory tract infections other than the common cold including tonsillitis , pharyngitis , laryngitis , sinusitis , otitis media , bronchitis , pneumonia and influenza . as soon as any patient in the caravans was identified , a data collection form was completed for that individual . considering that the level of presence in saudi arabia and the amount of contact with all types of viruses and bacteria are regarded as risk , on the day of identifying each patient , two pilgrims in the same caravan were randomly selected as control group . each control pilgrim was not affected by the mentioned outcome at the time of investigation , although these pilgrims may be enrolled as a case in future days . every pilgrim who become ill with studied disease and had consent to participate , was enrolled in the study . all pilgrims who did not get the studied disease at time of selection and had consent to participate , could be enrolled as controls . body mass index ( bmi ) is calculated by dividing the weight in kilograms to height square in meter . if another patient with studied disease existed in the same room then the pilgrim considered as having room contacty . if the pilgrims used face masks in crowding , even occasionally were considered as using face mask . pilgrims considered as vaccinated against influenza if they vaccinated by this vaccine since two years before traveling to saudi arabia . pilgrims , who washed their mouth and throat with salt water at least once a day , were considered as having salt water gargling . pilgrims with a past history of at least one systemic diseases including asthma , diabetes mellitus , hypertension , copd and cardiovascular diseases were considered as having systemic diseases . present or past smoker with any type and amount of smoking were considered as smokers . if the pilgrims used personal prayer carpet in holy places were considered as using personal prayer carpet . the effect of each preventive measure on the occurrence of respiratory tract infections was studied through a univariable model . at the next stage , all variables which were significant in the first stage ( p < 0.2 ) , were entered into the multiple models , and the adjusted odds ratio was obtained for each one of the variables . during the hajj pilgrimage , 32 people ( 9.5% ) of the cohort ( 338 pilgrims ) were affected by respiratory tract infections . according to the explained method description of cases and controls , in terms of age , sex , education and other factors which influence the incidence of respiratory tract infections , are presented in table 1 . distribution of factors affecting respiratory tract infections in hajj pilgrims , 2010 sixty nine percent of the patients were treated with symptomatic treatment and oral antibiotics , and 27.5% required treatment through intravenous antibiotics as well . in addition , 79.3% of the patients fully recovered through the treatment administered in the caravan , but 17.2% were referred to the hajj medical center , and finally 3.4% of the total patients were hospitalized in that center . univariable logistic regression analysis showed that among the variables studied , only gargling with salt water , presence of other patients in the room , age and level of education , have p < 0.2 and can be associated with respiratory tract infections . among these variables , gargling with salt water with or = 2.4 and p = 0.08 had the strongest relationship [ table 2 ] . the role of protective measures and other individual factors on respiratory tract infections among hajj pilgrims , 2010 the multivariable logistic regression model showed that none of the factors listed above were significantly associated with the outcome being studied . for example , presence of the other patient in the room increases the risk of outcome by 2.67 times , but this effect is not significant , according to the confidence intervals of or [ table 2 ] . the results also showed that the current preventive measures including wearing face masks , seasonal flu vaccination , and use of personal prayer carpet were not effective enough in the prevention of these infections . however , the seasonal flu vaccination has been recommended , especially in high - risk individuals or those over 65 years of age . the influenza vaccine has been proved by other researchers to have no effect in the prevention of respiratory infections , and it has even been seen that the use of seasonal influenza vaccine prolongs the period of sore throat . however , methodological problems of the studies and the applied methods , including laboratory or demographic studies , should be considered when comparing these results . it should also be noted that recommendations to take a seasonal influenza vaccine is due to its preventive effect on the incidence of flu and reduction of the risk of pandemic swine flu . other studies , like the results of this study , have shown that wearing face masks has no effect on respiratory infections . although the cdc does not recommend wearing it , the saudi ministry of health recommends that pilgrims wear face masks . the recommendation to wear masks is probably because of its role in the prevention of tuberculosis , which has not been studied among the pilgrims . the effect of using a personal prayer carpet has not been studied in other reports . probably , the close contact of the pilgrims with each other , and their presence in crowded places are the main causes for transmission of respiratory infections , so it hinders a possible protective role played by the personal prayer carpet . although , the effect of this action on the prevention of respiratory infections was not significant in the multiple logistic model , it seems that the reason for it is the relatively small sample size in the study . in other words , through studies with a larger sample size , it may be concluded that lack of gargling with salt water increases the risk of respiratory infections by 2.3 times . similarly , it can be said that staying with other patients in the same room increases the risk of respiratory infections by 2.67 times . as can be seen in table 2 , factors such as age , sex , level of education , area of the room , smoking , systemic diseases , obesity , and the average daily hours of gathering together in the holy places have no effect on respiratory tract infections . in other words , any age and sex group with different individual conditions is likely to get exposed to viral and bacterial causes of these infections . as mentioned above , the relatively small sample size is one of the limitations of this study . an increase in the sample size would make it possible to study the role of factors associated with respiratory infections in any of the holy places ( mecca , medina , mina , etc . ) . in this study , the cases were of those who were diagnosed clinically by a caravan physician . if the rapid test diagnosis is applied along with the acceptable sensitivity and specificity to diagnose cases , the accuracy of the study will increase . flu vaccinations wearing face masks , personal prayer carpet , smoking , obesity , etc . , do not affect the incidence of respiratory infections during the hajj , and if this study is to be undertaken with a larger sample size , according to the appropriate selected methodology , some evidence will be obtained showing the prophylactic effect of gargling with salt water in the prevention of these infections . care coordination is an important aspect of nursing care especially for elderly patients admitted to an acute care setting . in singapore care coordination and transitional care nursing is a new concept of care nevertheless important but unexplored . the objective of this paper is to explore the characteristics of elderly patients receiving care coordination , determine care gaps and intervention during home visit and telephonic review . a designed questionnaire was used to collect information on the patient s demography , social and clinical profile and determine post discharge activities using eric coleman s four pillars tool . the retrospective data from the patient s index admission from the last six months ( nov 08april 09 ) was analyzed using spss version 16 . majority , 69% were above 70 years old of which 57% female and 76% lives with their children . clinical information demonstrates that 53% had 36 co - morbidities and 58% were taking more than five medications . the abbreviated mental test score were 6.2 , 6% were depressed and delirium was present in 14% of patients . only 65 patients ( 0.1% ) had home visits and telephonic review done whilst 97% of the remaining had only telephonic review done . those who had both telephonic and home visit review , medications advice and compliance were checked only in 0.8% ( at one week ) and 1.6% ( at one month ) whilst during home visit this was done in 12.2% of patients as medication discrepancy were apparent at home . as for appointment compliance and compilation were done in 0.8% at one week and 51% at four weeks of telephonic review compared to during home visit only 4.8% . caregivers education was emphasized in 14% of patients at home visit , 2% at one week and 4% at one month of telephonic review . the result showed that home visit is effective in exploring medication compliance , advice and emphasizing caregiver education , managing appointments can be effectively done through telephone review . this study demonstrates the vital role of home visit for elderly patient to safely transit between hospitals to home .

background : respiratory tract infections are very common among the hajj pilgrims . some preventive measures including influenza vaccination , using face mask and salt water gargling have been considered to control these infections and the reports show conflicting results about the effects of each one of these measures . this study is trying to assess the effects of these recommendations on respiratory tract infections.methods:according to nested case - control design , in a cohort consisting of 338 iranian pilgrims , the outcome examined , was all types of respiratory tract infections other than common colds . with occurrence of any patient in convoy , data collection form was completed for that person . on the same day , two people were randomly selected as control group from among pilgrims who have not affected so far.results:during hajj , 32 pilgrims ( 9.5% ) were affected by respiratory tract infections other than common colds . in univariable logistic regression analysis , salt water gargling ( or = 2.4 , p = 0.08 ) , existence of other patient in the room ( or = 2.14 , p = 0.19 ) , age over 60 years ( or = 1.84 , p = 0.15 ) and the education more than or equal to 3 years ( or = 1.93 , p = 0.16 ) were effective in the respiratory tract infections ( p < 0.2 ) . however , multivariable logistic regression analysis showed that none of the above mentioned factors are significantly associated with these infections.conclusions:this study showed that measures such as seasonal influenza vaccination , use of face masks and personal prayer carpet have no effect on the incidence of respiratory tract infections . however , washing throat and mouth with salt water can be considered the most effective preventive measures . introductioncare coordination is an important aspect of nursing care especially for elderly patients admitted to an acute care setting . in singapore care coordination and transitional care nursing is a new concept of care nevertheless important but unexplored.aim/objectivesthe objective of this paper is to explore the characteristics of elderly patients receiving care coordination , determine care gaps and intervention during home visit and telephonic review.research design and samplinga designed questionnaire was used to collect information on the patient s demography , social and clinical profile and determine post discharge activities using eric coleman s four pillars tool . a pilot study of ten questionnaires was conducted . the retrospective data from the patient s index admission from the last six months ( nov 08april 09 ) was analyzed using spss version 16.resulttotal of 517 patients were recruited from october 2008 to march 2009 . majority , 69% were above 70 years old of which 57% female and 76% lives with their children . clinical information demonstrates that 53% had 36 co - morbidities and 58% were taking more than five medications . the abbreviated mental test score were 6.2 , 6% were depressed and delirium was present in 14% of patients.only 65 patients ( 0.1% ) had home visits and telephonic review done whilst 97% of the remaining had only telephonic review done . those who had both telephonic and home visit review , medications advice and compliance were checked only in 0.8% ( at one week ) and 1.6% ( at one month ) whilst during home visit this was done in 12.2% of patients as medication discrepancy were apparent at home . as for appointment compliance and compilation were done in 0.8% at one week and 51% at four weeks of telephonic review compared to during home visit only 4.8% . caregivers education was emphasized in 14% of patients at home visit , 2% at one week and 4% at one month of telephonic review.discussionthe result showed that home visit is effective in exploring medication compliance , advice and emphasizing caregiver education , managing appointments can be effectively done through telephone review.conclusionthis study demonstrates the vital role of home visit for elderly patient to safely transit between hospitals to home .

the irrevocable aim of endodontics is a three - dimensional unblemished seal of the root canal system which is achieved by perfect designing of the canal diameter and canal form . the biomechanical preparation is one of the major steps for removal of bacteria and debris in the root canal so as to achieve a successful endodontic treatment . during root canal instrumentation there are complications such as perforations , ledge formation , transportation of canal , and formation of cracks in the root dentin . at times , in the zeal of biomechanical preparation of the canal we inevitably end up damaging the root dentin , which becomes a gateway to dentinal cracks and minute intricate fractures ; thereby , causing failure of treatment . as a result of craze lines or microcracks , there might be occurrence of root fracture that propagates due to repeated application of stress by the occlusal forces . shemesh et al . , observed more dentinal defects in teeth which were obturated with spreader than teeth obturated without spreader . in different degrees , dentinal damage can occur due to procedures like biomechanical preparation , obturation , and retreatment . complexities in the preparation of root canal may be attributed to variation in the design of the cutting instrument , taper , or composition of the material from which it is made . hand instrumentation the milestone of endodontic practice in the past though have lost popularity , still remain the integral part of canal preparation . rotary instrument by its innate behavior in the canal may result in more friction , which may increase dentinal defects and microcracks formation in comparison to hand instruments . possible relationship between the design of niti rotary instruments and the incidence of the vertical root fractures was found by kim et al . , and it was concluded that the design of the file affects strain concentration and the apical stress during instrumentation of root canal . recently , the protaper next ( ptn , dentsply , maillefer ) files were introduced in the family of niti rotary instruments with a completely new design comprising of unique swaggering movement , greater flexibility , the m - wire technology , the 5 generation of continuous improvement , and its offset design . whether it is rotary or hand files ( hfs ) , they are assumed to cause limited frictional forces within the canal , hence creating dentinal defects . so there is need to study the behavior of different niti rotary instruments and the newly developed rotary system , ptn , on root dentin . teeth with curved roots , calcified canals , extracanals , and teeth with developmental anomaly or resorption were excluded from the study . the teeth were decoronated at coronal portions by using a diamond disc , leaving roots approximately of 10 mm in length . all the roots were inspected with transmitted light for detecting any preexisting cracks or any craze - lines by using a stereomicroscope under 12 , to exclude teeth with such findings from this study . patency of the canal was established using a # 10 k - file ( mani , japan ) in the canal . the specimens were then divided into four groups ; each group containing 15 specimens each . hfs upto file # 40 were used for canal preparation . in the pt ( dentsply , maillefer ) , hs ( micro - mega , besancon , france ) , and ptn ( dentsply , maillefer ) groups ; preparation of the canals was done using speed and torque controlled motor ( x - smart ; dentsply , maillefer ) . in the hand files group , step - back technique was used upto file # 40 . in the pt group , the following sequence of pt rotary niti files were used for preparation of canals at 300 rpm : the shaping file x for coronal enlargement , and s1 , s2 , f1 , f2 , and f3 files , corresponding to apical size 30 , used at the working length . in the hs group , the hs niti files were used upto file # 30 at 300 rpm in crown - down sequence . in ptn group , the ptn rotary system files were used at 300 rpm in the following sequence : x1 , x2 and x3 , corresponding to apical size # 30 . the ptn rotary files were used in a constant rotation at a speed of 300 rpm with light apical pressure ( recommended torque is 2.0 ncm , adjustable up to 5.2 ncm according to practitioner experience ) . flutes of the instruments were cleaned frequently to check any signs of distortion or wear . the ptn instruments are recommended to be used mechanically ( manually in very severe curvatures ) in a clockwise continuous motion with a brushing motion , away from external root concavities , to facilitate flute unloading and apical file progression . in all the experimental groups , sectioning of all the roots was done perpendicular to the long axis at 9 , 6 , and 3 mm using a diamond disc under water cooling . digital images of each sectioned root was captured using a 40 stereomicroscope by using a digital camera ( olympus , tokyo , japan ) . roots were classified as no defect , fracture , and other defects as described in table 1 . classification for identification of defects in the specimens the results were expressed as the number and percentage of defects in each group . chi - square test was used for the statistical analysis of the groups . the level of significance was set at p = 0.05 using statistical package for social sciences ( spss ) 20.0 . sectioning of all the roots was done perpendicular to the long axis at 9 , 6 , and 3 mm using a diamond disc under water cooling . digital images of each sectioned root was captured using a 40 stereomicroscope by using a digital camera ( olympus , tokyo , japan ) . roots were classified as no defect , fracture , and other defects as described in table 1 . classification for identification of defects in the specimens the level of significance was set at p = 0.05 using statistical package for social sciences ( spss ) 20.0 . figure 1 is a bar chart representing the number of root defects in each group . hfs group showed lowest defect ( 1/15 ) followed by pt ( 6/15 ) , hs ( 10/15 ) , and ptn ( 4/15 ) . statistical significant difference was seen between hfs and hs group and between hs and ptn groups ( p < 0.05 ) . no significant difference was found between the pt and hs ( p > 0.05 ) . bar chart representing number of root defects in each group the stereomicroscopic images of group i , ii , iii and iv are shown in figure 2 . stereomicroscopic images showing dentinal defects seen in groups i , ii , iii , and iv , showing craze lines seen in group i. fracture and other defects in group ii craze lines and partial crack in group iii and craze lines in group iv in the present study ; in hfs , pt , hs , and ptn , the number and incidence of defects observed in the root dentin was found to be 1/15 ( 6.67% ) , 6/15 ( 40% ) , 10/15 ( 66.67% ) , and 4/15 ( 26.7% ) , respectively . group i ( hfs ) showed the lowest incidence of defects ( 6.67% ) ; whereas , hs group showed the maximum incidence of defects ( 66.67% ) as compared to other groups . the results of our study are in accordance with imam , who reported lowest number of defects ( 1/20 ) by hfs ; and yold as et al . , observed highest incidence of defects ( 12/20 ) by hss rotary files . , in the present study the number and percentage of defects shown by the ptn rotary files were 4/15 , that is , 26.7% . the results of the present study are not in accordance with the results by bier et al . , where the hfs showed no defect and pt rotary files showed highest incidence of dentinal damage ( 16% ) . excess removal of root dentin during root canal preparation and obturation of the canal with spreader may create fracture in the teeth . the important goal in endodontics is resistance to tooth fracture because such fractures might cause decrease in the long - term survival rate . in the presents the number of rotations required for complete root canal preparation is more with niti rotary instruments than with the hfs . kim et al . , stated that taper of the files is the responsible for increase of stress on the walls of the root canal ; whereas , bier et al . , stated taper of the files as one of the contributing factor for crack formation in root dentin . pt have more taper ( 0.07 , 0.08 , and 0.09 , respectively ) than the hss ( 0.04 and 0.06 ) and the ptn ( x1 , x2 , and x3 ; 0.04 , 0.06 , and 0.07 , respectively ) . this explains that there can be formation of cracks in the pt group , as reported earlier by bier et al . , liu et al . , barreto et al . , and liu et al . furthermore , relatively low flexibility of the hs may have contributed to the maximum number of defects in hs group in the present study . rotational force is applied to the canals of the root by niti rotary instruments , thus creating craze line or microcracks in root dentin . formation of such defects may be associated with the design of tip , cross - sectional geometry , taper type ( constant or progressive ) , flute form , and pitch ( constant or variable ) . the pt files have a triangular cross - sectional geometry , hs having a triple helix cross - sectional geometry ; whereas , the ptn is rectangular . thus , it can be stated that design of the rotary files is not the only factor for defect formation in root dentin . lam et al . , stated that forces shaping the root dentin can be affected by the file design . risk of root fracture is increased due to the forces generated during the root canal preparation . ptn files have m - wire technology with off - centered rectangular cross - section , giving the file a snake - like swaggering movement as it moves along the root canal , thus reducing the screw effect , the unwanted taper lock , and torque on any of the given file ; thus decreasing the file - root dentin contact . m - wire alloy niti material with controlled memory niti wire are flexible than those made from conventional niti wire . thus , such flexibility of ptn rotary files may have contributed in less number of dentinal defects formation as compared to pt and hs . capar et al . , concluded that the swaggering motion and less taper of the ptn instruments could change the root canal volume to an extent as that of the higher tapered instruments . use of different speed and torque settings for each rotary system could be the limitation of our study . increase in the rotational speed is associated with increased cutting efficiency.simulation of periodontal ligament was not done in the present study . capar i d et al . stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . it plays an important role in stress dissipation created by application of load to the teeth . use of different speed and torque settings for each rotary system could be the limitation of our study . capar i d et al . stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . it plays an important role in stress dissipation created by application of load to the teeth . use of different speed and torque settings for each rotary system could be the limitation of our study . increase in the rotational speed is associated with increased cutting efficiency.simulation of periodontal ligament was not done in the present study . capar i d et al . stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . it plays an important role in stress dissipation created by application of load to the teeth . use of different speed and torque settings for each rotary system could be the limitation of our study . capar i d et al . stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . it plays an important role in stress dissipation created by application of load to the teeth . within the limitations of this in vitro study , ptn rotary system can induce less dentinal defects than pt and hs . batch processes are used in production of many low - volume but high - value - added products ( such as speciality chemicals , health care , food , agrochemicals, etc . ) because of operation flexibility in today 's market - driven environment . in addition , if two or more products require similar processing steps , the same set of equipment is considered for at least economical reason . a batch plant producing multiple products is categorized as either a multiproduct plant or a multipurpose plant . in such a plant , all the products follow the same path through the process and only one product is manufactured at a time . processing of other products is carried out using the same equipment in successive production runs or campaigns . in a multipurpose plant , each product follows one or more distinct processing paths ; so more than one product may be produced simultaneously in such plants . the present work is directed toward the optimal design problems of multiproduct batch plants . in conventional optimal design of a multiproduct plant , production requirements of each product and a total production time for all products are available and specified . the number , the required volume , and size of parallel equipment units in each stage are then determined to minimize the investment . it should be emphasized that batch plantsdesign has been for long identified as a key problem in chemical engineering as reported in literature [ 29 ] . formulation of batch plant design generally involves mathematical programming methods , such as linear programming ( lp ) , nonlinear programming ( nlp ) , mixed - integer linear programming ( milp ) or mixed - integer nonlinear programming ( minlp ) . mathematical programming or different optimization techniques , such as branch and bound , heuristics , genetic algorithm , simulated annealing , are thoroughly used to derive optimal solutions . however , in reality the multiproduct design problem can be formulated as a multiobjective design optimization problem in which one seeks to minimize investment , operation cost , and total production time , and , simultaneously , to maximize the revenue . recall that not much work has been reported in the literature on the multiobjective optimal design of a multiproduct batch plant . huang and wang introduced a fuzzy decision - making approach for multiobjective optimal design problem of a multiproduct batch plant . a monotonic increasing or decreasing membership function is used to define the degree of satisfaction for each objective function and the problem is then represented as an augmented minmax problem formulated as minlp models . to obtain a unique solution , presented the development of a two - stage methodology for multiobjective batch plant design and retrofit according to multiple criteria . the authors used a multiobjective genetic algorithm based on the combination of a single - objective genetic algorithm and a pareto sort procedure for proposing several plant structures and a discrete event simulator for evaluating the technical feasibility of the proposed configurations . in the case of multiple objectives , an optimum solution with respect to all objectives may not exist . in most cases , the objective functions are in conflict , because in order to decrease any of the objective functions , we need to increase other objective functions . recently , solimanpur et al . developed a sophisticated multiobjective integer programming model where the objectives considered were the maximization of total similarity between parts , the minimization of the total processing cost , the minimization of the total processing time and the minimization of the total investment needed for the acquisition of machines . the presence of multiple objectives in a problem usually gives rise to a family of nondominated solutions , largely known as pareto - optimal solutions , where each objective component of any solution along the pareto front can only be improved by degrading at least one of its other objective components . since none of the solutions in the nondominated set is absolutely better than any other , any one of them is then an acceptable solution . as it is difficult to choose any particular solution for a multiobjective optimization problem without iterative interaction with the decision maker ( dm ) one general approach is to establish first the entire set of pareto - optimal solutions , where an external decision maker ( dm ) direct intervention gives interactive information in the multiobjective optimization loop . so , a satisfactory solution of the problem is found as soon as the knowledge is acquired . promethee ii ( preference ranking organisation method for enrichment evaluations2nd version ) is a popular decision method that has been successfully applied in the selection of the final solution of multiobjective optimization problems . it generates a ranking of available points , according to the dm preferences , and the best ranked one is considered the favourite final solution . it is based on the concept of outranking relation , which is a binary relation defined between every pair ( a , b ) of alternatives , in such way that , if a is preferred to b ( according to the dm interests ) , then it is said that a outranks b. when these relations are defined between all pairs of alternatives , they are exploited according to some rules in order to rank all solutions from the best to the worst . this is a nonpareto based approach based on the selection of several relevant groups of individuals , each group being associated to a given objective . it is reported that the method tends to crowd results at extremes of the solution space , often yielding to poor convergence of the pareto front . a more recent algorithm , based on scalarization with a weighted sum function , many successful evolutionary multiobjective optimization algorithms were developed based on the two ideas suggested by goldberg : pareto dominance and niching . pareto dominance is used to exploit the search space in the direction of the pareto front and niching technique explores the search space along the front to keep diversity . the well - known algorithms in this category include multiobjective genetic algorithm : ( moga ) , niched pareto genetic algorithm : ( npga ) , strength pareto evolutionary algorithm : ( spea ) , multiobjective evolutionary algorithm : ( moea ) , the nondominated sorting genetic algorithm ( nsga ) proposed by srinivas and deb was one of the first evolutionary algorithm for solving multiobjective optimization problems . although nsga has been successfully applied , the main criticisms of this approach has been its high computational complexity of nondominated sorting , lack of elitism , and need for specifying a tuneable parameter called sharing parameter . recently , deb et al . reported an improved version of nsga , which they called nsga - ii , to address all the above issues . the purpose of this study is to extend this methodology for solution of multiobjective optimal control problems under the framework of nsga - ii . the problem of multiproduct batch plant covered in this paper can be defined by assuming that the plant consists of a sequence of m batch processing stages that are used to manufacture n different products . at each stage j there are nj identical units in parallel operating out of phase , each with a size vj . batches are transferred from one stage to the next without any delay , that is , we consider a zero - wait operating policy . in the conventional design of a multiproduct batch plant , one seeks to minimize the investment cost by determining the optimal number , required volume and size of parallel equipment units in each stage for a specified production requirement of each product and the total production time . however , in reality the designer considers not only minimizing the investment but also minimizing the operation cost and total production time while maximizing the revenue , simultaneously ( 1)maxnj , vj , bi , tli , qi , h revenue = f1=i=1ncpiqi , ( 2)minnj , vj , bi , tli , qi , hinvestment cost = f2=j=1mnjjvjbj , ( 3)minnj , vj , bi , tli , qi , h operation cost = f3=i=1n j=1mcejqjbi+coiqi , ( 4)minnj , vj , bi , tli , qi , h total production time = f4=h . so , the multiobjective problem consists of determining the following parameters : nj the number of parallel units in stage j , vj the required volume of a unit in stage j , bi size of the batch of product i at the end of the m stages , tli the cycle time for product i , qi the production requirement of product i and , h the total production time , while satisfying certain constraints such as volume , time , and so forth . volume vj has to be able to process all the products i : ( 2 ) time constraint . the summation of available production time for all products is not more than the net total time for production ( 6)i=1nqibitlih . ( 3 ) the limiting cycle time for product i : ( 7)ijnjtli , i=1, ,n ; j=1, ,m . every unit has restricted allowable range ( 8)vjlvjvju , j=1, ,m , bjlbjbju , j=1, ,n . in contrast to the simple genetic algorithms that look for the unique solution , the multiobjective genetic algorithm tries to find as many elements of the pareto set as possible . for the case of the nsga - ii , this one is provided with operators who allow it to know the level of nondominance of every solution as well as the grade of closeness with other solutions ; which allows it to explore widely inside the feasible region . in a brief form , the functioning of the multiobjective genetic algorithm nsga - ii can be described through the following steps . fast nondominated sorta very efficient procedure , is used to arrange the solutions in fronts ( nondominated arranging ) , in accordance with their aptitude values . a domination count np , the number of solutions which dominates the solution p , and a set ( sp ) , that contains the solutions that are dominated for p. the solutions of the first front have the higher status of nondominance in the pareto sense . a very efficient procedure , is used to arrange the solutions in fronts ( nondominated arranging ) , in accordance with their aptitude values . a domination count np , the number of solutions which dominates the solution p , and a set ( sp ) , that contains the solutions that are dominated for p. the solutions of the first front have the higher status of nondominance in the pareto sense . diversity preservationthis is achieved , by means of the calculation of the crowding degree or closeness for each of the solutions inside the population . this quantity is obtained , by calculating the average distance of two points on either side of a particular solution along each of the objectives . this quantity serves as an estimate of the cuboid perimeter , formed by using the nearest neighbours as the vertices . there is also , an operator called crowded - comparison ( n ) , which guides to the genetic algorithm , towards the pareto optimal front , in accordance with the following criterion : ( 9)in j if ( irank < jrank),or ( irank = jrank ) and ( ididtance > jdistance ) . in accordance with the previous criterion , between two nondominated solutions , we prefer the solution with the better rank . otherwise , if both solutions belong to the same front , then , we prefer the solution that is located in a lesser crowded region . this is achieved , by means of the calculation of the crowding degree or closeness for each of the solutions inside the population . this quantity is obtained , by calculating the average distance of two points on either side of a particular solution along each of the objectives . this quantity serves as an estimate of the cuboid perimeter , formed by using the nearest neighbours as the vertices . there is also , an operator called crowded - comparison ( n ) , which guides to the genetic algorithm , towards the pareto optimal front , in accordance with the following criterion : ( 9)in j if ( irank < jrank),or ( irank = jrank ) and ( ididtance > jdistance ) . in accordance with the previous criterion , between two nondominated solutions , we prefer the solution with the better rank . otherwise , if both solutions belong to the same front , then , we prefer the solution that is located in a lesser crowded region . initial loopinitially , a random parent population ( po ) of size n is created . then the usual binary tournament selection , recombination and mutation operators are used to create a new population ( q0 ) , of size n. initially , a random parent population ( po ) of size n is created . then the usual binary tournament selection , recombination and mutation operators are used to create a new population ( q0 ) , of size n. main loopthe nsga - ii procedure can be explained , by describing the th generation just as it is showed in figure 1 . the procedure begins with the combination of pt and qt forming a new population called rt , then the population rt is sorted using the nondomination criterion . since all previous and current population members are included in rt , the population rt has a size of 2n , later , the different fronts of nondominated solutions are created , being f1 the front that contains the better rank solutions . figure 4 shows that , during the process of forming the new population pt+1 , the algorithm takes all members of the fronts f1 and f2 , and some elements of the front f3 ; this is , because n solutions are needed exactly for the new population pt+1 to find them exactly n solutions , the last front is ordained , which for this description is the number 3 , arranging the solutions in descending order by means of the crowded comparison ( n ) , and selecting the best solutions needed to fill all population slots . after having the population pt+1 , the genetic operators of selection , crossing and mutation , are used to create the new population qt+1 of size n. finally it is mentioned that the selection process , the crowded comparison operator is used . the nsga - ii procedure can be explained , by describing the th generation just as it is showed in figure 1 . the procedure begins with the combination of pt and qt forming a new population called rt , then the population rt is sorted using the nondomination criterion . since all previous and current population members are included in rt , the population rt has a size of 2n , later , the different fronts of nondominated solutions are created , being f1 the front that contains the better rank solutions . figure 4 shows that , during the process of forming the new population pt+1 , the algorithm takes all members of the fronts f1 and f2 , and some elements of the front f3 ; this is , because n solutions are needed exactly for the new population pt+1 to find them exactly n solutions , the last front is ordained , which for this description is the number 3 , arranging the solutions in descending order by means of the crowded comparison ( n ) , and selecting the best solutions needed to fill all population slots . after having the population pt+1 , the genetic operators of selection , crossing and mutation , are used to create the new population qt+1 of size n. finally it is mentioned that the selection process , the crowded comparison operator is used . to demonstrate the effectiveness of nsga - ii on batch plant processes two examples are given here . the first example is about a batch plant consisting of three processing stages ( mixer , reactor , and centrifuge ) to manufacture two products , a and b. the second example treats four processing stages ( mixer , reactor , extraction and centrifuge ) to manufacture three products a , b and c. the data for examples 1 and 2 are illustrated , respectively , in tables 1 and 2 ( the processing times , size factor for the units and cost for each product ) . the set of decision variables consists of the batch size , the total production time , the number of parallel units at each stage , the cycle time for each product , and the required volume of a unit in each stage . since the number of parallel units at each stage is an integer decision variable , we code this variable as a binary variable . in addition to the constraints expressed by ( 5)(8 ) , we consider bounds on objective functions as additional constraints to generate feasible nondominated solutions in the range desired by the decision - maker , to have 19 constraints in all ( 10)filfifiu , i = 1 ,4 . then nsga - ii is employed to solve the optimization problem with the following parameters : maximum number of generation up to 200 , population size 500 , probability of crossover 0.85 , probability of mutation 0.05 , distribution index for the simulated crossover operation 10 and distribution index for the simulated mutation operation 20 . the revenue ( f1 ) increases with the increase in operation cost ( f3 ) , while the investment cost ( f2 ) decreases . when all the four objective functions are considered simultaneously , solutions obtained in the present study show improvement as by huang and wang results for the same problem . for example , let us consider the solution presented by huang and wang with unit reference membership level for all objectives : f1 = 121 350 , f2 = 171 624 , f3 = 77 299 , f4 = 5667 . the solution ( 1 ) presented in table 3 of the present study improves the above solution f1 , f3 , f4 while f2 is comparable . the large set of multiple optimal solutions provides the decision maker with immediate information about the relationship among the several objective criteria and a set of feasible solutions . thus , it helps the decision - maker to select a highly confident choice of solution . the fixed optimal plant structure as 221 corresponds to a two mixers , two reactors , and one centrifuge design . the batch plant in this case consists of four processing stages to manufacture three products a , b , and c , with four - objective optimization problem as expressed in ( 1)(4 ) . but we deal with 4 integer variables , 14 real variables , and 31 constraints which includes bounds on objective functions . the same model equations of example 1 are used here except the processing time , ij in ( 7 ) . the time required to process one batch of product i in stage j is expressed as : ( 11)ij=ij+cijbij , i , j , where ij 0 , cij 0 and j are constants and bi is the batch size for product i. thus , the processing time is not a constant , but depends on the decision parameters of the batch size . the constrained multiobjective minlp problem is solved by nsga - ii with the same set of nsga - ii parameters as used in example 1 . as in example 1 , the revenue ( f1 ) increases as operation cost ( f3 ) increases , while the investment cost ( f2 ) decreases following operation cost ( f3 ) . let mention that when all four objective functions are considered simultaneously , the solutions obtained in the present study improve significantly the results presented by huang and wang for the same problem . for example , the solution presented by huang and wang with unit reference membership level for all objectives ( f1 = 274 312 , f2 = 375 688 , f3 = 175 688 , f4 = 5639 ) the solution ( 1 ) presented in table 4 of the present study improves the above solution f1 , f3 , f4 while f2 is comparable . in this example , the plant structure evolved as optimal is : two mixers , two reactors , two extractors , and one centrifuge as presented in figure 7 . implementation of a trade - off analysis is dependent upon the availability of the decision - maker 's preferences . a multiobjective decision in a batch plant process design is considered and a non dominating sorting genetic algorithm ( nsga - ii ) is developed to get an optimal zone containing solutions under the concept of pareto set . nsga - ii capability has been proved in evolving the entire set of nondominating solutions along the pareto front in a single run of the algorithm . thus , the decision maker ( dm ) is provided with the best trade - off operating zone . furthermore , a better confident choice of design among several compromises of the decision maker can be achieved if the decision variables effects on the objective functions are known . finally , the large set of solutions presents a useful base for further alternative approaches to fulfil the dm targets . the inherent dynamic nature of batch processes allows for their ability to handle variations in feedstock and product specifications and provides the flexibility required for multiproduct or multipurpose facilities . they are thus best suited for the manufacture of low - volume , high - value products , such as specialty chemicals , pharmaceuticals , agricultural , food , and consumer products , and most recently the constantly growing spectrum of biotechnology - enabled products . reduced time to market , lower production costs , and improved flexibility are all critical success factors for batch processes .

introduction : the objective of this study was to evaluate dentinal defects formed by new rotary system protaper next ( ptn).materials and methods : sixty single - rooted premolars were selected . all specimens were decoronated and divided into four groups , each group having 15 specimens . group i specimens were prepared by hand k - files ( mani ) , group ii with protaper universal ( pt ; dentsply maillefer ) , group iii with hero shaper ( hs ; micro - mega , besancon , france ) , and group iv with ptn ( dentsply maillefer ) . roots of each specimen were sectioned at 3 , 6 , and 9 mm from the apex and were then viewed under a stereomicroscope to evaluate presence or absence of dentinal defects.results:in roots prepared with hand files ( hfs ) showed lowest percentage of dentinal defects ( 6.7% ) ; whereas in roots prepared with pt , hs , and ptn it was 40 , 66.7 , and 26.7% , respectively . there was significant difference between the hs group and the ptn group ( p < 0.05).conclusion : all rotary files induced defects in root dentin , whereas the hand instruments induced minimal defects . optimal design problem are widely known by their multiple performance measures that are often competing with each other . in this paper , an optimal multiproduct batch chemical plant design is presented . the design is firstly formulated as a multiobjective optimization problem , to be solved using the well suited non dominating sorting genetic algorithm ( nsga - ii ) . the nsga - ii have capability to achieve fine tuning of variables in determining a set of non dominating solutions distributed along the pareto front in a single run of the algorithm . the nsga - ii ability to identify a set of optimal solutions provides the decision - maker dm with a complete picture of the optimal solution space to gain better and appropriate choices . then an outranking with promethee ii helps the decision - maker to finalize the selection of a best compromise . the effectiveness of nsga - ii method with multiojective optimization problem is illustrated through two carefully referenced examples .

since the introduction of extra - oral implants in reconstruction of craniofacial defects , achieving proper prosthesis retention has become more promising . these problems include ulceration of hard and/or soft tissues used for retention , lack of retention due to prosthesis movement , and tissue irritation caused by adhesives . the ideal position and number of implants for restoring orbital defects would be three non - linear implants in lateral , supraorbital , and infra - orbital rims . however , such implant arrangement is not always conceivable considering the extension of the defect , and bone quality and quantity of defect s walls . two of the most common retention systems used in reconstruction of orbital defects include freestanding abutments with magnetic retention and bar - clip retention . magnetic abutments are more common because they resolve the potential problems associated with bar - clip attachment including difficulty in insertion and removal of prosthesis by the patient , difficulty in regular hygiene measurements , and rigidity of the attachment resulting in implant overloading . however , magnetic attachment might not provide sufficient retention if implants have been placed adjacently . the presence of implant in the defective area might complicate the usual impression - taking procedures used in fabrication of conventional craniofacial prostheses . accuracy of the impression is affected by defect shape , retention system , number , and divergence of the implants . moreover , anatomical undercuts in the defect , and proximity or remoteness of the implants could complicate the impression - taking procedure . use of multiple trays , elastomeric impression materials , and dual impression technique have been suggested to overcome such problems [ 2,1214 ] . the purpose of this article was to present a case treated with an implant - supported prosthesis to reconstruct a relatively large orbital defect using three adjacent implants in the lateral orbital rim . a 60-year - old woman with a left orbital defect due to removal of periocular basal cell tumor was referred to the implant department of tehran university of medical sciences , school of dentistry , for prosthetic reconstruction of the eye . three implants ( superline , dentium , seoul , south korea ) , 8 mm in length and 3.6 mm in diameter were placed in the lateral rim of the orbit . although the most suitable sites for orbital implants are the superior and lateral rims , in the present case the implants have been placed adjacently , due to insufficient bone thickness in superior and inferior orbital rims . the defect was relatively deep with undercuts in the medial wall which could complicate impression making . the preferred prosthesis design was an implant - supported prosthesis with a custom bar containing properly distributed magnetic components . the healing abutments were unscrewed and three hexed direct - casting abutments ( implantium , dentium , seoul , south korea ) with 4.5 mm diameter were directly secured to the implants . the medial undercuts were blocked out , using a gauze pack to avoid the penetration of acrylic resin . an auto - polymerizing acrylic resin ( pattern resin , gc , tokyo , japan ) pattern was formed directly on the abutments in a manner that cobalt samarium ( co5sm ) magnets ( implantium , dentium , seoul , south korea ) , with 5.5 mm diameter and retention force of 700 gram could be placed at proper distances in the superior , inferior and lateral segments of the acrylic bar ( fig . the acrylic resin bar was casted using base metal alloy ( aalba dent inc . ; cordelia , c.a , usa ) and the magnet keepers were cemented in corresponding sites with panavia f 2.0 resin cement ( kurary medical inc , japan ) . acrylic resin pattern of bar containing indentations for magnets ( a ) , try - in of metal bar on the implants with magnet keepers in place ( b ) . the space beneath the superstructure and also the undercuts in defect walls were blocked out with gauze packs . the final impression was made in order to pick up the magnets and simultaneously record the rest of the orbital defect . light viscosity addition silicone ( panasil , kettenbach , germany ) was used as the first layer to cover the entire defect as well as the intact side of the midface . afterwards , regular viscosity addition silicone ( panasil , kettenbach , germany ) was used over the light viscosity material to create mechanical retention projections for the gypsum layer ( herostone vigodent inc . the wax pattern of the orbit was formed containing an ocular prosthesis which simulated the properties of a healthy eye . the pattern was tried on the patient and some modifications were made to improve its esthetic and adaptation . the prosthesis was made of a combination of heat - cured acrylic resin for holding the magnets , and high - temperature vulcanizing silicone with internal / external staining and other characterizations of the skin , such as wrinkles , eye brow and eye lashes . the final prosthesis was delivered to the patient and necessary home care instructions were provided such as removing the prosthesis during night , cleaning the eye defect with damp gauze , and the need for regular biannual follow - ups [ 69 ] . tissue side of the prosthesis with three magnets ( a ) , delivery of the prosthesis ( b ) the patient presented here has been treated with an implant - supported orbital prosthesis with bar - magnetic attachment . this retention mechanism might minimize the risk of mechanical overload on the implants compared to a conventional bar - clip attachment with cantilever arms . despite the proximity of implants , the mentioned distribution of magnetic attachments has increased the retention through creating a tripod . furthermore , since the acrylic resin pattern of the bar was made directly in the defective area , no implant or abutment analogues were used in final impression procedure . prolonged chair - side time is a disadvantage of the stated method which could be justified considering the mentioned advantages . hepatocellular carcinoma ( hcc ) is one of the most common cancers worldwide especially in china [ 13 ] . the high hcc rates in china largely reflect the elevated prevalence of chronic hepatitis b virus ( hbv ) infection . in developed countries such as united states , nonalcoholic fatty liver disease ( nafld ) , associated with obesity , less study focused on the effect of obesity on hcc especially in the relationship between bmi and hcc . no study has focused on the effect of waistline on the surgical outcome of hcc patients . so , this study is about the influence of body mass index ( bmi ) and waistline on complications , postoperative death and long - time survival in patients undergoing surgery for hcc . between january 2007 and december 2012 , we retrospectively reviewed the database of 136 patients underwent hepatic resection for hcc at the department of surgery of the fourth affiliated hospital of zhejiang university school of medicine . this retrospective study had been performed with the approval of the ethics committee of the fourth affiliated hospital of zhejiang university school of medicine and was in compliance with the helsinki declaration . written informed consent was given by participants for their clinical records to be used in this study . we enrolled 136 patients and divided them into 4 groups : group a , bmi < 25 ; group b , bmi 25 ; group c , waistline < 90 cm in males or waistline < 80 cm in females ; group d , waistline 90 cm in males or waistline 80 cm in females . there were 61 patients ( 44.9% ) in group a , 75 patients ( 55.1% ) in group b , 58 patients ( 42.6% ) in group c , and 78 patients ( 57.4% ) in group d. all patients followed the same preoperative evaluation protocol , including blood biochemistry , ultrasonography , and computed tomography of the liver . the criteria for resection included good general condition , absence of essential organ dysfunction and distant metastasis , and liver function ( child a or b , icg r15 < 15% ) . follow - up data were obtained by direct communication with patients after they underwent hepatic resection . survival analysis , including overall survival and disease - free survival , was estimated by the kaplan - meier survival method and compared using the log - rank test . all statistical analyses were performed using statistical software ( spss 13.0 for windows , spss , chicago , il ) at a significant level of p<0.05 . there were 110 ( 80.9% ) men and 26 ( 19.1% ) women who underwent hepatectomy . the median age was 43 years ; 118 ( 86.8% ) patients were ages < 60 years and 18 ( 13.2% ) patients were older than 60 years . there were 61 ( 44.9 ) patients with normal bmi ( < 25 ) and 75 ( 55.1% ) patients were overweight or obese ( bmi 25 ) . fifty - eight patients ( 42.6% ) belong to group c and 78 patients ( 57.4% ) belong to group d. ninety - seven ( 71.3% ) patients had positive hepatitis b and 39 ( 28.7% ) patients without hbv . histopathology of the hcc confirmed vascular invasion in 41 patients ( 30.1% ) and liver cirrhosis in 65 ( 47.8% ) patients . surgical results confirmed capsular formation in 54 ( 39.7% ) patients and 51 ( 37.5% ) patients with solitary hcc ( table 1 ) . the intraoperative and postoperative data including major complications of 136 patients with hcc are shown in table 2 and supplementary table 1 . the median hospital stay was 11 days in group a and 12 days in group b , with no significant difference between the 2 groups ( p=0.373 ) . a , 23 ( 37.7% ) patients had blood loss 1000 ml compared to 29 ( 38.7% ) patients in group b. there was no hospital death in group a and 1 ( 1.3% ) hospital death in group b. however , no significant difference was found between the 2 groups . there were no significant differences in overall postoperative complication rate between group a and group b , although pulmonary infection showed a significant difference between these 2 groups ( p=0.017 ) . seven ( 11.5% ) patients had complications in group a compared to 9 ( 12% ) patients in group b. the most common complications in group a were ascites and wound infection , while the most common complications in group b were pulmonary infection and wound infection . variables that might affect overall survival of patients with hcc after hepatic resection were analyzed in this study . on univariate analysis of the prognostic factors , we found that patients with bmi 25 , waistline 90 cm ( or 80 cm in women ) and vascular invasion had poor overall survival than that without these variables ( p=0.032 , p=0.017 , p=0.009 ) . the other variables such as sex , afp level , and blood loss were not the significant prognostic factors of overall survival . on multivariate analysis , however , only vascular invasion and waistline were significantly associated with overall survival ( p=0.031 , p=0.039 ) . the other variables , including bmi , were not independent prognostic factors of overall survival on multivariate analysis ( table 3 ) . the overall survival curves of hcc patients according to the bmi are shown in figure 1 . the 1- , 3- , and 5-year overall survival rates of group a were 95% , 81% , and 16% , respectively . the 1- , 3- , and 5-year disease - free survival rates of these patients were 78% , 38% and 4% , respectively ( figure 1 ) . however , there was no significant difference between the 2 groups in disease - free survival ( p=0.235 ) . the overall and disease - free survival curves of hcc patients affected by waistline were shown in figure 2 . the 1- , 3- , and 5-year overall survival rates of group c were 97% , 88% , respectively , and 15% compared to 96% , 68% , 15% , respectively , in group d. the 1- , 3- , and 5-year disease - free survival rates of group c were 84% , 40% and 6% , respectively . group c had significant better overall survival and disease - free survival than group d ( p=0.028 , p=0.048 ) . there were 110 ( 80.9% ) men and 26 ( 19.1% ) women who underwent hepatectomy . the median age was 43 years ; 118 ( 86.8% ) patients were ages < 60 years and 18 ( 13.2% ) patients were older than 60 years . there were 61 ( 44.9 ) patients with normal bmi ( < 25 ) and 75 ( 55.1% ) patients were overweight or obese ( bmi 25 ) . fifty - eight patients ( 42.6% ) belong to group c and 78 patients ( 57.4% ) belong to group d. ninety - seven ( 71.3% ) patients had positive hepatitis b and 39 ( 28.7% ) patients without hbv . histopathology of the hcc confirmed vascular invasion in 41 patients ( 30.1% ) and liver cirrhosis in 65 ( 47.8% ) patients . surgical results confirmed capsular formation in 54 ( 39.7% ) patients and 51 ( 37.5% ) patients with solitary hcc ( table 1 ) . the intraoperative and postoperative data including major complications of 136 patients with hcc are shown in table 2 and supplementary table 1 . the median hospital stay was 11 days in group a and 12 days in group b , with no significant difference between the 2 groups ( p=0.373 ) . a , 23 ( 37.7% ) patients had blood loss 1000 ml compared to 29 ( 38.7% ) patients in group b. there was no hospital death in group a and 1 ( 1.3% ) hospital death in group b. however , no significant difference was found between the 2 groups . there were no significant differences in overall postoperative complication rate between group a and group b , although pulmonary infection showed a significant difference between these 2 groups ( p=0.017 ) . seven ( 11.5% ) patients had complications in group a compared to 9 ( 12% ) patients in group b. the most common complications in group a were ascites and wound infection , while the most common complications in group b were pulmonary infection and wound infection . variables that might affect overall survival of patients with hcc after hepatic resection were analyzed in this study . on univariate analysis of the prognostic factors , we found that patients with bmi 25 , waistline 90 cm ( or 80 cm in women ) and vascular invasion had poor overall survival than that without these variables ( p=0.032 , p=0.017 , p=0.009 ) . the other variables such as sex , afp level , and blood loss were not the significant prognostic factors of overall survival . on multivariate analysis , however , only vascular invasion and waistline were significantly associated with overall survival ( p=0.031 , p=0.039 ) . the other variables , including bmi , were not independent prognostic factors of overall survival on multivariate analysis ( table 3 ) . the overall survival curves of hcc patients according to the bmi are shown in figure 1 . the 1- , 3- , and 5-year overall survival rates of group a were 95% , 81% , and 16% , respectively . the 1- , 3- , and 5-year disease - free survival rates of these patients were 78% , 38% and 4% , respectively ( figure 1 ) . however , there was no significant difference between the 2 groups in disease - free survival ( p=0.235 ) . the overall and disease - free survival curves of hcc patients affected by waistline were shown in figure 2 . the 1- , 3- , and 5-year overall survival rates of group c were 97% , 88% , respectively , and 15% compared to 96% , 68% , 15% , respectively , in group d. the 1- , 3- , and 5-year disease - free survival rates of group c were 84% , 40% and 6% , respectively . group c had significant better overall survival and disease - free survival than group d ( p=0.028 , p=0.048 ) . there are many reports about the relationship between bmi , the size of waistline , and cancer risk [ 714 ] . research conducted by the cancer council of victoria , australia shows that a waist measurement of over 100 cm for men and over 85 cm for women can significantly increase the risk of cancer including breast , bowel , and aggressive prostate cancer . however , there were few reports about the relationship between bmi , waistline , and hcc . the high hcc rates in china are largely due to the prevalence of chronic hepatitis b virus infection . however , with the increase in prevalence of obesity , nafld , which can progress to cirrhosis and subsequently to the development of hcc , is now recognized as one of the most common liver diseases in china . although many reports had affirmed that patient with higher bmi tend to show a higher incidence of liver cirrhosis , there are few reports about the relationship between the bmi and the surgical outcome of hepatectomy for hcc [ 1517 ] . regarding the relationship between bmi , waistline , and obesity , we hypothesized that waistline might be related to surgical outcome of hepatectomy for hcc , as well . in this study , there were no significant differences in postoperative complication rate and postoperative death between group a and group b. however , pulmonary infection ( 13.3% ) showed a significant difference between the 2 groups ( p=0.017 ) . the observation that the obese patients had more pulmonary infection after hepatectomy for hcc can be explained by the fact that obesity can reduce pulmonary function through diminishing exercise capacity and lung volume , and increasing the resistance of breathing . vascular invasion , waistline , and bmi are the independent prognostic factors for long - term survival . group c had better overall and disease - free survival than group d , and group a had the better overall survival than group b. this result showed that bmi and waistline are both independent prognostic factors for long - term survival . waistline may be more important than bmi in predicting the prognosis of hcc after hepatectomy . obesity , which can lead to nafld and finally progress to cirrhosis , is thought to account for the majority of hcc [ 1821 ] . with the increase in prevalence of obesity , we recommend that individuals whose bmi or waistline exceeds normal limits should cultivate a healthy lifestyle , such as eating foods that help to lose weight , regular physical exercise , and getting enough sleep . in conclusion , overweight and obesity have been shown to be significant predictors of an adverse long - time survival after hepatectomy for hcc . waistline is more important than bmi in predicting the disease - free survival of hcc after hepatectomy .

implant - supported craniofacial prostheses are made to restore defective areas in the face and cranium . this clinical report describes a technique for fabrication of an orbital prosthesis with three adjacent implants in the left lateral orbital rim of a 60-year - old woman . selection of appropriate attachment system ( individual magnetic abutments versus bar - clip attachment ) for implant - supported orbital prostheses depends upon the position of implants . bar - magnetic attachment has been selected as the retention mechanism in the present case . backgroundhepatocellular carcinoma ( hcc ) is one of the most common cancers worldwide especially in china . this article aimed to evaluate the influence of body mass index ( bmi ) and waistline on complications , postoperative death , and long - term survival in patients undergoing surgery for hcc.material/methods136 patients were enrolled and divided into 4 groups : group a , bmi < 25 ; group b , bmi 25 ; group c , waistline < 90 cm in males or waistline < 80 cm in females ; group d , waistline 90 cm in males or waistline 80 cm in females . clinical pathological features and surgical outcomes of these patients were analyzed retrospectively.resultsthere were no significant differences in postoperative complication rate and postoperative death between group a and group b , although pulmonary infection showed a significant difference between 2 groups ( p=0.017 ) . vascular invasion , waistline , and bmi are the independent prognostic factors for long - term survival . the disease - free survival curves after hepatectomy showed no statistically significant difference between group a and group b. group c had the better overall survival than group d , and group a had the better overall survival than group b.conclusionsbmi and waistline are both independent prognostic factors for long - term survival of hcc after hepatectomy . waistline is more important than bmi in predicting the disease - free survival of hcc after hepatectomy .

professional mononuclear phagocytes , such as polymorphonuclear neutrophils ( pmn ) , monocytes , and macrophages , are considered as the first line of defence of the early host innate immune response [ 1 , 2 ] . their main function has been classically understood to kill invasive pathogens by a variety of potent intracellular microbicidal effector mechanisms [ 37 ] . after the first contact with pathogens , mononuclear phagocytes engulf and internalize them into their phagosomes . by the fusion with intracellular granules and the formation of phagolysosomes the pathogens may be killed intracellularly by a combination of non - oxidative and oxidative mechanisms [ 1 , 8 ] . actions of potent antimicrobial peptides , such as defensins , cathelicidins , cathepsins , pentraxin , and lactoferrin , are parts of non - oxidative killing mechanisms , while oxidative killing relies exclusively on the production of antimicrobial reactive oxygen species ( ros ) via the nadph oxidase ( nox ) complex . within blood circulating phagocytes , pmn are by far the most abundant cell population representing 5080% of the total white blood cells in different vertebrates . moreover , after being released from the bone marrow into the blood circulation , pmn are highly mobile and short - lived phagocytes , being densely packed with secretory granules [ 4 , 8 ] . pmn granules are categorized into three different types based on their contents : primary ( azurophilic ) , secondary ( specific ) , and tertiary ( gelatinase ) granules . the types of granules to be found in circulating pmn depend on their maturation stage . thus , pmn maturation starts with the formation of primary granules , followed by secondary and tertiary granules [ 4 , 9 , 10 ] . the content of primary granules includes myeloperoxidase ( mpo ) , neutrophil elastase ( ne ) , cathepsin g , proteinase 3 , defensins , and lysozyme ; secondary granules contain collagenase , gelatinase , cystatin , lysozyme , and lactoferrin ; and tertiary granules comprise gelatinase , lysozyme , and arginase amongst others . following granule maturation , pmn will possess all three types of granules displaying full killing capacity not only in the blood but also in tissues / organs and gut lumen . in addition , pmn act against pathogens by actively participating in complex inflammatory networks such as the release of a broad panel of proinflammatory chemokines , cytokines , and survival- and growth - factors which trigger both downstream proinflammatory effects and the transition into adaptive immune reactions . as such , several proinflammatory cytokines / chemokines were found enhanced in activated pmn in response to apicomplexan parasites , such as tnf- , il-1 , cc , and cxc chemokines ( e.g. , il-8 , ip-10 , gro- , rantes , and mip-1 ) [ 1115 ] . several of pmn - derived immunomodulatory molecules can augment the production of various chemokines and cytokines to further regulate phagocyte functions [ 16 , 17 ] . more importantly , by this means activated pmn recruit not only other innate immune cells but also t cells to the site of infection [ 1820 ] or even induce sterile inflammation [ 21 , 22 ] . beginning with the landmark study of brinkmann et al . , the paradigm of how pmn fight and kill pathogenic bacteria has profoundly been changed . the discovery of dna - based antimicrobial neutrophil extracellular traps ( nets ) changed the current knowledge on innate immune reactions not only on the level of the pathogen killing but also on the pathophysiology of metabolic , autoimmune , reproductive , and inflammatory diseases , as well as cancer progression [ 3237 ] . nets are released by activated pmn by a novel cell death process , called netosis , which can be stimulated by a variety of molecules and invasive pathogens . microorganisms such as bacteria [ 31 , 3941 ] , fungi [ 4244 ] , viruses [ 4549 ] , and parasites [ 5055 ] were identified as net inducers . also different molecules or cellular structures such as gm - csf / complement factor 5a [ 56 , 57 ] , activated platelets [ 40 , 58 ] , pma and zymosan [ 24 , 26 , 31 , 59 ] , singlet oxygen , lps [ 31 , 61 ] , and fc receptor trigger netosis . in addition , il-8 as well - known chemoattractant for pmn was demonstrated as net inducer [ 31 , 62 ] . efficient netosis requires mature pmn and in most cases nox , mpo , ne , and peptidylarginine deiminase type iv ( pad4 ) activities [ 14 , 24 , 59 , 6365 ] . furthermore , the process of netosis obviously requires intracellularly signalling pathways of which raf - mek - erk kinases as well as p38 mapk are being the most frequently reported to be involved in this process [ 14 , 23 , 33 , 6669 ] . in addition , calcium release is needed for optimal net formation in different vertebrate species [ 14 , 23 , 7072 ] . upon stimulation of pmn , the nuclear envelope disintegrates permitting the mixture of chromatin with granular proteins / peptides . ne and mpo degrade histones ( h1 , h2a / h2b , h3 , and h4 ) and promote chromatin decondensation , mediated by pad4 via hypercitrullinating of specific histones to allow electrostatic coiling of the chromatin [ 64 , 73 , 74 ] . the total of the dna complexes being decorated with granular proteins / peptides and specific histones ( h1 , h2a / h2b , h3 , and h4 ) are finally extruded as nets to the extracellular environment by dying pmn . net formation is primarily a nox - dependent mechanism [ 14 , 24 , 59 , 75 , 76 ] . however , nox - independent netosis was also reported [ 29 , 60 , 67 , 68 , 77 ] . this mode of netosis is accompanied by a substantially lower level of erk activation and rather moderate level of akt activation , whereas activation of p38 is similar in both kinds of net formation [ 67 , 68 ] . as an example irrespectively of nox - dependency , pathogens may either be immobilised within sticky dna fibres [ 55 , 78 , 79 ] or be killed via the local high concentration of effector molecules [ 31 , 42 , 51 , 53 ] . meanwhile , other types of leukocytes of the innate immune system , such as macrophages [ 8083 ] , monocytes [ 26 , 28 ] , mast cells [ 84 , 85 ] , eosinophils [ 55 , 86 , 87 ] , and also basophils , have been reported to release net - like structures which are now collectively entitled as extracellular traps ( ets ) . described already many years ago that enucleated pmn may remain vital and are even capable of killing invasive microbes . more recent studies corroborated these findings proving that leukocytes do not necessarily die after et extrusion [ 56 , 68 , 86 ] . in this context , yousefi et al . [ 56 , 86 ] demonstrated that eosinophils and certain pmn subpopulations release ets of mitochondrial origin without dying . furthermore , yipp et al . verified that pmn which had released nets were still viable and retained their capability to engulf bacteria via phagocytosis . however , it appears to be nonlethal for pmn and faster than nox - dependent net formation and to rely on a vesicular - based pathway releasing nuclear dna [ 33 , 68 ] . additionally , different molecular pathways will lead in a stimulus - dependent manner to the extrusion of different types of ets in vitro and in vivo . different morphological forms of ets were for the first time described in the human gout disease in vivo proving that monosodium urate crystals ( msu ) induced aggregated ( aggets ) , spread ( sprets ) , and diffused ( diffets ) et formation . as such , the parasitic nematode haemonchus contortus larvae triggered in ruminant pmn and eosinophils aggets , spreets , and diffets . while most net- and et - related studies focused on bacterial , viral , and fungal pathogens , little attention was paid to protozoan parasites . as such , the first ever published study on parasite - triggered netosis was published in 2008 by baker et al . 4 years after the discovery of this novel effector mechanism and reported on plasmodium falciparum - triggered net formation . parasites are mosquito - borne pathogens that cause malaria , a serious public health disease worldwide in the tropic and subtropics . globally , an estimated 3.3 billion people are at risk of being infected with malaria of whom approximately 1.2 billion are at high risk ( > 1 in 1000 chance ) of developing malarial disease . the first report on p. falciparum - induced nets referred to p. falciparum - infected children and demonstrated in vivo net - entrapped trophozoite - infected erythrocytes in blood samples . moreover , baker and colleagues provided first evidence on the involvement of parasite - triggered nets in the pathogenesis of malaria since the high levels of anti - dsdna antibodies were above the predictive levels for autoimmunity . interestingly , a recent study also indicates the capacity of p. falciparum to inhibit net formation which may be of relevance in immunopathogenesis . thus , a mosquito - derived salivary protease inhibitor ( agaphelin ) induced by p. falciparum infection inhibited vertebrate elastase and net formation . whether this represents a true anti - net mechanism remains to be elucidated . parasites of the genus eimeria are worldwide of high veterinary and economic importance in livestock , especially in chicken , cattle and small ruminants [ 95100 ] . coccidiosis is a disease with high morbidity in animals of all ages , nonetheless inducing pathogenicity especially in young animals and occasionally causing death of heavily infected animals [ 99 , 102 , 103 ] . several studies showed that pmn infiltrate intestinal mucosa in response to eimeria infections and are occasionally found in close contact to the parasitic stages in vivo [ 102 , 104107 ] . pmn have also been shown to directly interact with e. bovis stages and antigens in vitro , resulting in release of proinflammatory cytokines , chemokines , and inos . additionally , their phagocytic and oxidative burst activities were enhanced in response to eimeria stages in vitro and in vivo . first indications on eimeria spp . as potent net inducers came from behrendt and colleagues who reported on sporozoites to be entangled by an extracellular network of delicate dna fibres being extruded from pmn in vitro ( figure 1(a ) ) . using extracellular dna measurements and dnase treatments other studies confirmed typical characteristics of nets , such as the colocalization of ne , mpo , and histones in the dna backbone of eimeria - induced net - like structures . meanwhile , also other pathogenic ruminant eimeria species were shown to induce netosis , such as e. arloingi ( figures 2(a ) and 2(b ) ) [ 24 , 27 ] and e. ninakohlyakimovae ( prez , personal communication ) . importantly , muoz - caro and colleagues proved nets also to occur in vivo in eimeria - infected gut mucosa . the current data suggest that eimeria - induced netosis is a species- and stage - independent mechanism , since it was induced by sporozoites , merozoites i , or oocysts of different eimeria species [ 23 , 24 ] . given that pmn were described to act even in the intestinal lumen via different effector mechanisms [ 27 , 108 , 109 ] , it appears likely that interactions of luminal pmn with ingested eimeria oocysts or newly excysted sporozoites may occur [ 6 , 23 , 24 ] . in particular , net - related reactions against oocysts would have a high impact on the ongoing infection since they may hamper proper excystation of infective stages ( sporozoites ) and , in consequence , dampen the degree of infection at the earliest possible time point in the host . since e. arloingi sporozoites must egress from the oocyst circumplasm through the micropyle , nets covering this area of the oocyst will have a detrimental effect on proper excystation [ 6 , 24 ] . the same explanation seems feasible for e. bovis and e. ninakohlyakimovae , regardless of the fact that excystation occurs by rupture of the oocyst walls prior to sporozoites egress from sporocysts . although all eimeria species tested so far equally induced nets , significant differences in entrapment effectivity were reported amongst different host species , parasite species , and stages . thus , caprine nets immobilised a high proportion of e. arloingi sporozoites ( 72% ) , whilst in the bovine system considerably less parasite stages ( e. bovis sporozoites : 43% , b. besnoiti tachyzoites : 34% ) were found entrapped in net structures [ 23 , 59 ] . so far , it remains to be elucidated whether the varying effectivity of nets is based on the pmn origin ( goats are generally considered as strong immune responders ) or on the parasite species . the molecular basis of eimeria - induced netosis is not entirely understood , so far . enzyme activity measurements and inhibition studies revealed a key role of nox , ne , and mpo in eimeria - triggered net formation ( see table 1 ) which is in agreement to bacterial , fungal , and parasitic pathogens [ 14 , 25 , 59 , 65 , 75 , 110 ] . referring to signal cascades , analyses on the grade of phosphorylation revealed a key role of erk1/2 and p38 mapk in sporozoite - exposed bovine pmn . since respective inhibitor experiments led to decreased parasite - mediated net formation , muoz - caro et al . this finding is in agreement with data on t. gondii - mediated net formation . referring to ca influx , further inhibition experiments proved e. bovis - mediated netosis as dependent on intracellular ca mobilization , since 2-abp ( inhibitor of store - operated ca entry ) and bapta - am ( binding intracellular ca ; muoz - caro , unpublished data ) but not egta ( inhibitor of ca influx from the extracellular compartment ; muoz - caro , unpublished data ) significantly blocked parasite - triggered netosis . so far , . reported on enhanced cd11b surface expression on pmn following e. bovis sporozoite exposure . by antibody - mediated cd11b blockage leading to a significant reduction of parasite - triggered netosis bacteria and fungi netosis was reported as a lethal effector mechanism [ 31 , 42 ] . however , killing effects of nets were not observed in the case of eimeria spp . so far . given that eimeria spp . are obligate intracellular parasites , the main function of nets rather seems to be the extracellular immobilisation of infective stages hampering them from host cell invasion . accordingly , reduced host cell infections rates were reported for e. bovis and e. arloingi sporozoites when previously exposed to pmn [ 23 , 24 ] . the same feature was reported for monocyte - preexposed e. bovis sporozoites indicating that this leukocyte cell type also casts ets in response to this parasite stage and that etosis had an impact on parasite invasion . besides e. bovis , e. arloingi ( silva , unpublished data ) , and e. ninakohlyakimovae ( prez et al . , submitted manuscript ) were also shown to induce monocytes - derived ets . furthermore , e. ninakohlyakimovae - induced monocytes - etosis showed a rapid induction of ets release upon viable sporozoites , sporocysts , and oocysts encounters , corroborating a stage - independent process in monocyte - derived etosis . in addition , it was found that caprine monocyte - derived - etosis is nox - dependent . with the upregulation of the genes transcription encoding for il-12 and tnf- , relevant immunoregulatory cytokines with transition properties into the adaptive immunity were also demonstrated in e. ninakohlyakimovae - exposed caprine monocytes ( prez et al , submitted manuscript ) . since the reduction in infection rates early after infection automatically results in decreased proliferation of the parasite , this indirect et - mediated effect should have a beneficial impact on the outcome of the disease . despite advantageous properties of ets , their ineffective clearance and/or poor regulation might also bear adverse pathological implications , leading to tissue damage in addition to enhanced local proinflammatory reactions [ 112 , 113 ] . toxoplasmosis is caused by the facultative heteroxenous apicomplexan polyxenous protozoan t. gondii representing one of the most common parasitic zoonoses worldwide . toxoplasma gondii is well known to affect almost all warm - blooded mammals including a wide range of domestic animals , wild mammals , marine mammals , marsupials , and humans [ 115 , 116 ] . in response to t. gondii infections , pmn are promptly recruited to the site of infection producing a variety of proinflammatory cytokines and chemokines [ 11 , 117 ] . in addition , pmn are capable of killing t. gondii tachyzoites via phagocytosis [ 118 , 119 ] . besides this effector mechanism , human , murine , bovine , and harbour seal ( phoca vitulina ) pmn additionally perform netosis in reaction to t. gondii tachyzoites ( figures 1(c ) and 1(d ) ) [ 25 , 26 ] . abi abdallah et al . showed that netosis was triggered by tachyzoites in a parasite strain - independent fashion as an invasion / phagocytosis - independent process . interestingly , in the murine toxoplasmosis model , tachyzoites - induced nets were not the result of a random cell lysis , but of a controlled dna release process since lysozyme was still present in pmn after performing netosis [ 25 , 120 ] . in contrast to eimeria spp . , t. gondii - triggered netosis had modest toxoplasmacidal effects by killing up to 25% of the parasites . considering the obligate intracellular life style of t. gondii and its enormous proliferative capacity in mammalian host cells , parasite entrapment via nets might be of particular importance in vivo based on its interference with host cell invasion . consistently , harbour seal pmn - promoted nets significantly hampered host cell invasion of t. gondii tachyzoites in vitro . in vivo evidence of t. gondii - induced netosis was reported in a murine pulmonary infection model , revealing an increase of dsdna contents in the bronchoalveolar lavage fluids of t. gondii - infected mice . as equally reported for several other coccidian parasites [ 14 , 23 ] , t. gondii - induced nets were also proven to be nox- , ne- , mpo- , and ca- ( soce ) dependent and to be mediated by an erk 1/2-related signalling pathway in pmn ( see table 1 ) [ 25 , 26 ] . additionally , in earlier studies , not only the pivotal role of pmn but also the important role of monocytes in toxoplasmosis was clearly demonstrated [ 121123 ] ; however , their capacity to also induce ets in response to tachyzoite stages was just recently demonstrated . exposure of harbour seal - derived monocytes to viable t. gondii tachyzoites resulted in a significant induction of monocyte - ets and tachyzoites were firmly entrapped and immobilised within harbour seal monocyte - et structures , hampering parasite replication . bovine besnoitiosis caused by besnoitia besnoiti is an endemic disease in africa and asia [ 124126 ] and considered as emergent in europe . during the acute phase of cattle besnoitiosis , b. besnoiti tachyzoites mainly replicate in host endothelial cells of different organs [ 28 , 128 ] and , upon release , may be exposed to circulating leukocytes . besnoitia besnoiti tachyzoites were recently reported as effective inducers of pmn- and monocyte - derived ets ( figures 1(e ) , 1(g ) , and 1(h ) ) [ 28 , 59 ] . in the latter case , a high proportion of pmn was found to be involved in netosis , since up to 76% of encountered pmn were found to participate in netosis leading to the immobilisation of approximately one - third of the parasites . besnoitia besnoiti - triggered netosis furthermore proved as vitality - independent process that was even induced by soluble parasite molecules ( homogenates ) , though at lower levels . regarding pmn - derived effector molecules , nox , ne , and mpo proved as essential for efficient b. besnoiti - triggered netosis . thus , respective enzyme activities were encountered in tachyzoite - exposed pmn and chemical blockage of these enzymes via inhibitors blocked parasite - triggered netosis [ 28 , 59 ] . in contrast to tachyzoites of t. gondii , entrapped b. besnoiti tachyzoites were neither killed by nets nor ets since their host cell infectivity was entirely restored upon dnase i treatments [ 28 , 59 ] . given that b. besnoiti tachyzoites mainly proliferate within endothelial cells during the acute phase , these parasitic stages are released via cell lysis in close proximity to endothelium and are exposed to blood contents , such as leukocytes . several reports have shown that nets themselves interact with endothelium and may cause endothelial damage or dysfunction [ 129131 ] . since activated endothelial cells may produce a broad panel of immunomodulatory molecules with il-8 or p - selectin having been identified as potent net inducers [ 129 , 132 ] , interactions between infected endothelial cells , b. besnoiti tachyzoites , and nets are quite likely . recently reported on infection - induced upregulation of endothelial - derived il-8 and p - selectin gene transcription and furthermore presented indications on net formation occurring adjacent to infected endothelium after pmn adhesion assays being performed under physiological flow conditions as the ones present in small vessels . recent net - related investigations on the closely related cyst - forming apicomplexan protozoa neospora caninum have shown that bovine pmn exposed to viable tachyzoites also result in strong netosis ( figure 1(f ) ) . with regard to molecular mechanisms , n. caninum - triggered netosis seems to be p2y2- , nox- , soce- , mpo- , ne- , erk1/2- , p38 mapk- , and pad4-dependent ( villagra - blanco et al . , submitted manuscript ) . cryptosporidium parvum is an euryxenous apicomplexan parasite with worldwide distribution and high zoonotic potential , mainly affecting young children , immunocompromised humans , and neonatal livestock . typically , cryptosporidiosis is a water- and food - borne enteric disease that causes diarrhoea , dehydration , weight losses , and abdominal pain and leads to significant economic losses in the livestock industry [ 133 , 134 ] . after ingestion , sporozoites are released from oocysts into the intestinal lumen and infect small intestine epithelial cells . recent studies reported on a significant contribution of pmn and macrophages to inflammatory responses in cryptosporidiosis in vivo [ 136 , 137 ] . muoz - caro and colleagues reported on nets being cast by both bovine and human pmn in response to c. parvum stages . parasite - triggered netosis proved stage - independent since it was induced by both sporozoites and oocysts ( figure 1(b ) ) . especially in the latter case parasite stages were occasionally entirely covered with net structures thereby most probably hampering proper sporozoite excystation . given that pmn were shown as active even within the intestinal lumen [ 108 , 109 , 138 , 139 ] , these reactions should have a significant impact on ongoing in vivo infection . in vitro infection experiments additionally showed the negative impact of nets on host cell invasion since infection rates were significantly reduced when using pmn - preexposed c. parvum stages . the fact that these reactions were entirely reversible via dnase i treatments rather argued against any cryptosporidicidal effects of nets . the colocalization of ne , histones , and mpo with dna in parasite - mediated extracellular fibres proved classical characteristics of nets and inhibitor experiments emphasized the key role of ne , nox , and mpo in efficient net formation . in agreement with findings on eimeria - induced netosis , inhibition experiments revealed c. parvum - triggered net formation as dependent on intracellular ca release and erk 1/2 and p38 mapk - mediated signalling pathways . interestingly , c. parvum sporozoite - exposed bovine pmn showed increased gene transcription of proinflammatory molecules , some of which were recently shown as potent net inducers ( e.g. , il-8 and tnf- ) [ 140 , 141 ] and may have potentiated net reactions . infections with leishmania spp . represent a major health problem and according to the who 10% of the human world population is at risk of infection , meaning that approximately 12 million people in 98 countries are infected , and 2 million new cases occur each year [ 142 , 143 ] . leishmaniasis is a vector - transmitted zoonosis caused by more than 25 different obligate intracellular protozoan leishmania species [ 142144 ] . particularly pmn have been implicated in the immunopathogenesis of leishmaniasis [ 145149 ] and recent studies examined the potential role of nets during the early phase of the disease of different leishmania species . . showed for the first time that promastigotes of leishmania amazonensis , l. major , and l. chagasi were capable of triggering net formation . additionally , leishmania - triggered netosis seems not entirely stage - specific , since both promastigotes ( l. amazonensis , l. major , l. chagasi , l. donovani , l. mexicana , and l brasiliensis ) and amastigotes ( l. amazonensis , l. braziliensis ) promoted net formation in vitro and in vivo [ 51 , 147 , 150152 ] . more importantly , guimares - costa et al . provided first indications on possible parasite - specific ligands being responsible for leishmania - mediated netosis . thus , leishmania - derived lipophosphoglycans ( lpg ) were suggested as the main trigger of net release since these molecules also induced nets in a purified form . the former authors showed that nets possessed detrimental effects on parasites as net - entrapped l. amazonensis promastigotes exhibited decreased viability . authors also demonstrated that the extracellular dna and histones found on nets were involved in the parasite inactivation / killing process . the leishmanicidal effects of histones were proven in promastigotes cocultures with purified h2a histones leading to the killing of parasites and by a significant reduction of leishmanicidal effects when cocultured in the presence of anti - histone antibodies . demonstrated that also the histone h2b could directly and efficiently kill promastigotes of l. amazonensis , l. major , l. braziliensis , and l. mexicana . in case of l. donovani , gabriel et al . reported netosis as a ros - dependent process which was equally triggered in human and murine pmn ( see table 1 ) . however , leishmania - lipophosphoglycan- ( lpg- ) dependent net induction reported by guimares - costa et al . was not observed with l. donovani . when using genetically modified l. donovani promastigotes gabriel et al nonetheless , in this infection system , lpg appeared to be involved in the resistance to nets - mediated killing , since the wild type of l. donovani maintained its viability in the presence of nets , whilst mutant parasites lacking lpg were efficiently killed by these extracellular structures . a more recent study revealed that leishmania parasites trigger not only the classical ros - dependent netosis as previously demonstrated but also a ros - independent form , named as early / rapid vital netosis . during this early / rapid leishmania - triggered netosis , in which net formation takes place after 515 min of activation without affecting pmn viability [ 29 , 68 ] , the parasites are also being efficiently entrapped . regarding net - related evasion strategies of trypanosomatidae parasites , leishmania spp . seem capable of evading net killing by firstly blocking the oxidative burst activity of pmn or even by resisting microbicidal activity of nets [ 145 , 150 ] . moreover , guimares - costa et al . showed that l. infantum promastigotes express the enzyme 3-nucleotidase / nuclease which was previously described to be involved in parasite nutrition and infection and was proven to be part of the ability of promastigotes to escape net - mediated killing . a recent investigation has shown that a salivary component of the sand fly insect that transmits leishmaniasis may also play a role in the survival of leishmania in the definitive hosts , by modulating their innate immune system . a molecule named lundep from the salivary gland of lutzomyia longipalpis was recently described as an endonuclease with net - destroying properties in humans . in the presence of lundep , measured the ne release from nets as an indicator of net destruction , since ne is normally decorating nets backbone structures and found at low concentrations in culture supernatants , as previously demonstrated . lundep was responsible for the significant increase of ne concentration in the supernatants when compared to negative controls . in conclusion , these experiments showed degradation of dna scaffold of nets , destroying their functional integrity , and increasing promastigote survival and exacerbating l. major infection . approximately eight million people are affected by this tropical disease in the americas and an average of 12,000 deaths per year is known to occur due to american trypanosomiasis . it is well known that macrophages , eosinophils , monocytes , and pmn are implicated in the control of early infection [ 30 , 155 ] . . demonstrated in vitro that t. cruzi is able to trigger nets in a dose- , time- , and ros - dependent manner . in agreement with reports on eimeria spp . and b. besnoiti [ 23 , 24 , 59 ] but in contrast to observations on t. gondii and leishmania spp . [ 25 , 51 ] , the viability of t. cruzi stages was not affected by nets , but netosis significantly impaired the parasite host cell infectivity . in fact , nets components as ne may affect t. cruzi infectivity , since this enzyme appears to be involved in increased trypanocidal activity and in the reduction of trypomastigote release by prestimulated infected macrophages [ 30 , 156 ] . additionally , the authors showed via antibody - mediated blockage that t. cruzi - triggered netosis is a tlr2- and tlr4-dependent process . moreover , the study showed that not only viable t. cruzi trypomastigote forms but also soluble antigens and killed t. cruzi parasites induced net release in human pmn . in vivo murine studies indicated the relevance of netosis for the outcome of trypanosomiasis since significantly decreased parasites numbers were found in the blood system of those animals which had previously been infected with nets - pretreated parasites . during the last years a vast amount of data on protozoan - mediated etosis was published strengthening the role of this effector mechanism in the defence of parasitic infections . several in vivo data have now proven the existence and importance of this early host innate effector mechanism . however , there is still a total lack of information on parasite - derived ligands triggering etosis . taking into account that in most cases et formation is considered as a species- and stage - independent process , rather ubiquitary occurring molecules may represent parasite - derived target molecules of ets . moreover , recent data revealed that other leukocytes such as monocytes , macrophages , basophils , mast cells , and eosinophils also perform etosis upon pathogen encounter . furthermore , et - related research mainly focused on the leukocytes aptitude to impact the parasites life cycle , but not on the propensity of parasitic stages to develop counter mechanisms for ets avoidance . while a bunch of data is available on bacterial nucleases or other counter mechanisms , taken together , we call for more parasite - related studies in the exciting field of etosis . the national tb prevalence survey in eritrea was conducted from february through october 2005 ( 6 ) . in 40 selected villages , a census ( which included information about sex and age ) was taken of 875 persons in each village . all persons > 15 years of age were asked to provide a morning and a spot sputum sample . persons who had 2 positive sputum samples were informed about the test results and referred for treatment . those who had 1 positive sputum sample were referred to a nearby healthcare facility for further smear examination . if results of smear examination were negative , thoracic radiographs were taken and evaluated by 2 experienced radiologists . the case definition for a sputum smear positive case was at least 2 sputum specimens positive for acid - fast bacilli by ziehl - neelsen staining and microscopy or at least 1 sputum specimen positive for acid - fast bacilli and radiographic abnormalities consistent with active pulmonary tb ( classification of the national tuberculosis control program in eritrea ) . using the prevalence estimate obtained from the survey and 2 different models , we calculated the cdr for 2004 . in model 1 , described by styblo , cdr = ( notification rate / prevalence rate ) / ( 0.5 + 0.83 [ notification rate / prevalence rate ] ) ( 7,8 ) . in model 2 , described by dye et al . , cdr = ( notification rate / prevalence rate ) / ( [ notification rate / prevalence rate ] + 0.5 ) ( 9,10 ) . we then compared the calculated cdr with the cdr estimated by the world health organization ( who ) to evaluate whether comparable conclusions about tb case detection would be obtained . a total of 38,047 persons were included in the prevalence survey . of those > 15 years of age , 18,152 ( 94.6% ) provided at least 1 sputum sample ( figure ) . the prevalence of new smear - positive tb was estimated at 90/100,000 ( 95% confidence interval [ ci ] 35145/100,000 ) in persons > 15 years of age . in 2005 , 44.7% of the eritrean population was < 15 years of age ( 11 ) , which resulted in an overall new smear - positive tb prevalence of 50/100,000 ( 95% ci 1980/100,000 ) under the assumption of no cases in persons < 15 years of age . summary of tuberculosis prevalence survey in eritrea , 2005 . in 2004 , 17/100,000 new smear - positive cases were reported ( 2 ) . for eritrea , the cdr provided by who is considerably lower than that calculated from the results of the national tb prevalence survey . both estimates indicate that eritrea has not reached the 70% target for case detection . however , the who estimate suggests that the program needs to improve case detection by a factor of 5 , whereas the survey estimate suggests that case detection needs to be improved by a factor of 1.6 . two explanations may account for the large difference : 1 ) the cdr derived from the tb prevalence survey is too high because of an underestimation of the prevalence of smear - positive tb , or 2 ) the cdr estimate published by who is too low because of an overestimation of the incidence of smear - positive tb . in the national tb prevalence survey , measures were taken to ensure high quality of the results ; e.g. , training of data collectors , repeat census taking , reexamination of all slides found positive on fluorescence microscopy , and reexamination of a 5% random sample of the negative slides . persons who had smear - positive tb may have been missed because they did not provide a specimen ; however , because only 5% of eligible persons did not provide a specimen , this can explain only a slight underestimation . furthermore , recorded reasons for not providing a specimen seem to be unrelated to a higher chance of having tb . the quality of the provided specimens may have been suboptimal because instructing and motivating persons to provide a sputum sample is challenging . for diagnosis of tb , microscopic examination of saliva is less sensitive than examination of sputum ; however , in 50% of saliva samples from patients with a positive sputum sample , bacilli can be demonstrated ( 12,13 ) . for 27,647 samples that appeared to be saliva , smear assuming that only 50% were detected , a maximum of 12 smear - positive tb patients may have been undetected . taking this into account results in a prevalence of 87/100,000 . using this estimate , model 1 provides a cdr of 30% and model 2 a cdr of 28% the possibility that persons who provided a saliva sample were not able to produce a sputum sample because they did not have pathologic pulmonary changes should also be taken into consideration . estimation of the incidence of smear - positive tb in eritrea is complicated by the fact that no data from tuberculin or prevalence surveys were available . the only data available for eritrea were reporting data , which experts assessed as being of low quality ( 14 ) . use of this limited information will result in an uncertain incidence estimate , which may result in an unreliable cdr . for most countries in africa , little information is available for estimating the prevalence of disease and progress towards the millennium development goals ( http://unstats.un.org/unsd/mi/mi_goals.asp , accessed 2006 aug 30 ) . on the basis of case reporting , tb was rightly declared an emergency by african health ministers at the who africa regional committee in maputo in 2005 ( 15 ) . to be able to fight this emergency , more reliable information about the prevalence of tb in africa is needed . furthermore , for global tb control , reliable information about the tb epidemic in africa is needed because 28% of the incident smear - positive cases occurred in the who african region in 2004 ( 2 ) . in conclusion , the example of eritrea shows that a large gap may exist between available estimates of tb prevalence and actual tb prevalence in africa . national tb prevalence surveys in africa would help provide better information on tb prevalence and case detection .

professional mononuclear phagocytes such as polymorphonuclear neutrophils ( pmn ) , monocytes , and macrophages are considered as the first line of defence against invasive pathogens . the formation of extracellular traps ( ets ) by activated mononuclear phagocytes is meanwhile well accepted as an effector mechanism of the early host innate immune response acting against microbial infections . recent investigations showed evidence that etosis is a widely spread effector mechanism in vertebrates and invertebrates being utilized to entrap and kill bacteria , fungi , viruses , and protozoan parasites . ets are released in response to intact protozoan parasites or to parasite - specific antigens in a controlled cell death process . released ets consist of nuclear dna as backbone adorned with histones , antimicrobial peptides , and phagocyte - specific granular enzymes thereby producing a sticky extracellular matrix capable of entrapping and killing pathogens . this review summarizes recent data on protozoa - induced etosis . special attention will be given to molecular mechanisms of protozoa - induced etosis and on its consequences for the parasites successful reproduction and life cycle accomplishment . we used results from a national tuberculosis prevalence survey in eritrea to calculate case detection rate ( cdr ) and compared it with the published cdr . the cdr obtained from the survey was 40% , whereas the cdr published by the world health organization was 3 lower ( 14% ) .

spermatogenesis is a complex process of development that occurs in most mammals throughout adulthood period . this process happens in seminiferous tubules , lined with epithelial tissue that contains sertoli cells . sertoli cells are surrounded by a thin septum of peritubular cells that hold keep germ cells ( 1 , 2 ) . the spermatogonia undergo mitotic divisions , meiotic alternations , and morphologically transform to highly developed cells , spermatozoa , with a unique function and structure ( 3 ) . such a complex process needs a unique program to control and regulate the involved genes . this gene regulation is accomplished by cell surface molecules which participate in cell to cell and cell to extracellular matrix interactions ( 4 , 5 ) . on the other hand , when sperm is passing through the male and female genital tracts , it gains fertilization ability ( capacitation ) and adopts maturity characteristics . maturity is initiated in the epididymis , where sperm communicates with the cell surface molecules of the pathway ( 6 ) . therefore , the investigation of stage and/or cell - specific molecular expression in testis or post - testicular maturation system can improve our understanding of germ cell differentiation and sperm formation ( 7 ) . first identified a 38kda protein ( mtex101 ) in adult mouse testis which is detectable on the surface of spermatocytes and spermatids in testis that it is absent on somatic cells , including sertoli or interstitial cells , like leydig cells ( 3 ) . there is a temporary expression of mtex101 on oogonia , but the molecule disappears in the mature ovary . in embryonic testis , mtex101 is detected on the prosperm - atogonia ( 8) . upon puberty , mtex101 mrna is expressed in spermatocytes and in steps 1 - 9 spermatids during spermatogenesis , but not during spermatogonia . the tex101 protein remains on the cell surfaces in steps 10 - 16 spermatids and testicular sperm , but hides from epididymal sperm after passing the caput epididymis ( 6 ) . the expression of mtex101 homologous protein has been reported in a leukemia cell line and lung cancer in rats and humans , respectively ( 9 , 10 ) . the exact structure and function of mtex101 is not completely understood yet , but it has been speculated that this protein is associated with several transmembrane proteins like ly6k ( 11 ) and cellubrevin ( 12 ) that transduce the extra - cellular signals to the intracellular molecules . the present study was undertaken to produce recombinant mtex101 , which might pave the way for the production of specific antibodies for further structural and functional characterization of the molecule . total rna was isolated from male gonadal organs ( four to 6 week - old mice from pasteur institute of iran ) . rna concentration was measured by a biophotometer ( eppendorf , hamburg , germany ) at 260 nm . one microgram of the total rna was reversetranscribed to cdna by using 200u of molony murine leukemia virus ( rtm - mulv ) reverse transcriptase enzyme ( fermentas , vilnius , lithuania ) , and 20pmol of random hexamer primers ( cybergene , stockholm , sweden ) . pcr amplifications for mtex101 transcript was performed in a volume of 25l using 10 - 20ng of testis cdna , forward and reverse primers ( 10pmol each ) , specific for mtex101 transcript , 10x pcr buffer ( 2.5l ) , dntp mixture ( 0.2 mm each ) , 1 mm mgcl2 , and 1 unit of taq dna polymerase ( roche , mannheim , germany ) . pcr reactions were carried out using a thermal cycler ( eppendorf mastercycler gradient ) as indicated below : a preheating cycle at 95c for 3min , 35 cycles of denaturation at 95c for 30sec , annealing at 60c for 30sec , extension at 72c for 60sec , and finally a 7-min cycle at 72c . the forward and reverse mtex101 orf primers used ( forward : 5ccg aat tca tgg gag cct gcc gca tcc ag 3 , reverse : 5 agg gaa gtg ggt gag ggg gga gca gag cgg ccg c 3. ) , contained ecori and noti restriction enzyme ( re ) sites , respectively . the pcr products were resolved by ethidium bromide - stained 1.5% agarose gel electrophoresis , which revealed a single distinct band at 750bp ( figure 1 ) . the 750bp band was purified using qia quick gel extraction kit ( qiagen , germantown , md , usa ) . pcr optimization on mouse testis cdna by mgcl2 gradients . 1 - 4 : mgcl2 concentrations from 1 to 4 mm , respectively , 5 : 1 kb dna ladder , 6 : negative control ( no dna ) . the pgem - t easy vector ( promega , madison , wi , usa ) was used for the cloning of the purified pcr product . ligation reac tion was set up by 50ng of pgem - t easy vector ( promega ) , three units of t4 dna ligase , 75ng of mtex101 purified fragment , and 6l of rapid 2x ligation buffer ( promega ) . competent cells of e. coli jm109 strain were used for transformation through heat shock method ( 17 ) . the transformed bacteria were left for 1 hr in lb broth at 37c for recovery , and later 100l of the transformation culture was plated onto an ampicillin ( 100mg / ml ) ( sigma , louis , mo , usa ) , iptg ( sigma ) ( 0.5 mm ) and x - gal ( sigma ) , ( 80g / ml ) containing lb agar plate and was cultured for 16hrs at 37c . the recombinant plasmids were isolated from confirmed colonies by miniperp kit ( qiagen ) and then digested by ecori and noti restriction enzymes . plasmid dna ( 800ng ) was used in 25l of the total volume , including noti ( 15units ) , ( invitrogen , carlsbad , ca , usa ) , ecori ( 15 units ) , ( invitrogen ) , and 2.5l of 10 x reaction buffer 3 ( invitrogen ) and incubated for 1.5 hr at 37c . the mtex101 fragment in pgem - t easy was extracted form agarose gel and then subcloned into a pet-28a ( + ) expression vector ( merck , darmstadt , germany ) and was digested by the same restriction enzymes above . ligation reaction was performed by 125ng of digested pet-28a ( + ) , 0.5pmol of mtex101 purified fragments , 2l of rapid 2x ligation buffer ( pro - mega ) , and 3 units of t4 dna ligase ( promega ) in 10l of the total volume and it was incubated overnight at 4c . ligation products were used for transformation of e. coli jm109 strain by heat shock method ( 17 ) . after a recovery time of 1 hour in lb broth , the transformed bacteria were cultured on lb agar and kanamycin ( 50g / ml ) ( sigma , usa ) containing plates for 16 hrs at 37c . the obtained colonies were screened by colony pcr and one of them was confirmed by dna sequencing . recombinant plasmid was purified and was then used for transformation of bl21 ( de3 ) , which is a protease deficient strain of e. coli . a single transformed colony was inoculated into 50ml of lb broth , including kanamycin ( 50g / ml ) ( sigma , usa ) and untransformed bl21 ( de3 ) bacteria were cultured into 50 ml of lb medium . incubation was performed under shaking at 37c until the optical density ( od ) at 600 nm reached 0.6 . iptg ( sigma ) was added to the remainder with the final concentration of 1 mm and incubation continued for 2 - 3 more hours . cell lysates were prepared by sonication of pellets in pbs buffer including 1% protease inhibitor ( roche ) . protein solutions were obtained from bacteria by boiling them in a sample buffer that contained tris - hcl ( ph=6.8 , 0.5 m ) , sds ( 10% w / v ) , glycerol ( 50% v / v ) , and bromo phenol blue ( 0.5% w / v ) , for 5min . samples ( 50l of each ) were separated by sds - page ( 8% ) ( bio - rad , hercules , ca , usa ) under non - reducing conditions . the protein constituents were then electro - phoretically blotted onto a polyvinylidene difluoride ( pvdf ) membrane ( millipore , billerica , ma , usa ) , as described by towbin et al . ( the membrane was blocked in 5% skim milk / 0.1% tween 20 in pbs ( ph 7.4 ) , and then the reactivity of the transferred protein(s ) with 1g / ml of anti mtex101 pab ( avicenna research institute , iran ) was assessed using sheep anti rabbit ig hrp ( avicenna research institute , iran ) . finally , the membranes were visualized using an ecl system ( ge healthcare , biotech , bucking - hamshire , uk ) . total rna was isolated from male gonadal organs ( four to 6 week - old mice from pasteur institute of iran ) . rna concentration was measured by a biophotometer ( eppendorf , hamburg , germany ) at 260 nm . one microgram of the total rna was reversetranscribed to cdna by using 200u of molony murine leukemia virus ( rtm - mulv ) reverse transcriptase enzyme ( fermentas , vilnius , lithuania ) , and 20pmol of random hexamer primers ( cybergene , stockholm , sweden ) . pcr amplifications for mtex101 transcript was performed in a volume of 25l using 10 - 20ng of testis cdna , forward and reverse primers ( 10pmol each ) , specific for mtex101 transcript , 10x pcr buffer ( 2.5l ) , dntp mixture ( 0.2 mm each ) , 1 mm mgcl2 , and 1 unit of taq dna polymerase ( roche , mannheim , germany ) . pcr reactions were carried out using a thermal cycler ( eppendorf mastercycler gradient ) as indicated below : a preheating cycle at 95c for 3min , 35 cycles of denaturation at 95c for 30sec , annealing at 60c for 30sec , extension at 72c for 60sec , and finally a 7-min cycle at 72c . the forward and reverse mtex101 orf primers used ( forward : 5ccg aat tca tgg gag cct gcc gca tcc ag 3 , reverse : 5 agg gaa gtg ggt gag ggg gga gca gag cgg ccg c 3. ) , contained ecori and noti restriction enzyme ( re ) sites , respectively . the pcr products were resolved by ethidium bromide - stained 1.5% agarose gel electrophoresis , which revealed a single distinct band at 750bp ( figure 1 ) . the 750bp band was purified using qia quick gel extraction kit ( qiagen , germantown , md , usa ) . pcr optimization on mouse testis cdna by mgcl2 gradients . 1 - 4 : mgcl2 concentrations from 1 to 4 mm , respectively , 5 : 1 kb dna ladder , 6 : negative control ( no dna ) . the pgem - t easy vector ( promega , madison , wi , usa ) was used for the cloning of the purified pcr product . ligation reac tion was set up by 50ng of pgem - t easy vector ( promega ) , three units of t4 dna ligase , 75ng of mtex101 purified fragment , and 6l of rapid 2x ligation buffer ( promega ) . competent cells of e. coli jm109 strain were used for transformation through heat shock method ( 17 ) . the transformed bacteria were left for 1 hr in lb broth at 37c for recovery , and later 100l of the transformation culture was plated onto an ampicillin ( 100mg / ml ) ( sigma , louis , mo , usa ) , iptg ( sigma ) ( 0.5 mm ) and x - gal ( sigma ) , ( 80g / ml ) containing lb agar plate and was cultured for 16hrs at 37c . the recombinant plasmids were isolated from confirmed colonies by miniperp kit ( qiagen ) and then digested by ecori and noti restriction enzymes . plasmid dna ( 800ng ) was used in 25l of the total volume , including noti ( 15units ) , ( invitrogen , carlsbad , ca , usa ) , ecori ( 15 units ) , ( invitrogen ) , and 2.5l of 10 x reaction buffer 3 ( invitrogen ) and incubated for 1.5 hr at 37c . the mtex101 fragment in pgem - t easy was extracted form agarose gel and then subcloned into a pet-28a ( + ) expression vector ( merck , darmstadt , germany ) and was digested by the same restriction enzymes above . ligation reaction was performed by 125ng of digested pet-28a ( + ) , 0.5pmol of mtex101 purified fragments , 2l of rapid 2x ligation buffer ( pro - mega ) , and 3 units of t4 dna ligase ( promega ) in 10l of the total volume and it was incubated overnight at 4c . ligation products were used for transformation of e. coli jm109 strain by heat shock method ( 17 ) . after a recovery time of 1 hour in lb broth , the transformed bacteria were cultured on lb agar and kanamycin ( 50g / ml ) ( sigma , usa ) containing plates for 16 hrs at 37c . the obtained colonies were screened by colony pcr and one of them was confirmed by dna sequencing . recombinant plasmid was purified and was then used for transformation of bl21 ( de3 ) , which is a protease deficient strain of e. coli . a single transformed colony was inoculated into 50ml of lb broth , including kanamycin ( 50g / ml ) ( sigma , usa ) and untransformed bl21 ( de3 ) bacteria were cultured into 50 ml of lb medium . incubation was performed under shaking at 37c until the optical density ( od ) at 600 nm reached 0.6 . iptg ( sigma ) was added to the remainder with the final concentration of 1 mm and incubation continued for 2 - 3 more hours . cell lysates were prepared by sonication of pellets in pbs buffer including 1% protease inhibitor ( roche ) . protein solutions were obtained from bacteria by boiling them in a sample buffer that contained tris - hcl ( ph=6.8 , 0.5 m ) , sds ( 10% w / v ) , glycerol ( 50% v / v ) , and bromo phenol blue ( 0.5% w / v ) , for 5min . samples ( 50l of each ) were separated by sds - page ( 8% ) ( bio - rad , hercules , ca , usa ) under non - reducing conditions . the protein constituents were then electro - phoretically blotted onto a polyvinylidene difluoride ( pvdf ) membrane ( millipore , billerica , ma , usa ) , as described by towbin et al . ( 13 ) . the membrane was blocked in 5% skim milk / 0.1% tween 20 in pbs ( ph 7.4 ) , and then the reactivity of the transferred protein(s ) with 1g / ml of anti mtex101 pab ( avicenna research institute , iran ) was assessed using sheep anti rabbit ig hrp ( avicenna research institute , iran ) . finally , the membranes were visualized using an ecl system ( ge healthcare , biotech , bucking - hamshire , uk ) . after cloning mtex101 fragments in pgem - t easy vector , several white colonies with probable target fragment inclusion were screened by colony pcr ( figure 2 ) . a right - sized pcr product and a pet-28a(+ ) vector were cut by noti and ecori restriction enzymes to obtain the required fragments for the next step ( figures 3 and 4 ) . coloy pcr on transformed jm109 clones by pgem - t easy vector carrying mtex101 gene . 1 - 5 : 5 selected white colonies , 6 : negative control ( blue colony ) , 7 : positive control ( pcr product on testis cdna ) , 8 : dna ladder viii ( roche ) . double digestion of pgem - t easy vector containing mtex101 fragment with ecori and noti restriction enzymes . 1 : digested vector with mtex101 750bp insert cut out of the vector , 2 : dna ladder viii . double digestion of pet-28a ( + ) expression vector with restriction enzymes . 1 : digested pet-28a ( + ) with ecori and noti , 2 : uncut pet-28a ( + ) , 3 : 1 kb dna ladder . e. coli j m109 strains were transformed using recombinant pet-28a(+ ) containing mtex101 fragment and the undertaken transformation was verified by colony pcr ( figure 5 ) . one of the confirmed colonies ( clone 2 in figure 5 ) was picked for detailed analysis by dna sequencing . alignment of dna sequencing results with mtex101 orf by genbee site ( 14 ) confirmed the cloned sequence . the verified fragment was used for the next round of transformation using e - coli bl21 ( de3 ) strain . colony pcr on transformed jm109 clones by pet-28a ( + ) vector containing mtex101 fragment . 1 and 2 represent two selected colonies , 3 : dna ladder viii , 4 : negative control ( no dna ) , 5 : positive control ( pgem - t easy vector containing mtex101 fragment ) . western blot analysis using anti - mte x 101 peptide polyclonal antibody revealed the correct size of mtex101 recombinant protein production ( 27kda ) in bl-21 ( de3 ) bacteria ( figure 6 ) . western blot analysis of production of the recombinant mtex101 in bl21 ( de3 ) bacteria . 1 : bl21 ( de3 ) containing mtex101 gene after induction , 2 : bl21 ( de3 ) containing mtex101 gene before induction , 3 : untransfected bl21 ( de3 ) , 4 : protein marker ( see blue - invitrogen ) . after cloning mtex101 fragments in pgem - t easy vector , several white colonies with probable target fragment inclusion were screened by colony pcr ( figure 2 ) . a right - sized pcr product and a pet-28a(+ ) vector were cut by noti and ecori restriction enzymes to obtain the required fragments for the next step ( figures 3 and 4 ) . coloy pcr on transformed jm109 clones by pgem - t easy vector carrying mtex101 gene . 1 - 5 : 5 selected white colonies , 6 : negative control ( blue colony ) , 7 : positive control ( pcr product on testis cdna ) , 8 : dna ladder viii ( roche ) . double digestion of pgem - t easy vector containing mtex101 fragment with ecori and noti restriction enzymes . 1 : digested vector with mtex101 750bp insert cut out of the vector , 2 : dna ladder viii . double digestion of pet-28a ( + ) expression vector with restriction enzymes . 1 : digested pet-28a ( + ) with ecori and noti , 2 : uncut pet-28a ( + ) , 3 : 1 kb dna ladder . e. coli j m109 strains were transformed using recombinant pet-28a(+ ) containing mtex101 fragment and the undertaken transformation was verified by colony pcr ( figure 5 ) . one of the confirmed colonies ( clone 2 in figure 5 ) was picked for detailed analysis by dna sequencing . alignment of dna sequencing results with mtex101 orf by genbee site ( 14 ) confirmed the cloned sequence . the verified fragment was used for the next round of transformation using e - coli bl21 ( de3 ) strain . colony pcr on transformed jm109 clones by pet-28a ( + ) vector containing mtex101 fragment . 1 and 2 represent two selected colonies , 3 : dna ladder viii , 4 : negative control ( no dna ) , 5 : positive control ( pgem - t easy vector containing mtex101 fragment ) . western blot analysis using anti - mte x 101 peptide polyclonal antibody revealed the correct size of mtex101 recombinant protein production ( 27kda ) in bl-21 ( de3 ) bacteria ( figure 6 ) . western blot analysis of production of the recombinant mtex101 in bl21 ( de3 ) bacteria . 1 : bl21 ( de3 ) containing mtex101 gene after induction , 2 : bl21 ( de3 ) containing mtex101 gene before induction , 3 : untransfected bl21 ( de3 ) , 4 : protein marker ( see blue - invitrogen ) . the researchers immunized female mice with 8-week old testis lysates and obtained 12 monoclonal antibody producing clones ( tes101 - 112 ) . immunohisto - chemistry analyses revealed that only tes101 was able to recognize a novel testicular protein as determined by searching the expressed sequence tag ( est ) database ( 3 ) . all studies on mtex101 protein are performed solely by tes101 antibody and presently , no commercial antibodies are available to carry out research on this protein . in this study , we produced mtex101 recombinant protein that is useful for antibody development for further investigations . mtex101 mrna contains 750 bases , which encodes 250 amino acids . after the removal of the putative 25-amino acid signal peptide at n - terminus reported a 38kda band by western blot analysis under non - reducing conditions and no 24 kda band was observed ( 3 ) . this large difference in the molecular mass was speculated to be due to glycosylation of the peptide and it was proved to be true by jin et al . glycosylation is known as the prime cause of post - translational modifications ( ptm ) in proteins ( 16 ) . the asn - x - ser / thr is the basic sequence for n - linked glycosylation , however , the secondary structure of the protein can affect the final addition . no consensus sequence has been established for o - linked glycosylation yet ( 17 ) . jin et al , found four putative sites for n - glyco - sylation and several possible sites for o - glyco - sylation in mtex101 amino acid sequence ( 15 ) . in fact , mtex101 is a highly glycosylated protein and it has been clarified that most of the oligosaccharide chains on this peptide are n - linked carbohydrates ( 12 ) . de - n - glycosylation of mtex101 created an 20kda band which is close to or even smaller than estimated molecular weight of mtex101 amino acid backbone ( 15 ) , confirming glycosylation as the cause for the higher mass of native mtex101 . the cellular role of glycoprotein sugar components can be investigated via several methods , like protein production in a host lacking oligo - saccharide addition system , such as prokaryotes ( 18 ) . we succeeded to produce mtex101 recombinant protein in a prokaryotic system , which may be useful in clarifying the role of sugar components in protein structures . however , proteins which are produced by this method may vary in their structures and functions ( 19 ) . western blot analysis showed that this protein had a molecular mass of approximately 27kda under non - reducing conditions that is concordant with the various post - translational modification processes in prokaryotic systems as compared to those in eukaryotes . the size of the recombinant protein ( 27kda ) is in conformity with the backbone protein ( 24kda ) and a 3kda peptide derived from the digested vector by noti and ecori restriction enzymes . in this study , we successfully cloned mtex101 into a his - tagged expression vector ( pet-28a(+ ) ) , followed by an efficient production of the relevant recombinant protein . this protein can be used in antibody production to find out the role of mtex101 in spermatogenesis , egg fertilization and further investigation about the critical role of glycosylation in the function of this protein . most malignancies of the upper aerodigestive tract ( figure 1 ) , comprising the naso- , oro- , hypo- , and laryngopharynx , are squamous cell carcinomas . head and neck squamous cell carcinomas ( hnsccs ) are the primary tumor type in head and neck cancer ( hnc ) , characterized by local tumor aggressiveness , high rate of early recurrences , metastasis , and development of second primary tumors , which are the major cause of morbidity and mortality in hnscc ( details in [ 14 ] ) . more than 90% of hnc cases are induced by chronic exposure to carcinogens enclosed in all forms of tobacco , synergized by heavy alcohol consumptions and poor diet ( see [ 5 , 6 ] ) . it is estimated that about 5%10% of suspicious lesions arising in the mucous membranes of the mouth , pharynx , and larynx undergo malignant transformation . cure rates of early disease ( stage i and ii ) range between 70% and 80% , and chemoprevention strategies seem promising to control potentially malignant oral lesions ( reviewed in [ 13 ] ) . however , long - term survival rates , especially for advanced hnc , have not improved significantly over the last decades . despite modern therapeutic strategies and sophisticated surgical management of the tumor , the estimated five - year survival rate for advanced disease ( 30%40% ) remains poor ( [ 13 ] and references therein ) . currently , rational therapeutic strategies targeting growth factor receptors by specific antibodies or kinase inhibitors have gained increasing clinical relevance in particular for the treatment of locally advanced cancer with the intent of preserving speech and swallowing ( see [ 13 ] ) . thus , developing new therapeutic strategies and defining novel target proteins for the treatment of advanced hnc is of particular importance . in this respect , nuclear receptors ( nrs ) are transcription factors implicated in cancer development and are recently attracting major interest as therapeutic targets ( see [ 7 , 8 ] ) . as nrs modulate cell proliferation , apoptosis , invasion , and migration , clearly representing hallmarks of cancer cells , several highly successful cancer drugs target this receptor family [ 811 ] . since several nrs have been shown to be expressed also in head and neck cancer cells , nrs are most likely also contributing to hnscc development and progression [ 12 , 13 ] . nrs belong to a large superfamily of transcription factors and based on sequence comparison are currently classified into seven subfamilies ( table 1 ) . these transcription factors are able to modulate transcription of a variety of target genes by several distinct mechanisms , including both transcriptional activation and repression [ 7 , 8 , 14 , 15 ] . transcriptional regulation can either be ligand - dependent or -independent , genomic or nongenomic , allowing nrs to mediate gene repression or its release , gene activation , or gene trans - repression ( details in [ 7 , 8 , 16 ] ) . in particular , the large group of so - called orphan nuclear receptors , for which natural ligands are still unknown , do not exist at all ( true orphans ) , or have only recently been identified ( adopted orphans ) is adding additional complexity to the field ( table 1 ) ( [ 8 , 17 ] , and references within ) . in contrast to cell surface growth factor receptors , such as the epidermal growth factor receptor ( egfr ) , which activate genetic programs through complex intracellular signaling cascades , nrs are able to directly bind to specific dna - sequences , so - called hormone response elements ( hres ) . thus , nrs are composed of an n - terminal regulatory domain ( activation function 1 = af1 ) , followed by a dna - binding domain ( dbd ) , a ligand - binding domain ( lbd ) , and another c - terminal regulatory domain ( activation function 2 = af2 ) ( figure 2 ) [ 7 , 8 ] . despite their conserved structural organization , nevertheless , two major modes of nr action can be assigned , depending on their intracellular steady - state localization in the absence of ligands ( figure 3 ) . one group of nrs is confined to the cytoplasm within multiprotein - complexes in the absence of ligand . upon ligand binding , they actively enter the nucleus and bind to hres as homo- or heterodimers ( figure 4 , details in [ 7 , 8 ] ) . other nrs already reside in the nucleus in a complex with corepressor proteins , while ligand binding triggers corepressor dissociation allowing the recruitment of coactivators [ 18 , 19 ] . however , in order to fulfill multiple biological tasks minor to major deviations from these two modes of nr action exist [ 7 , 8 ] . nrs are not only implicated in a broad spectrum of physiological processes but are associated with many human diseases including metabolic and cardiovascular disorders as well as cancer . beside their proven clinical relevance for hormone regulated malignancies , there is rather limited information on their pathophysiological role as well as their prognostic and therapeutic potential for head and neck cancer [ 7 , 8 , 12 , 2022 ] . most studies were investigating members of two classes of the nr superfamily , the thyroid hormone receptor - like and the estrogen receptor - like receptors ( table 1 ) . thus , we will focus on relevant members of these subfamilies , summarize their potential diagnostic / prognostic value , and discuss their therapeutic potential . within the thyroid hormone receptor - like receptor subfamily , the peroxisome proliferator - activated receptors ( ppars ) show the highest disease relevance for hnscc . to date , three isoforms of the ppar ( , / , and ) have been identified , all able to form heterodimers with retinoid x receptors ( rxrs ) ( see [ 23 , 24 ] ) . ppars are expressed in different cell types and activate the transcription of several genes involved in a variety of biological processes , including lipid metabolism and insulin sensitivity ( see [ 23 , 24 ] ) . furthermore , a role in limiting inflammation has also been reported [ 24 , 25 ] . as tumor cell metabolism and inflammation appear to be critical for tumorigenesis and clinical outcome , nrs may thus directly or / and indirectly modulate malignancies [ 26 , 27 ] . as such , ppar is overexpressed in many epithelial malignancies [ 22 , 28 , 29 ] including oral squamous cell carcinoma . in the absence of ligand , ppars are complexed with corepressor proteins , thus acting as transcriptional repressors . ligand binding induces conformational changes facilitating heterodimerization with rxr , thus leading to the attraction of transcriptional coactivators ( figures 3 and 4 ) ( see [ 19 , 24 ] ) . natural and synthetic ligands for ppars include lipophilic molecules such as fatty acids and eicosanoids as well as thiazolidinedione ( tzd ) drugs and derivates thereof ( overview in [ 7 , 24 , 31 ] ) . ppar ligands seem to exert their effects in a dosage - dependent manner , although the detailed mechanism is currently not yet resolved . the postulated cancer modulating mechanisms are diverse , including effects on wnt signaling , inhibition of nfb , as well as the modulation of cell cycle regulators and pro- and antiapoptotic proteins , which have been linked with head and neck cancer ( see [ 4 , 23 ] ) . clinical aspects of peroxisome proliferator - activated receptors in hnsccin hnscc , overexpression on the protein level has been convincingly demonstrated for ppar and ppar [ 12 , 30 , 33 ] . agonist binding to ppar can induce cell differentiation , growth arrest , and apoptosis of cancer cells . additionally , such activating ppar ligands were shown to exert antiproliferative on human colon and breast cancers ( details in [ 23 , 24 ] ) and were also suggested as potential chemopreventive agents for oral carcinogenesis [ 12 , 35 , 36 ] . of note , since at least 1.6 million patients take antidiabetic drugs that function as ppar ligands , epidemiological data on their long - term effects on tumor prevention would therefore be of value to rationally design cancer chemoprevention trials . paradoxically , not only ppar agonists are considered as potential therapeutic agents in cancer therapy but also antagonists were studied in this respect . ppar inhibition was shown to induce apoptosis and anoikis and inhibit tumor cell invasion in squamous cell carcinomas . moreover , the results of several studies indicated that the growth - inhibiting activity of ppar ligands in oscc may be ppar independent . others showed that the observed effects were strongly dependent on ppar-expression [ 12 , 38 ] as well as on the type and concentration of the agonist . in the majority of oscc cases , ppar mrna could be detected by rt - pcr . by immunohistochemical analysis of primary tumors , ppar was often found in low - grade tumors , especially in tumor endothelium , and a favorable impact of ppar expression on relapse - free survival of the patients could be demonstrated . the beneficial effects of ppar ligands on malignancies were tested in several clinical trials , but outcomes proved to be highly diverse . some trials revealed 40% partial response rates , whereas others could not show any significant beneficial effect [ 41 , 42 ] . moreover , one may speculate that the tumor modulating effects of ppar ligands are mediated indirectly by affecting the tumor microenvironment , such as cancer - associated fibroblasts or tumor endothelial cells . in fact , ppar ligands have been shown to affect endothelial cell proliferation and migration and hence to regulate angiogenesis . also hypoxia - induced angiogenesis appears to be affected by ppar ligands in cancer therapy , even if the precise mechanisms still remain unclear . as angiogenesis is a crucial aspect for tumor development , therapy resistance and metastasis and inhibition of angiogenesis may hence have contributed to the clinical benefit observed.in sum , ppar ligands appear to be of clinical benefit for the treatment of head and neck cancer , in particular for oscc . nevertheless , a more detailed molecular knowledge on ppar biology is clearly required . increasing knowledge about the mode of action , specificity , and dosage - dependence of ppar agonistic and antagonistic ligands will hopefully allow a better modeling of ppar receptor function and thus lead to a more effective design of combinatorial application schemes for cancer treatment and cancer prevention in the future . in hnscc , overexpression on the protein level has been convincingly demonstrated for ppar and ppar [ 12 , 30 , 33 ] . agonist binding to ppar can induce cell differentiation , growth arrest , and apoptosis of cancer cells . additionally , such activating ppar ligands were shown to exert antiproliferative on human colon and breast cancers ( details in [ 23 , 24 ] ) and were also suggested as potential chemopreventive agents for oral carcinogenesis [ 12 , 35 , 36 ] . of note , since at least 1.6 million patients take antidiabetic drugs that function as ppar ligands , epidemiological data on their long - term effects on tumor prevention would therefore be of value to rationally design cancer chemoprevention trials . paradoxically , not only ppar agonists are considered as potential therapeutic agents in cancer therapy but also antagonists were studied in this respect . ppar inhibition was shown to induce apoptosis and anoikis and inhibit tumor cell invasion in squamous cell carcinomas . moreover , the results of several studies indicated that the growth - inhibiting activity of ppar ligands in oscc may be ppar independent . others showed that the observed effects were strongly dependent on ppar-expression [ 12 , 38 ] as well as on the type and concentration of the agonist . in the majority of oscc cases , ppar mrna could be detected by rt - pcr . by immunohistochemical analysis of primary tumors , ppar was often found in low - grade tumors , especially in tumor endothelium , and a favorable impact of ppar expression on relapse - free survival of the patients could be demonstrated . the beneficial effects of ppar ligands on malignancies were tested in several clinical trials , but outcomes proved to be highly diverse . some trials revealed 40% partial response rates , whereas others could not show any significant beneficial effect [ 41 , 42 ] . moreover , one may speculate that the tumor modulating effects of ppar ligands are mediated indirectly by affecting the tumor microenvironment , such as cancer - associated fibroblasts or tumor endothelial cells . in fact , ppar ligands have been shown to affect endothelial cell proliferation and migration and hence to regulate angiogenesis . also hypoxia - induced angiogenesis appears to be affected by ppar ligands in cancer therapy , even if the precise mechanisms still remain unclear . as angiogenesis is a crucial aspect for tumor development , therapy resistance and metastasis and inhibition of angiogenesis may hence have contributed to the clinical benefit observed . in sum , ppar ligands appear to be of clinical benefit for the treatment of head and neck cancer , in particular for oscc . increasing knowledge about the mode of action , specificity , and dosage - dependence of ppar agonistic and antagonistic ligands will hopefully allow a better modeling of ppar receptor function and thus lead to a more effective design of combinatorial application schemes for cancer treatment and cancer prevention in the future . another group of thyroid hormone receptor - like receptors implicated in hnscc is the retinoid acid receptors ( rars ) . rars are characterized by their activation via vitamin a derivatives . upon activation , rars are able to heterodimerize with retinoid x receptors ( rxr ) and to bind to specific hormone response elements ( hres ) , thereby modulating transcription of target genes ( figures 3 and 4 ) [ 8 , 26 , 46 ] . to date , a variety of coactivator- and corepressor - proteins have been identified , allowing the fine - tuning of target gene transcription , ranging from repression to full activation . however , the molecular details are just beginning to be uncovered [ 8 , 26 , 46 ] . rar activation often leads to differentiation , cell - cycle arrest , or apoptosis , culminating in the inhibiting of tumor growth . hence , its ligand retinoid acid ( ra ) or derivates thereof are currently tested as therapeutic agents in several cancer types ( table 2 ) . paradoxically , in some malignancies ra rather promotes cell survival , which may be due to the ability of ra to also activate ppars , and as a consequence expression of prosurvival genes is induced . could also show that the channeling of ra between these two nuclear receptor heterodimers is mediated by the cytoplasmic ra transporters crabp2 and fabp5 and thus is strongly depending on the fabp5/crabp2 ratio . thus , the channeling of ra to different receptor heterodimers appears to be crucial for the regulation of cell - proliferation , positively or negatively affecting tumor growth . interestingly , both proteins were found differentially expressed in metastatic and hpv - associated hnscc , but their biological and clinical effects remain to be investigated [ 51 , 52 ] . an additional way of biological regulation is epigenetic modulation playing an important role in cancer development ( reviewed in ) . gene silencing caused by aberrant hypermethylation of cpg islands has not only been detected in promoter regions of several tumor suppressor genes , but several studies show hypermethylation of the rar promoter in colon , breast , and lung cancers [ 54 , 55 ] . in head and neck carcinogenesis , hypermethylation of the rar promoter was found to be indeed associated with rar downregulation and hence appears to be biologically relevant . clinical aspects of retinoid acid receptors in hnsccas outlined above , a rationale for the use of retinoids in chemoprevention and cancer therapy was provided experimentally by different cellular and animal models . moreover , this strategy was supported by epidemiological data as well as by clinical trial outcomes [ 26 , 5961 ] . several clinical chemoprevention trials including patients with increased risk for developing cancer have shown that treatment with retinoids resulted in the suppression of precancerous lesions ( see [ 26 , 60 ] ) . also , certain retinoids inhibited the development of second primary tumors in patients who had been previously treated for an early - stage cancer but remained at high risk to relapse ( [ 26 , 60 ] and references within ) . however , other studies using isotretinoin or other retinoids ( e.g. , retinyl palmitate ) did not observe any benefit in second primary tumor development , recurrence , or mortality of hnscc or lung cancer [ 26 , 62 ] . current trials ( table 2 ) are therefore aiming to resolve these controversies by recruiting appropriate study populations as well as by the use of novel drugs and improved treatment protocols.reduced rar mrna levels have been observed not only in several malignant tumors ( [ 26 , 56 ] and references therein ) but also in premalignant oral lesions ( and references within ) . thus , most of the studies demonstrating rar downregulation were based on in situ hybridization and could therefore only show a decrease in the amount of mrna . were the first to demonstrate decreased expression of the rxr and rar// on protein level correlating with different stages of oscc development and progression . the molecular mechanism leading to downregulation or loss of rar is poorly understood , but it was suggested that expression of rar could depend on the intracellular level of retinoids . several studies demonstrated a decrease in the amount of rar during vitamin a deficiency as well as its upregulation by ra . additionally , there is evidence that retinoic acid induces the expression of rar mrna in certain cell lines , but not in the malignant counterparts of these cells . thus , transformed cells may have developed an aberrant response to retinoic acid due to the deregulated expression of coactivator / repressor proteins . ralhan et al . found a significant association between the increase in rar mrna levels and clinical responses of premalignant oral lesions to isotretinoin [ 65 , 66 ] . hence , rar indeed seems to contribute to the suppression of the premalignant phenotype and malignancy and may be causally linked to the clinical outcome in chemoprevention trials with retinoids [ 26 , 67 ] . if so , rar may indeed serve as a useful diagnostic marker in retinoid trials ( table 2 ) for the prevention of oral carcinogenesis . rar modulation by its agonist ligand all - trans retinoic acid ( atra ) represents a successful example of how targeting of an nr contributes to an impressive clinical benefit in liquid tumors ( and references therein ) . lessons learned from these studies clearly show that the therapeutic benefit could be further enhanced by combining atra with chromatin modulating agents , such as histone deacetylase inhibitors . nevertheless , the design of receptor specific drugs as well as an in depth understanding of the molecular regulation of rar biology is required in order to fully exploit its therapeutic benefit and minimize potential side - effects in the area of head and neck cancer [ 7 , 26 ] . as outlined above , a rationale for the use of retinoids in chemoprevention and cancer therapy was provided experimentally by different cellular and animal models . moreover , this strategy was supported by epidemiological data as well as by clinical trial outcomes [ 26 , 5961 ] . several clinical chemoprevention trials including patients with increased risk for developing cancer have shown that treatment with retinoids resulted in the suppression of precancerous lesions ( see [ 26 , 60 ] ) . also , certain retinoids inhibited the development of second primary tumors in patients who had been previously treated for an early - stage cancer but remained at high risk to relapse ( [ 26 , 60 ] and references within ) . however , other studies using isotretinoin or other retinoids ( e.g. , retinyl palmitate ) did not observe any benefit in second primary tumor development , recurrence , or mortality of hnscc or lung cancer [ 26 , 62 ] . current trials ( table 2 ) are therefore aiming to resolve these controversies by recruiting appropriate study populations as well as by the use of novel drugs and improved treatment protocols . reduced rar mrna levels have been observed not only in several malignant tumors ( [ 26 , 56 ] and references therein ) but also in premalignant oral lesions ( and references within ) . unfortunately , until recently no antibodies convincingly detecting rar were available . thus , most of the studies demonstrating rar downregulation were based on in situ hybridization and could therefore only show a decrease in the amount of mrna . were the first to demonstrate decreased expression of the rxr and rar// on protein level correlating with different stages of oscc development and progression . the molecular mechanism leading to downregulation or loss of rar is poorly understood , but it was suggested that expression of rar could depend on the intracellular level of retinoids . several studies demonstrated a decrease in the amount of rar during vitamin a deficiency as well as its upregulation by ra . additionally , there is evidence that retinoic acid induces the expression of rar mrna in certain cell lines , but not in the malignant counterparts of these cells . thus , transformed cells may have developed an aberrant response to retinoic acid due to the deregulated expression of coactivator / repressor proteins . found a significant association between the increase in rar mrna levels and clinical responses of premalignant oral lesions to isotretinoin [ 65 , 66 ] . hence , rar indeed seems to contribute to the suppression of the premalignant phenotype and malignancy and may be causally linked to the clinical outcome in chemoprevention trials with retinoids [ 26 , 67 ] . if so , rar may indeed serve as a useful diagnostic marker in retinoid trials ( table 2 ) for the prevention of oral carcinogenesis . rar modulation by its agonist ligand all - trans retinoic acid ( atra ) represents a successful example of how targeting of an nr contributes to an impressive clinical benefit in liquid tumors ( and references therein ) . lessons learned from these studies clearly show that the therapeutic benefit could be further enhanced by combining atra with chromatin modulating agents , such as histone deacetylase inhibitors . nevertheless , the design of receptor specific drugs as well as an in depth understanding of the molecular regulation of rar biology is required in order to fully exploit its therapeutic benefit and minimize potential side - effects in the area of head and neck cancer [ 7 , 26 ] . this subfamily is composed of the estrogen receptors ( er and er ) , the estrogen - related receptor , and the 3-ketosteroid receptors . besides the estrogen receptors themselves , many of the genes regulated by the er / estrogen - axis are critical for cell proliferation , inhibition of apoptosis , stimulation of invasion and metastasis , as well as for the promotion of angiogenesis ( see [ 10 , 11 ] and references within ) . since these processes clearly state hallmarks of cancer cells , it is well accepted that ers are implicated in various cancer types [ 9 , 21 ] . sex hormone receptors are expressed not only in sexual organs but , amongst others , also in the vascular epithelium , the lung epithelium , and the larynx . the expression of sex hormone receptors could also be demonstrated in hnscc by several studies [ 12 , 13 ] . both er isoforms as well as the progesterone receptor ( pr ) were detectable in cancer cells of the oral cavity , the salivary gland , and in laryngeal / hypopharyngeal cancers , whereas the tumor stroma was mostly negative [ 12 , 13 ] . expression of er inversely correlated with that of er in esophageal carcinoma , and a correlation of er levels with tumor dedifferentiation and staging was suggested [ 73 , 74 ] . clinical aspects of estrogen receptors in hnsccconsidering the impressive benefit of endocrine therapy in breast cancer , targeting sex steroid hormone receptor as a potential therapeutic strategy is also discussed for hnscc [ 12 , 75 ] . currently , two main strategies are pursued in endocrine therapy of er - positive tumors . one is based on steroidal antiestrogens like tamoxifen , which bind to the er , block its function , and ultimately induce receptor degradation [ 8 , 11 ] . the other is based on aromatase inhibitors and luteinizing hormone - releasing hormone agonists , which reduce the level of circulating estrogen , thereby inhibiting er activation by depriving the receptor of its ligand . tamoxifen was already shown to inhibit proliferation and invasion of hnscc cell lines , resulting in apoptosis , which could be further enhanced upon combination with cisplatin [ 7678 ] . thus , a therapeutic role of antiestrogens or aromatase inhibitors in the clinical management of hnscc is currently under investigation , and the results of just completed clinical trials ( table 2 ) are eagerly awaited.however , the precise molecular roles and impact of estrogen receptor - like receptors for the onset and/or progression of head and neck cancer remain to be clarified . this knowledge will be required , in order to rationally decide whether to further investigate the potential of modern endocrine therapy also for this tumor entity . considering the impressive benefit of endocrine therapy in breast cancer , targeting sex steroid hormone receptor as a potential therapeutic strategy is also discussed for hnscc [ 12 , 75 ] . currently , two main strategies are pursued in endocrine therapy of er - positive tumors . one is based on steroidal antiestrogens like tamoxifen , which bind to the er , block its function , and ultimately induce receptor degradation [ 8 , 11 ] . the other is based on aromatase inhibitors and luteinizing hormone - releasing hormone agonists , which reduce the level of circulating estrogen , thereby inhibiting er activation by depriving the receptor of its ligand . tamoxifen was already shown to inhibit proliferation and invasion of hnscc cell lines , resulting in apoptosis , which could be further enhanced upon combination with cisplatin [ 7678 ] . thus , a therapeutic role of antiestrogens or aromatase inhibitors in the clinical management of hnscc is currently under investigation , and the results of just completed clinical trials ( table 2 ) are eagerly awaited . however , the precise molecular roles and impact of estrogen receptor - like receptors for the onset and/or progression of head and neck cancer remain to be clarified . this knowledge will be required , in order to rationally decide whether to further investigate the potential of modern endocrine therapy also for this tumor entity . nrs are associated with head and neck cancer and hence seem to be at least partially amenable for prevention and/or treatment strategies . so far , three nr groups have mainly been linked with hnscc , the retinoic acid and the peroxisome proliferator - activated and the estrogen receptors . also , target genes activated by these nr subfamilies ( table 3 ) have been implicated as key elements in the molecular circuits involved in head and neck cancer development and progression . reports on other members of the nr superfamiliy are rather scarce for this tumor entity , suggesting that they have not been investigated so far . taking the thyroid hormone receptor as an example , many studies on its relevance for various malignancies have been conducted , whereas its role in head and neck cancer , including even thyroid carcinomas , has not been analyzed in detail . likewise , data on the cancer - related biological functions of orphan nrs are still missing for this tumor entity [ 7 , 8 ] . cancer 's achilles ' heel , it may be conceivable to speculate that molecules present in diet , tobacco , or beetle nut might deregulate the cell 's metabolism by affecting nrs and as such contribute to head and neck carcinogenesis [ 27 , 80 ] . of note , the development of novel nr ligands with improved specificity and activity is currently intensively pursued in the area of metabolic diseases ( see [ 7 , 8 , 81 ] ) . hence , an interdisciplinary exploitation of the existing knowledge of nr pharmacobiology may result in novel hnscc treatment approaches . in sum , keeping in mind the enormous success of nr targeting therapeutics in several malignancies , a systematic investigation of nr biology as well as of its clinical relevance is highly desirable also for head and neck cancer . together with the outcomes of current clinical trials ( table 2 ) , such improved knowledge will hopefully result in strategies with improved benefit for the patient .

introductionproduction of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis , study of infertility problems and determination of the probable role of these proteins as cancer - testis antigens . murine testis specific recombinant protein 101 ( mtex101 ) is a 38kda , gpi - anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues . the structure and function of mtex101 is not completely understood yet , but it is speculated that it may transduce biochemical signals into the cytoplasm since mtex101 does not have an intracellular domain but the precise mechanisms are still ambiguous.materials and methodsrna was extracted from three adult mice testis . the rna was used in rt - pcr , employing a pair of specific primers for mtex101 orf region . ta - cloning technique was performed by the insertion of mtex101 into a pgem - t easy vector , followed by its subcloning into a his - tagged expression vector , pet-28a ( + ) . the recombinant mtex101 was then produced by transfection of the expression vector into bl 21 ( de3 ) e. coli strain.resultsa recombinant protein , weighing 27kda , was produced upon iptg - induction of the bacterial host . the presence of mtex101 protein was detected through western blot analysis by anti - mtex101 peptide antibodies.conclusionwe produced mtex101 recombinant protein that could be used for the production of mono and polyclonal antibodies . head and neck squamous cell carcinomas are among the most common neoplasms worldwide and characterized by local tumor aggressiveness , high rate of early recurrences , development of metastasis , and second primary cancers . despite modern therapeutic strategies and sophisticated surgical management , overall survival - rates remained largely unchanged over the last decades . thus , the need for novel treatment options for this tumor entity is undeniable . a key event in carcinogenesis is the uncontrolled modulation of genetic programs . nuclear receptors belong to a large superfamily of transcription factors implicated in a broad spectrum of physiological and pathophysiological processes , including cancer . several nuclear receptors have also been associated with head and neck cancer . this review will summarize their mode of action , prognostic / therapeutic relevance , as well as preclinical and clinical studies currently targeting nuclear receptors in this tumor entity .

alcoholic liver disease ( ald ) represents a spectrum of clinical illness and morphological changes that range from fatty liver , hepatic inflammation , and necrosis ( alcoholic hepatitis ) to progressive fibrosis ( alcoholic cirrhosis ) . many of the toxic effects of ethanol in the liver have been associated with its metabolism . ethanol oxidation generates toxic products such as acetaldehyde , and reactive oxygen species result in oxidative stress that initiates apoptosis and cell injury [ 25 ] . it is activated by endotoxin , cytokines , and oxidative stress . in unstimulated cells , nf-b is a heterodimeric complex that is sequestered in the cytoplasm by its interaction with ib family of inhibitors . when these cells are stimulated , ib is phosphorylated with subsequent release of nf-b resulting in the translocation of nf-b from the cytoplasm to the nucleus where it activates the expression of target genes [ 7 , 8 ] . activation of nf-b increased expression of proinflammatory cytokines and chemokines that were key factors in ethanol - induced liver injury rats [ 912 ] . peroxisome proliferators activated receptors gamma ( ppar ) is a family of ligand - activated nuclear transcriptional factor which regulates cell differentiation , apoptosis , lipid metabolism , and inflammation . more recently , decreased expression of ppar has been found in rats with alcoholic liver fibrosis . these suggested that ppar may play an important role in the development of hepatocellular inflammation , necrosis , and fibrosis in rats with ethanol consumption . curcumin ( diferuloylmethane ) , an antiinflammatory and antioxidant compound , is isolated from the rhizomes of the plant curcuma longa linn . importantly , it has been showed that curcumin suppressed the activation of nf-b in ethanol - induced liver injury in rats . activation of ppar by curcumin resulted in inhibition of nf-b trans activating activity and increased expression of ppar at both the transcriptional and translational levels in activated hepatic stellate cells ( hscs ) . however , it is unclear whether curcumin had any effect in early stage of ethanol - induced liver injury . therefore , the present study determined the effect of curcumin on early stage of ethanol - induced liver inflammation and improved pathology in rats . female sprague - dawley rats , weighing 180220 grams , purchased from the national laboratory animal center , mahidol university , salaya , nakorn pathom , were used . the rats were kept in a controlled temperature room at 25 1c under standard conditions ( 12-hour day - night rhythm ) . all rats were received well care in accordance with the ethical committee , faculty of medicine , chulalongkorn university , thailand . curcumin in powder form ( cayman chemical company , usa ) is dissolved in 50% ethanol that freshly prepared for the experiment . all rats were fed with the controlled diet which contained 35% of energy from fat , 18% from protein , and 47% from carbohydrate for 4 weeks ad libitum . group 1 ( control , n = 8) : rats were fed distilled water ( 2.0 ml ) orally via an intragastric tube once per day for 4 weeks . group 2 ( ethanol , n = 8) : rats were fed 50% ethanol ( 7.5 g / kg bw a day ) orally via an intragastric tube twice a day for 4 weeks . group 3 ( ethanol + curi , n = 6 ) : rats were fed curcumin ( 200 mg / kg bw ) dissolved in 50% ethanol ( 7.5 g / kg bw a day ) via intragastric tube twice a day for 4 weeks . group 4 ( ethanol + curii , n = 7 ) : rats were fed curcumin ( 600 mg / kg bw ) dissolved in 50% ethanol ( 7.5 g / kg bw a day ) by using intragastric tube twice a day for 4 weeks . at the end of the study three small pieces of livers were collected , frozen in liquid nitrogen , and stored at 80c for mda analysis , sod activity , and ppar protein expression . the remaining of liver was fixed in 10% formalin solution to determine histopathology , nf-b activation , and hepatic apoptosis . after the liver samples were fixed in 10% formalin solution at room temperature , they were processed by the standard method . briefly , tissues were embedded in paraffin , sectioned at 5 m , and stained with hematoxylin - eosin , and then picked up on glass slides for light microscopy . all fields in each section were examined for grading of steatosis and necroinflammation according to colantoni et al . steatosis was scored as the percentage of parenchymal cells containing fat ( micro- or macrosteatosis ) : 0 = no parenchymal cells containing fat , 1 = < 20% of parenchymal cells containing fat , 2 = 2039% of parenchymal cells containing fat , 3 = 4050% of parenchymal cells containing fat , inflammation and necrosis were scored by the number of foci of inflammation and necrosis identified under low - power field of light microscope : 0 = no inflammation and necrosis , 1 = 1 focus per low - power field of inflammation and necrosis , 2 = 2 foci per low - power field of inflammation and necrosis , 3 = 3 or more foci per low - power field of inflammation and necrosis . mda was assayed by determining the rate of production of thiobarbituric acid - reactive components . an aliquot of 0.2 ml was mixed with solution containing 20% acetic acid , 0.8% thiobarbituric acid , and 8.1% sodium dodecyl sulfate , heated in water bath at 95c for 60 minutes . the solution was centrifuged for 10 minutes at 4 000 rpm , and the absorbance of the supernatant fraction was determined at a wavelength of 546 nm . sod was determined using the method of winterbourn , in which the light - triggered release of superoxide radicals from riboflavin leads to the formation of a blue complex through reaction with nitroblue tetrazolium . one gram of the liver was homogenized in 0.1 m phosphate buffer ph 7.4 on ice and cleared by centrifugation at 3 000 rpm at 4c for 15 minutes . the supernatant fraction was incubated in solution containing 0.067 m phosphate buffer ph 7.8 , 0.1 m edta , 1.5 mm nbt and 0.12 mm riboflavin for 10 minutes in an illuminated chamber with an 18 w fluorescent lamp . absorbance was recorded at 560 nm , and sod activity was expressed as units / mg protein . apoptosis was measured by the identification of apoptotic nuclei in sections of liver by fragment end labeling of dna ( apoptosis detection kit , chemicon , usa ) . in brief , the dna fragments were allowed to bind an antidigoxigenin antibody that was conjugated to a peroxidase . diaminobenzidine ( dab ) was applied to develop dark brown color and then the slides were counterstained with hematoxylin . the results were expressed as the number of positive stained cells per high - power field . the liver sections were deparaffinized with xylene and ethanol for ten minutes . after water washing , sections retrieved the antigen ( nf-b p65 , santa cruz , usa ) with citrate buffer ph 6.0 in microwave for thirteen minutes . next , 3% h2o2 and 3% normal horse serum were performed on the slides to block endogenous peroxidase activity for five minutes and blocked nonspecific binding for twenty minutes , respectively . then , the primary antibody used for nf-b p65 , a polyclonal antibody against the p65 subunit , was applied at a dilution of 1:150 for one hour at room temperature and incubated with the secondary antibody for thirty minutes . when the development of the color with dab was detected , the slides were counterstained with hematoxylin . under light microscopy , the positive stained cells presented dark brown in nucleus . the results were expressed as the number of positive stained cells per high - power field . liver sample ( 0.1 g ) was homogenized in 1 ml of lysis buffer for 30 minutes on ice and cleared by centrifugation at 12 000 rpm for 15 minutes at 4c . a 60 g of protein was applied to 10% sds - page gel , and the fractionated proteins were transferred to polyvinylidene fluoride membrane . membrane was blocked in tbst containing 5% dry nonfat milk for 1 hour and then incubated with ppar monoclonal antibodies ( 1:400 , santa cruz , usa ) overnight at 4c . then washed three times and incubated with secondary antibody , goat antimouse igg horseradish peroxidase ( 1:4,000 , cayman , usa ) for 1 hour . all data were presented as means and standard deviation ( sd ) . for comparison among all groups of animals , one way analysis of variance ( one - way anova ) and the histologic appearance of the liver in the control group was normal ( figure 1(a ) ) . in the ethanol - treated group , the histologic features showed mild to moderate steatosis and mild necroinflammation ( figure 1(b ) ) . rats treated with ethanol and curcumin 400 mg / kg bw a day improved the liver histopathology that showed only mild steatosis but not necroinflammation ( figure 1(c ) ) . the high dose of curcumin treatment ( 1,200 mg / kg bw a day ) also improved the liver histopathology that showed mild steatosis and mild necroinflammation ( figure 1(d ) ) . the summary of steatosis and necroinflammation score were shown in table 1 . the level of hepatic mda , a marker of lipid peroxidation , increased significantly in ethanol - treated group as compared with control group ( 3.42 1.36 versus 1.44 0.24 nmol / mg protein , p < .05 ) . curcumin treatment ( 400 or 1,200 mg / kg bw a day ) decreased the elevation of hepatic mda level significantly when compared with ethanol - treated group ( 1.43 0.14 versus 3.42 1.36 and 1.43 0.29 versus 3.42 1.36 nmol / mg protein , resp . ; our results showed that the level of hepatic sod activity of the control group was 1081.36 145.01 units / mg protein , while that of the ethanol - treated group was 1135.86 209.48 units / mg protein . in rats treated with ethanol and curcumin ( 400 or 1,200 mg / kg bw a day ) , the levels of hepatic sod activity were 966.28 139.44 and 967.84 116.66 units / mg protein , respectively . the number of apoptotic nuclei in the liver of control group was very low ( 0.38 0.28 cells / high - power field ) . in contrast , the numbers of apoptotic cells were observed frequently in centrilobular area in ethanol - treated group when compared with control group ( 2.43 2.68 versus 0.38 0.28 cells / high - power field , p < .05 ) ( figures 4 and 5 ) . there was a trend of decreased apoptosis in low dose of curcumin treatment , but the difference did not reach a statistical significance ( figure 4 ) . the number of positive stained cells in the liver of ethanol - treated group was significantly higher than control group ( 1.08 0.52 versus 0.04 0.04 cells / high - power field , p < .05 ) . in contrast , curcumin treatment ( 400 or 1,200 mg / kg bw a day ) decreased the number of positive stained cells significantly when compared with ethanol - treated group ( 0.15 0.02 versus 1.08 0.52 and 0.17 0.09 versus 1.08 0.52 cells / high - power field , resp . ; p < .05 ) ( figures 6 and 7 ) . in order to examine the change of ppar protein expression in early stage of ethanol - induced liver injury the ppar protein expression in control group was 0.57 0.19 , and ethanol group was 0.68 0.16 . rats treated with ethanol and curcumin ( 400 or 1,200 mg / kg bw a day ) had 0.44 0.03 and 0.54 0.23 , respectively . these data did not show a significant change in ppar protein expression in the liver in all groups ( figure 8) . ethanol oxidation generates toxic metabolites , free radicals ; and induces a state of oxidative stress which contributes to the pathogenesis of ald . importantly , oxidation of ethanol through the cytochrome p450 2e1 ( cyp 2e1 ) generates superoxide anion radical and hydrogen peroxide [ 2 , 21 , 22 ] . these free radicals are capable of damaging many cellular components such as dna , protein , and lipid . one of the characteristic features of oxidative stress is enhancement of lipid peroxidation . a number of studies have been demonstrated that ethanol intake increased the formation of lipid peroxidation product , such as mda [ 2426 ] . we found that an increase in hepatic mda level as well as pathological changes were observed in ethanol - treated group . to counteract this oxidative stress , cells have a variety of antioxidant enzymes , including sod , catalase , and glutathione peroxidase . the effects of chronic ethanol exposure on activity of sod are controversial , with reports of decrease or no changes [ 25 , 28 ] . these studies may reflect variations in experimental design , diet , and duration of ethanol feeding . decreased sod activity in ethanol fed rats was associated with enhancement of lipid peroxidation and severe pathology of liver . therefore , the sod activity in liver did not change in early ethanol - induced liver injury . oxidative stress can also initiate or amplify inflammation through upregulation of several genes involved in the inflammatory response . one such gene is nf-b , whose activation results in the upregulation of proinflammatory cytokines . activation of nf-b and upregulation of cytokine production occurred in ethanol - induced liver injury and are associated with lipid peroxidation [ 9 , 10 ] . evidence was presented that curcumin prevented ethanol - induced liver injury in rats by inhibiting the expression of nf-b - dependent genes . although , a high dose of curcumin treatment ( 1,200 mg / kg bw ) was not better than low dose ( 400 mg / kg bw ) , the present study showed that curcumin improved ethanol - induced liver injury by reduction of oxidative stress and inhibition of nf-b activation . ethanol - induced liver injury has been linked to oxidative stress caused by the production of reactive oxygen intermediates that cause mitochondrial dysfunction , leading to a release of proapoptotic factors such as cytochrome c that can activate caspases and initiate the apoptotic cascade in hepatocytes . jin and coworker observed the pathological changes and investigated the correlation of hepatocyte apoptosis with cyp2e1 expression and oxygen free radical in rats with ald . using the tunel assay , we detected a difference in apoptosis between the control and ethanol - treated group that was similar to human alcoholic hepatitis and experimental rat model of ald [ 3335 ] . cells in centrilobular area are low o2 and nutrient supply thus the distribution of apoptotic cells is observed frequently in centrilobular area . in this study curcumin treatment did not detect a difference in hepatocyte apoptosis ; however , this was a trend of decreased apoptosis in low does of curcumin treatment . this stage showed severe liver injury and hsc activation . in normal liver , hscs undergo a process known as activation , which upregulate cytokines and growth factor . for instance , platelet - derived growth factor is capable of inhibiting ppar expression via mitogen - activated protein kinase - mediated phosphorylation of ppar . also , tnf- , inflammatory cytokine , is known to inhibit ppar expression in adipocytes and an early phase of hsc activation in liver fibrosis [ 38 , 39 ] , thus alcoholic liver fibrosis rats could decrease ppar expression . our model showed only mild steatosis , necroinflammation , and no hsc activation ; therefore , no change of ppar protein expression was found in ethanol - treated group . further studies should be determined roles of ppar in different stages of ald . in conclusion , our study demonstrated that curcumin , a representative phenolic antioxidant and antiinflammmation , could improve histopathology of liver in early stage of ethanol - induced liver injury by reduction of oxidative stress and inhibition of nf-b activation . for hepatocyte apoptosis , curcumin treatment might have a trend of decreased apoptotic cells in ethanol - fed rats . this study provides class iii evidence that more patients are seizure - free and have stopped aed treatment in the long term after resective epilepsy surgery than nonoperated epilepsy patients . in sweden , all epilepsy surgery procedures are reported to the snesur , which was initiated in 1990 . an internal control system rejects certain impossible combinations of data and regular external quality controls are performed by an independent controller . since 2005 , the follow - up has been extended from 2 years to 5 , 10 , and 15 years postoperatively . snesur contains baseline information on patient 's epilepsy history , preoperative seizure types and syndromes , mean monthly seizure frequency during the year preceding the presurgical investigation , aeds , preoperative investigations , psychosocial data , surgical data ( type and location of surgery ) , histopathologic diagnoses , and postoperative complications . two - year follow - up data cover seizure situation , aeds , and psychosocial data . the 5- , 10- , and 15-year follow - ups are structured telephone interviews regarding seizure situation , aeds , psychosocial aspects , and driving . in this study , we analyzed seizure outcome and aed medication 5 and 10 years after resective epilepsy surgery in patients who had 5- and 10-year follow - ups in 2005 to 2007 ( and hence were operated on in 2000 to 2002 and 1995 to 1997 ) . the cohort comprises the 327 patients who had resective surgery during these time periods . in 2005 to 2007 , 144/176 patients operated on in 1995 to 1997 ( 98/116 adults and 46/60 children 18 years ) had a 10-year follow - up , and 134/151 patients operated on in 2000 to 2002 ( 92/103 adults and 42/48 children 18 years ) had a 5-year follow - up . seventeen patients were reoperated before long - term follow - up and there were 11 deaths . twenty - one patients ( 6.4% ) were lost to follow - up ( for details , see figure e-1 on the neurology web site at www.neurology.org ) . as a control group , consecutive patients who underwent presurgical investigations during the same time periods but were not operated were identified at 3 of the 6 operating centers ( gteborg , uppsala , and lund ) . eighty adults and 13 children out of 94 adults and 13 children underwent a cross - sectional long - term follow - up in 2008 after a mean of 9.3 years ( adults ) and 8.8 years ( children ) using the same structured telephone interview as for the surgical group . thirteen adult patients had died ( 4 epilepsy - related deaths ) and 1 was lost to follow - up ( 1% ) . reasons for not having surgery were nonconclusive workup ( n = 41 ) , multifocality ( n = 27 ) , patient declined surgery ( n = 12 ) , seizure onset within eloquent cortex ( n = 11 ) , and neuropsychological reasons ( n = 2 ) . seizure freedom ( without or with aura , international league against epilepsy [ ilae ] class i and ii ) is reported for the year preceding the follow - up except for patients with sustained seizure freedom since surgery , which is separately reported . for patients with continuing seizures or seizure relapse postoperatively , the mean monthly seizure frequency in the last year of follow - up is categorized as follows : 75% reduction in seizure frequency ; 50%74% reduction in seizure frequency ; 0%49% reduction in seizure frequency ; and increased seizure frequency . for comparison between 2 groups , fisher exact test was used for dichotomous variables , mann - whitney u test for continuous variables , and mantel - haenszel test for ordered categorical variables . logistic regression analysis was performed for each independent variable to predict seizure - free outcome . a forward stepwise multiple logistic regression was used to select independent predictors to outcome . only univariate predictors attaining a p value of < 0.10 this study was approved by the regional board of medical ethics at the university of gothenburg . consent for research was obtained from all controls . for operated patients , the board considered long - term follow - up as a quality control measure not necessitating individual consent . primary research questions were as follows : are more patients seizure - free and without aed in the long term after resective epilepsy surgery compared to nonoperated patients ? this longitudinal observational study provides class iii evidence that 41% of adults and 44% of children have sustained seizure freedom in the long term after surgery compared to none of the nonoperated patients ( p < 0.0005 ) . also , 43% of seizure - free adults and 86% of seizure - free children had stopped aeds after 10 years compared to none of the nonoperated patients ( p < 0.0005 ) . this study was approved by the regional board of medical ethics at the university of gothenburg . consent for research was obtained from all controls . for operated patients , the board considered long - term follow - up as a quality control measure not necessitating individual consent . primary research questions were as follows : are more patients seizure - free and without aed in the long term after resective epilepsy surgery compared to nonoperated patients ? this longitudinal observational study provides class iii evidence that 41% of adults and 44% of children have sustained seizure freedom in the long term after surgery compared to none of the nonoperated patients ( p < 0.0005 ) . also , 43% of seizure - free adults and 86% of seizure - free children had stopped aeds after 10 years compared to none of the nonoperated patients ( p < 0.0005 ) . table 1 shows baseline characteristics of the operated patients and the controls , children ( 18 years ) and adults . the only adult who underwent hemispherectomy at age 20 was added to the pediatric hemispherectomy group . none of the baseline characteristics differed between operated and nonoperated patients , except for number of previously tried aeds in adults ( p < 0.001 ) and iq in children ( p = 0.042 ) . baseline data of operated patients and nonoperated controls there was no significant difference in seizure outcome between the operated patients who had follow - up after 5 years compared to 10 years . in order to enable comparison with the nonoperated group , who had a mean long - term follow - up of 9.1 years ( range 514 ) , the results from 5- and 10-year follow - ups in the operated patients were merged ( figure 1 ) . two - year and long - term ( mean follow - up time 7.6 years , range 510 ) seizure situation for patients after resective epilepsy surgery compared to long - term follow - up of nonoperated controls ( mean follow - up time 9.2 years , range 514 ) . seizure - free patients include those with sustained seizure freedom with or without aura since surgery ( blue ) and patients seizure - free at least the last year before follow - up ( green ) . overall , 117 ( 62% ) of the operated adults were seizure - free at long - term follow - up compared to 11 ( 14% ) of the controls ( p < 0.001 ) . fifty percent ( n = 93 ) of the operated adults had sustained seizure freedom at the 2-year and 41% ( n = 78 ) at the long - term follow - up . none of the controls was seizure - free for the whole time period . for the children , 44 ( 50% ) in the operated group were seizure - free the year before long - term follow - up , compared to 5 ( 38% ) in the control group ( not significant ) . fifty - three percent ( n = 46 ) of the operated children had sustained seizure freedom since surgery at the 2-year and 44% ( n = 39 ) at the long - term follow - up , compared to none in the control group . for both adults and children , the proportion with sustained seizure freedom at long term was significantly higher compared to the nonoperated patients ( p < 0.0005 ) . details on seizure outcome for the operated patients at 2 , 5 , and 10 years after epilepsy surgery are shown in table e-1 . eighty - seven percent of the adults as well as of the children who were seizure - free after 2 years were seizure - free at the 5- or 10-year follow - ups ( 95% confidence interval [ ci ] 7794 and 8095 , respectively ) . seizure outcomes for adults and children after different resection types are illustrated in table 2 . the most common resection type was tlr ( 81% in adults , 43% in children ) followed by frontal lobe resection ( flr ) in adults ( 12% ) and multilobe resection in children ( 20% ) . in adults , the best long - term seizure outcome was after tlr ( 63% seizure - free , sustained seizure freedom since surgery in 44% ) . in children the tlr outcome ( 60% seizure - free , 55% with sustained seizure freedom ) was matched by the long - term hemispherectomy results ( 60% seizure - free , all with sustained seizure freedom ) . if the extratemporal procedures are summed up , 31% of adults had sustained seizure freedom at long - term follow - up , compared to 36% of the children . seizure outcomes in the long term related to resection type the main histopathologic diagnoses were categorized as follows : hippocampal sclerosis ( hs , n = 45 ) , neurodevelopmental tumors ( n = 34 ) , low - grade astrocytomas ( n = 21 ) , cavernous hemangiomas ( n = 21 ) , malformations of cortical development ( n = 52 ) , gliosis ( n = 65 ) , and other ( n = 40 ) . highest seizure freedom rates were seen in hs ( 60% , 47% sustained since surgery ) and epileptogenic lesions ( 72% , 57% sustained since surgery ) ( table e-2 ) . the univariate relationship between long - term seizure freedom and the following baseline variables was explored for both operated patients and controls : epilepsy duration in percent of life length ( relative epilepsy duration , in odds ratio [ or ] increment units of 10% ) , 30 seizures / month , and presence of secondarily generalized tonic - clonic seizures ( sgtcs ) . for the operated patients , additional variables were positive mri , resection types , and histopathology ( figure 2 ) . for the controls , the univariate associations were nonsignificant although in the same direction as for the operated patients ( relative epilepsy duration , or 0.89 [ 95% ci 0.731.09 ] , 30 seizures / month , or 0.88 [ 95% ci 0.223.47 ] , and sgtcs , or 0.56 [ 95% ci 0.181.68 ] ) . in the surgical group , multivariate analysis was performed , where 30 seizures / month , or 0.40 ( 95% ci 0.230.69 ) , and relative epilepsy duration , or 0.91 ( 95% ci 0.831.00 ) , were negative predictors , while positive mri , or 1.96 ( 95% ci 1.083.55 ) , was a positive predictor of seizure - free long - term outcome . the auc value ( goodness of fit ) for the final multiple model was 0.66 . for the categorical variables with more than 2 categories ( resection type and histopathology ) , temporal lobe resections ( tlr ) and epileptogenic lesions ( epi les ) were chosen as reference categories . ci = confidence interval ; epi dur ( per 10% of life ) = epilepsy duration in percentage of life length , odds ratio units per 10% of life ; epileptogenic lesions = gangliogliomas , dysembryoplastic neuroepithelial tumors , cavernomas , and low - grade astrocytomas ; flr = frontal lobe resection ; he = hemispherectomy ; hs = hippocampal sclerosis ; mcd = malformations of cortical development ; mlr = multilobe resection ; or = odds ratio ; other = vascular malformations other than cavernomas , cysts , tuberous sclerosis , unknown histopathology ; p / olr = parietal or occipital lobe resection ; sgtcs = secondarily generalized tonic - clonic seizures . at baseline , 183 ( 66% ) of the patients had 2 aeds , 85 ( 31% ) had 1 aed , and 8 ( 3% ) had no aeds . the number of aeds used by the operated seizure - free patients decreased over time ( figure 3 ) . overall , 45 ( 54% ) patients who were seizure - free at 10-year follow - up had stopped aeds , compared to none of the seizure - free controls ( p < 0.0005 ) . more children than adults had stopped aeds ( 86% of the children and 43% of the adults , p = 0.002 ) . there was no significant association between the proportion of seizure - free patients who stopped aeds and resection type . among patients with seizures , the proportion with 2 aeds had increased from 77% preoperatively to 82% 10 years after surgery . number of antiepileptic drugs at the start of preoperative investigations , and after 2 , 5 , and 10 years for patients who were seizure - free at 5- or 10-year follow - up . table 1 shows baseline characteristics of the operated patients and the controls , children ( 18 years ) and adults . the only adult who underwent hemispherectomy at age 20 was added to the pediatric hemispherectomy group . none of the baseline characteristics differed between operated and nonoperated patients , except for number of previously tried aeds in adults ( p < 0.001 ) and iq in children ( p = 0.042 ) . there was no significant difference in seizure outcome between the operated patients who had follow - up after 5 years compared to 10 years . in order to enable comparison with the nonoperated group , who had a mean long - term follow - up of 9.1 years ( range 514 ) , the results from 5- and 10-year follow - ups in the operated patients were merged ( figure 1 ) . two - year and long - term ( mean follow - up time 7.6 years , range 510 ) seizure situation for patients after resective epilepsy surgery compared to long - term follow - up of nonoperated controls ( mean follow - up time 9.2 years , range 514 ) . seizure - free patients include those with sustained seizure freedom with or without aura since surgery ( blue ) and patients seizure - free at least the last year before follow - up ( green ) . overall , 117 ( 62% ) of the operated adults were seizure - free at long - term follow - up compared to 11 ( 14% ) of the controls ( p < 0.001 ) . fifty percent ( n = 93 ) of the operated adults had sustained seizure freedom at the 2-year and 41% ( n = 78 ) at the long - term follow - up . none of the controls was seizure - free for the whole time period . for the children , 44 ( 50% ) in the operated group were seizure - free the year before long - term follow - up , compared to 5 ( 38% ) in the control group ( not significant ) . fifty - three percent ( n = 46 ) of the operated children had sustained seizure freedom since surgery at the 2-year and 44% ( n = 39 ) at the long - term follow - up , compared to none in the control group . for both adults and children , the proportion with sustained seizure freedom at long term was significantly higher compared to the nonoperated patients ( p < 0.0005 ) . details on seizure outcome for the operated patients at 2 , 5 , and 10 years after epilepsy surgery are shown in table e-1 . eighty - seven percent of the adults as well as of the children who were seizure - free after 2 years were seizure - free at the 5- or 10-year follow - ups ( 95% confidence interval [ ci ] 7794 and 8095 , respectively ) . seizure outcomes for adults and children after different resection types are illustrated in table 2 . the most common resection type was tlr ( 81% in adults , 43% in children ) followed by frontal lobe resection ( flr ) in adults ( 12% ) and multilobe resection in children ( 20% ) . in adults , the best long - term seizure outcome was after tlr ( 63% seizure - free , sustained seizure freedom since surgery in 44% ) . in children the tlr outcome ( 60% seizure - free , 55% with sustained seizure freedom ) was matched by the long - term hemispherectomy results ( 60% seizure - free , all with sustained seizure freedom ) . if the extratemporal procedures are summed up , 31% of adults had sustained seizure freedom at long - term follow - up , compared to 36% of the children . seizure outcomes in the long term related to resection type the main histopathologic diagnoses were categorized as follows : hippocampal sclerosis ( hs , n = 45 ) , neurodevelopmental tumors ( n = 34 ) , low - grade astrocytomas ( n = 21 ) , cavernous hemangiomas ( n = 21 ) , malformations of cortical development ( n = 52 ) , gliosis ( n = 65 ) , and other ( n = 40 ) . highest seizure freedom rates were seen in hs ( 60% , 47% sustained since surgery ) and epileptogenic lesions ( 72% , 57% sustained since surgery ) ( table e-2 ) . the univariate relationship between long - term seizure freedom and the following baseline variables was explored for both operated patients and controls : epilepsy duration in percent of life length ( relative epilepsy duration , in odds ratio [ or ] increment units of 10% ) , 30 seizures / month , and presence of secondarily generalized tonic - clonic seizures ( sgtcs ) . for the operated patients , additional variables were positive mri , resection types , and histopathology ( figure 2 ) . for the controls , the univariate associations were nonsignificant although in the same direction as for the operated patients ( relative epilepsy duration , or 0.89 [ 95% ci 0.731.09 ] , 30 seizures / month , or 0.88 [ 95% ci 0.223.47 ] , and sgtcs , or 0.56 [ 95% ci 0.181.68 ] ) . in the surgical group , multivariate analysis was performed , where 30 seizures / month , or 0.40 ( 95% ci 0.230.69 ) , and relative epilepsy duration , or 0.91 ( 95% ci 0.831.00 ) , were negative predictors , while positive mri , or 1.96 ( 95% ci 1.083.55 ) , was a positive predictor of seizure - free long - term outcome . the auc value ( goodness of fit ) for the final multiple model was 0.66 . for the categorical variables with more than 2 categories ( resection type and histopathology ) , temporal lobe resections ( tlr ) and epileptogenic lesions ( epi les ) were chosen as reference categories . ci = confidence interval ; epi dur ( per 10% of life ) = epilepsy duration in percentage of life length , odds ratio units per 10% of life ; epileptogenic lesions = gangliogliomas , dysembryoplastic neuroepithelial tumors , cavernomas , and low - grade astrocytomas ; flr = frontal lobe resection ; he = hemispherectomy ; hs = hippocampal sclerosis ; mcd = malformations of cortical development ; mlr = multilobe resection ; or = odds ratio ; other = vascular malformations other than cavernomas , cysts , tuberous sclerosis , unknown histopathology ; p / olr = parietal or occipital lobe resection ; sgtcs = secondarily generalized tonic - clonic seizures . at baseline , 183 ( 66% ) of the patients had 2 aeds , 85 ( 31% ) had 1 aed , and 8 ( 3% ) had no aeds . the number of aeds used by the operated seizure - free patients decreased over time ( figure 3 ) . overall , 45 ( 54% ) patients who were seizure - free at 10-year follow - up had stopped aeds , compared to none of the seizure - free controls ( p < 0.0005 ) . more children than adults had stopped aeds ( 86% of the children and 43% of the adults , p = 0.002 ) . there was no significant association between the proportion of seizure - free patients who stopped aeds and resection type . among patients with seizures , the proportion with 2 aeds had increased from 77% preoperatively to 82% 10 years after surgery . number of antiepileptic drugs at the start of preoperative investigations , and after 2 , 5 , and 10 years for patients who were seizure - free at 5- or 10-year follow - up . since long - term results of epilepsy surgery can not be obtained from rcts , observational studies with defined quality criteria are needed . a number of requirements for studies on the prognosis after epilepsy surgery have been suggested . studies based on snesur meet many of these requirements , with a prospective design , representative study populations , a standard protocol for outcome measures , and prolonged longitudinal follow - up with few patients lost to follow - up . we found 63% of adults and 60% of children to be seizure - free after tlr at the long - term follow - up and most had continuous seizure freedom since surgery ( adults 41% , children 44% ) . as expected , the results were not as good after flr , with 44% of adults seizure - free ( sustained in 35% ) , and in children 27% seizure - free ( all sustained ) . this is still substantially better than for the patients who could not be operated ( 14% seizure - free adults , none sustained and 38% seizure - free children , none sustained ) . the adults who underwent other types of resections ( posterior or multilobar ) were few , and only 3 had sustained long - term seizure freedom . children who had undergone posterior resections or hemispherectomies had long - term seizure freedom in 55% and 60% , respectively ( sustained in 46% vs 60% ) . long - term outcome after resective epilepsy surgery is often reported cross - sectionally . in a meta - analysis from 2005 based on 78 studies , 66% of tlr patients , 46% of parietal or occipital lobe resection patients , and 27% of flr patients were seizure - free at follow - up 5 years postsurgery , but the authors point out that few studies reported sustained seizure freedom from surgery . a number of recent longitudinal long - term outcome studies report sustained seizure freedom after tlr . sustained seizure freedom is reported as engel 1 , engel 1a , or engel 1a+b and in a few studies as ilae class i and ii . sustained seizure freedom around 5 years postoperatively varies between 46% and 55% and 60% to 80% . among the studies with moderate rates of sustained seizure freedom , 2 of 4 are prospective , and our prospective study with 41% sustained seizure freedom at long - term follow - up after tlr in adults and 44% in children supports these results . a number of studies report cross - sectional long - term follow - up of pediatric epilepsy surgery , with seizure freedom in 50% to 82% of patients 5 to 12 years after tlr . only one study is longitudinal and reports sustained seizure freedom ( engel 1 ) in 54% of patients 5 years after tlr . there are few retrospective reports of long - term outcomes in patients after flr or other extratemporal resections . sustained seizure freedom 5 years after surgery varies in these studies from 15% and 27% to 52% , which can be compared to 31% ( adults ) vs 36% ( children ) in our cohort of 86 patients with varying extratemporal resections . long - term reports from population - based cohorts are sparse . in a retrospective cross - sectional comprehensive audit from ireland , national long - term seizure outcome data were reported . forty - four percent of patients were seizure - free 10 years after resective surgery , but the total number of operated patients could not be accounted for . a recent norwegian retrospective questionnaire study in children with a response rate of 70% reported 58% seizure - free after a mean of 7 years . however , sustained seizure freedom was not reported in these studies . in a recent systematic review , 20 studies comparing surgical results to those of a nonsurgical control group were identified ; 13 had a follow - up of at least 4 years . the controls were in general patients evaluated for surgery who for various reasons were not operated . overall 44% of surgical patients were seizure - free at follow - up ( in 90% of the studies , seizure freedom was reported at last follow - up or last year ) compared to 12% of medically treated patients . this is well in line with our findings of 58% seizure - free at last year of follow - up in the surgical group and 17% in the nonsurgical group , children and adults together . it must be remembered that patients who after preoperative investigations are considered ineligible for epilepsy surgery are not comparable to surgically treated patients , and they may have another disease course . it is difficult though to identify other reasonable controls for long - term follow - up studies , and from a clinical point of view they match the operated patients in many baseline variables . predictors for seizure freedom in the long term ( 4 years ) have been sought by several investigators . while some found no remaining predictors in multivariate analysis , the most commonly identified predictors are sgtcs at baseline and epilepsy duration or age at surgery . we found high baseline seizure frequency and relative epilepsy duration to be negatively and positive mri to be positively related with long - term seizure freedom . epilepsy duration before undertaking presurgical investigation has repeatedly been shown not to have shortened significantly over the years . these results from long - term outcome studies underline the importance of earlier identification of good candidates for resective epilepsy surgery . there is no common swedish protocol for discontinuation of aeds in seizure - free patients . most adults continue aeds the first 2 postoperative years ; further decisions about withdrawal are taken individually . we found that 43% of seizure - free adults and as many as 86% of seizure - free children had discontinued aeds after 10 years . the reasons for this difference are unknown ; possibly , social consequences of seizure relapse are of greater importance to adults ( e.g. , driving or occupation ) , who therefore may choose to continue aeds . by contrast , more concern about adverse effects of aeds on the developing brain in children may lead to earlier decisions about withdrawal . the proportion of seizure - free adults and children in whom aeds have been withdrawn after successful epilepsy surgery varies widely across studies . in a meta - analysis from 2007 , 9 studies were identified and a pooled analysis showed that 27% of seizure - free children and 19% of seizure - free adults had discontinued aeds at a mean follow - up of 7 years . however , in an indian study , aed withdrawal was systematically planned for all seizure - free patients after tlr and was successful in 63% of 258 patients who were followed for 5 years . very similar to our results in children , a canadian study found that 82% of seizure - free children had stopped aeds 10 years after surgery . possible reasons for these differences in patient decisions about aed cessation may include socioeconomic and cultural differences , as well as how doctors inform their patients , which partly depends on their own assessment of recurrence risk . the timetostop study showed that early withdrawal of aed in children did not influence the long - term seizure outcome but could unmask incomplete surgical success sooner , and the authors suggest that it is now time to plan an rct of aed withdrawal after epilepsy surgery in children . like most , if not all , observational outcome studies after epilepsy surgery , we had no masking in seizure outcome assessment . our nonoperated control group was recruited from the 3 largest of the swedish epilepsy surgery centers and is hence not a national sample . on the other hand , we have a regional referral system and it is reasonable to assume that this sample is representative of the population of patients who are evaluated for surgery in sweden but not operated . patients need detailed counseling before deciding to undergo irrevocable brain surgery . realistic expectations concerning long - term outcomes are part of the information they need to consider . this prospective population - based study contributes to this knowledge and supports seizure outcome data from prospective single - center studies , and also shows that a greater proportion of seizure - free patients than in most studies had stopped aed treatment 10 years after surgery . funded by grants from the swedish research council ( grant 521 - 2011 - 169 ) , the sahlgrenska academy at gothenburg university through the lua / alf agreement ( grant alfgbg137431 ) , the gothenburg foundation for neurological research , the gothenburg medical society , the margaretahem foundation , and an unconditional research grant from glaxosmithkline to anna edelvik . k. malmgren served on an educational advisory board for ucb and has received speaker 's honoraria from ucb .

to study the mechanism of curcumin - attenuated inflammation and liver pathology in early stage of alcoholic liver disease , female sprague - dawley rats were divided into four groups and treated with ethanol or curcumin via an intragastric tube for 4 weeks . a control group treated with distilled water , and an ethanol group was treated with ethanol ( 7.5 g / kg bw ) . treatment groups were fed with ethanol supplemented with curcumin ( 400 or 1 200 mg / kg bw ) . the liver histopathology in ethanol group revealed mild - to - moderate steatosis and mild necroinflammation . hepatic mda , hepatocyte apoptosis , and nf-b activation increased significantly in ethanol - treated group when compared with control . curcumin treatments resulted in improving of liver pathology , decreasing the elevation of hepatic mda , and inhibition of nf-b activation . the 400 mg / kg bw of curcumin treatment revealed only a trend of decreased hepatocyte apoptosis . however , the results of sod activity , ppar protein expression showed no difference among the groups . in conclusion , curcumin improved liver histopathology in early stage of ethanol - induced liver injury by reduction of oxidative stress and inhibition of nf-b activation . objective : to investigate prospective , population - based long - term outcomes concerning seizures and antiepileptic drug ( aed ) treatment after resective epilepsy surgery in sweden.methods : ten- and 5-year follow - ups were performed in 2005 to 2007 for 278/327 patients after resective epilepsy surgery from 1995 to 1997 and 2000 to 2002 , respectively . all patients had been prospectively followed in the swedish national epilepsy surgery register . ninety - three patients , who were presurgically evaluated but not operated , served as controls.results:in the long term ( mean 7.6 years ) , 62% of operated adults and 50% of operated children were seizure - free , compared to 14% of nonoperated adults ( p < 0.001 ) and 38% of nonoperated children ( not significant ) . forty - one percent of operated adults and 44% of operated children had sustained seizure freedom since surgery , compared to none of the controls ( p < 0.0005 ) . multivariate analysis identified 30 seizures / month at baseline and long epilepsy duration as negative predictors and positive mri to be a positive predictor of long - term seizure - free outcome . ten years after surgery , 86% of seizure - free children and 43% of seizure - free adults had stopped aeds in the surgery groups compared to none of the controls ( p < 0.0005).conclusions : this population - based , prospective study shows good long - term seizure outcomes after resective epilepsy surgery . the majority of the patients who are seizure - free after 5 and 10 years have sustained seizure freedom since surgery . many patients who gain seizure freedom can successfully discontinue aeds , more often children than adults.classification of evidence : this study provides class iii evidence that more patients are seizure - free and have stopped aed treatment in the long term after resective epilepsy surgery than nonoperated epilepsy patients .

the prevalence of obesity among adolescents in the united states has increased dramatically in the past 30 years , with particularly high rates among hispanic , african american , and native american youth [ 14 ] . currently 33.6% of all adolescents 1219 years of age in the united states are either overweight or obese . although the causes of obesity are complex , it is widely recognized that poor nutrition and physical inactivity play important roles . for this reason , public health interventions targeting youths frequently focus on health promotion programs in schools [ 69 ] , as well as calling for nutrition and exercise counseling in the health care setting [ 1012 ] . the potential for physicians to influence behavioral changes among patients through simple nutrition and exercise advice , as opposed to more time - intensive counseling is crucial . some studies have documented the value of physician - counseling either used as a stand - alone strategy or as part of a coordinated effort to help patients make changes in their diet and physical activity patterns . national health organizations have also recognized the importance of clinician counseling and have called for an increase in the provision of both nutrition and exercise counseling given to adolescents during physician visits [ 15 , 16 ] . the surgeon general 's recent vision for a healthy and fit nation ( 2010 ) encourages clinicians to recommend healthy eating and increased physical activity to their patients and recommends training for clinicians and health care students on effective ways to counsel patients on lifestyle behavior change . in california , where adolescent overweight and obesity rates are comparable to national levels , major programmatic and policy responses aimed at obesity prevention in adolescents have been undertaken at schools to limit nonnutritious foods and beverages sold on campus and in communities to increase access to nutritious foods and safer places to exercise [ 8 , 9 , 19 ] . however , little is known about the frequency of weight - related counseling given to different racial / ethnic groups or those with limited access to care , over the course of treating or preventing obesity in the health care environment . a recent study found that race was an important factor that explained the prevalence of nutrition or physical activity counseling among california adolescents : african americans compared to whites were more likely to receive nutrition counseling , while hispanics compared to whites were more likely to receive both nutrition and physical activity counseling . with regards to insurance type , data suggests that california adolescents who are uninsured or who qualify for low - cost / free insurance are at greatest risk for overweight or obesity , yet a recent national study found that adolescents with private insurance generally receive more counseling , compared to those who have low - cost insurance . changes in the frequency of obesity - related counseling overtime by race / ethnicity or insurance type are yet to be examined . previous research in california suggests that physician obesity - related counseling has been declining , with overall counseling rates between 2003 and 2007 , shifting from 75% to 59% for nutrition and 74% to 60% for physical activity . insufficient time , lack of resources , inadequate reimbursement , and patient noncompliance are typically cited as barriers to provision of routine advice by health care practitioners . the objectives of this current study were to document trends from 2003 to 2009 in either nutrition or exercise counseling or a combination of both among california adolescents by race / ethnicity and by insurance type . these findings can provide guidance for policies and programs in a state with high rates of adolescent overweight or obesity and large ethnic populations at particular risk . further , we hypothesized that physicians would favor dietary counseling over exercise counseling when providing counseling . in an earlier study , stafford et al . found that physicians offered dietary counseling to obese patients 41.5% of the time , while exercise counseling was offered only 32.8% of the time . among healthy weight participants , branner and colleagues found higher rates of nutrition compared with exercise counseling among children and adolescents ( 42.1% and 26.1% , resp . ) . data demonstrating trends in nutrition and exercise counseling by race / ethnicity and by insurance type were obtained using four biennial california health interview surveys ( 20032009 ) , the largest state surveys in the united states . the california health interview survey ( chis ) is a two stage sampling , weighted , random digital dialing telephone survey , representative of the california noninstitutionalized population . within households , an adult and adolescent were randomly selected and interviewed by trained chis interviewers ; adolescents were directly interviewed . the chis program obtained informed consent from all individuals participating in the survey and this current study was deemed exempt or waived for human subjects review by the university of california , berkeley , institutional review board . in this study , obesity - related counseling refers to simple advice about nutrition and/or exercise practices , as opposed to more time - intensive counseling . adolescents self - reported whether they discussed nutrition or exercise habits with their physician at their last routine exam : when you had your last routine physical exam , did you and a doctor talk about nutrition or healthy eating ? and when you had your last routine physical exam , did you and a doctor talk about exercise or physical activity ? data were analyzed using stata version 10 , with the svy module to account for weighting and the raking method in variance estimation . obesity - related counseling proportions are presented graphically by race / ethnicity and by insurance type for the period 20032009 . to better represent the obesity - related counseling construct , we categorized this variable as respondents having no discussions of nutrition or exercise with their physician , discussing either nutrition or exercise , or discussing both nutrition and exercise with their physician . participants ' self - reported their weight and height , which were used to generate the centers for disease control and prevention ( cdc ) bmi age and gender specific percentiles , categorized into underweight ( < 5th percentile ) , normal weight ( 5th<85th percentile ) , overweight ( 85th<95th percentile ) , and obese ( 95th percentile ) . in addition , insurance type variables ( uninsured , medicaid , healthy families , employer - based , privately owned insurance , and other public insurance ) were collapsed into the categories ( uninsured , low - cost / free , employer - based , and private insurance ) . medicaid is the united states health insurance program for certain low - income individuals and families , which is jointly funded by state and federal governments , while healthy families is a low - cost health insurance program for children and adolescents who do not have health insurance and who do not qualify for medicaid . table 1 presents the study sample characteristics , using the chis ( 2009 ) . participants ranged in age from 12 to 17 years , with 51.0% being male and 49.0% being female . the sample consisted primarily of hispanics ( 49.3% ) and non - hispanic whites ( 33.5% ) . most adolescents had some form of health insurance , with almost 60% being covered through their parents'/guardians ' employer - sponsored health insurance . less than half of all adolescents ( 44.8% ) were at or above 300% of the federal poverty level . based on self - reported data , 28.7% of california adolescents were either overweight or obese , while 48.2% of all california adolescents received counseling on both nutrition and exercise subjects ( table 1 ) . the majority of respondents ( 84.7% ) reported having a physical exam within the past year ( table 1 ) . previously published data indicate how california adolescent demographics have changed from 2003 to 2007 . figure 1 and table 2 present data for obesity - related counseling stratified by race / ethnicity . when examining nutrition or exercise counseling separately for the period from 2003 to 2009 , african americans generally reported higher levels of nutrition than exercise counseling , while whites generally reported higher levels of exercise than nutrition counseling . hispanics generally reported higher levels of nutrition than exercise counseling during 20032005 , after which counseling levels remained consistent . overall , trends show that counseling declined between 2003 and 2009 for all groups , except for hispanics and whites which started to increase again after 2007 ; american indians / alaska natives reported a sharp decline in 2009 . between 2003 and 2009 , the proportion of adolescents who reported counseling on both nutrition and exercise decreased from 66.8% to 53.7% among hispanics ; from 60.7% to 15.1% among american indians / alaska natives ; from 61.7% to 33.4% among asians ; from 58.8% to 42.9% among african americans ; and from 60.0% to 46.2% among whites ( figure 1 ) . figure 2 and table 3 present data for obesity - related counseling stratified by insurance type . those who had private insurance generally received exercise counseling more frequently than nutrition counseling over the study time period . there appeared to be no imbalance in frequency of nutrition or exercise counseling for the uninsured , low - cost / free or employer - based groups , except in 2003 when adolescents who were underinsured or had low - cost insurance reported more nutrition than physical activity counseling . counseling appeared to decline from 20032009 among all insurance types , although after 2007 , a slight increase was observed for the low - cost / free insurance group . among those who were uninsured counseling declined from 70.8% to 42.2% ; among the low - cost / free group , counseling declined from 64% to 53.4% ; among the employer group , counseling declined from 61.8% to 46.6% and among the private insurance group , counseling declined from 58.9% to 39.8% ( figure 2 ) . as early as the 1950s , the american medical association council on food and nutrition cited the benefits of nutrition counseling , as well as inadequacies in nutrition education in u.s . medical schools . further , counseling has been shown to be valuable in helping patients to change their behavior and to achieve weight loss and can be even more beneficial if used as part of a coordinated approach with health education materials . kreuter and colleagues reported that patients who received a combination of health education materials , followed by physician counseling were 51% more likely to increase their leisure time physical activity , and 35% more likely to reduce fat from dairy sources at follow - up . some groups that need obesity prevention counseling the most may still be missing out , including american indians / alaska natives , african americans , and the uninsured . time trend findings from 2003 to 2009 indicate that nutrition and exercise counseling decreased for all racial / ethnic groups except for hispanics and whites , for whom it started to increase after 2007 . our findings also suggest that counseling levels in california for racial / ethnic groups and for patients with different types of insurance are generally higher compared to national levels . the higher counseling levels reported in this study , compared with national figures , may have been due to fewer barriers or more public health awareness of the obesity epidemic in california and stronger health care leadership . previously reported barriers to counseling include insufficient reimbursement rates , lack of time , lack of training for medical providers in policy advocacy related to improved nutrition and activity environments , and the need for information on evidence - based obesity - related messages and referral networks for nutrition counseling . when examining nutrition or exercise counseling separately during the study period ( 20032009 ) , interesting differences were found by race / ethnicity : hispanic and african american chis adolescents generally reported higher levels of nutrition than exercise counseling , while whites generally reported higher levels of exercise , than nutrition counseling . meanwhile , participants who had private insurance generally received more exercise than nutrition counseling during the study period . further research is needed to investigate the underlying factors that may explain the differences in the findings for racial / ethnic groups . the chis surveys were only able to identify the existence of discussions or conversations that adolescents may have had with their physicians regarding nutrition and exercise messages , but were not able to ascertain the depth of these discussions . given the limited time physicians have in working with patients , it is unlikely that any advice given would be in - depth psychological advice . further , it is difficult to ascertain whether these conversations were initiated by the physician or the patient . these data were unable to measure specific evidence - based obesity - related messages ( limiting sugar sweetened beverages , increasing fruit and vegetables , reducing television viewing , and increasing moderate - to - vigorous physical activity ) [ 7 , 29 ] . the potential for recall bias also exists since adolescents were asked to self - report nutrition and/or exercise data that occurred during their last physical exam , however , most adolescents ( 84.7% ) had a physical exam at their physician visit within the past year . this is the first study to examine trends in obesity - related counseling by race / ethnicity and by insurance type among california adolescents . additionally , this is one of the first studies to examine trends in counseling among american indians / alaska natives , a group that is also disproportionately affected by overweight and obesity [ 3 , 4 ] . the findings from our study have demonstrated that the downward trend in obesity prevention counseling in california among racial / ethnic groups and health insurance groups has changed course and has begun to increase . future analysis of the biennial chis surveys will indicate if the trend continues in this direction . it is widely accepted that obesity prevention should follow a socioecological approach , combining multiple interventions tailored to specific demographic groups . while a vast amount of work has already been conducted in california to implement population - wide obesity prevention policies in schools and in low - income communities , this momentum must also be applied to california primary health care settings . however , for physician - based counseling to continue to increase among the general adolescent population and among vulnerable high - risk groups in particular , this will require institutional and policy changes . research on effective ways to support clinicians ' use of the aforementioned evidence - based obesity - related messages is still in its infancy , although preliminary data from clinic - based obesity interventions have begun to show promising results . at least one us state ( washington ) has adopted clinic - based programs to address the childhood obesity epidemic by establishing partnerships with hospitals , health care plans , and community - based organizations . other states may consider adopting such a program in the primary care setting in order to build on obesity prevention programs and policies previously implemented in schools and low - income communities . student perception of their dental school experience is an essential measure of the success of dental education . undergraduates ' feedback and suggestions are very important for improving the curriculum and learning process . this information also helps determine the students ' preferences regarding different elements of their educational experience . endodontics teaching can be considered complex , difficult , and stressful because of the complex anatomy of the root canal system , responsibility toward patients , and low self - confidence . however , teaching endodontics in recent years has improved as a result of the development of knowledge , techniques , and materials . dental students at taibah university , saudi arabia , take a preclinical full - year endodontic course in the 3 year of their 6-year bachelor of dental surgery degree . the course consists of 28 h of theoretical lectures and 28 3-h laboratory sessions , during which they perform technical aspects of root canal treatment on extracted single- and multi - rooted teeth . there is a one - semester clinical endodontic course in the 4 year that consists of 14 theoretical lectures and fourteen 3-h clinical sessions during which students treat single- and multi - rooted teeth . in the 5 year , endodontic treatments are performed as part of a comprehensive dentistry care course under the supervision of specialists . this study examined the endodontic experiences , perceptions of endodontic practice , and self - rated confidence of dental students enrolled in taibah university , saudi arabia . study approval was obtained from the research ethics committee of the college of dentistry , taibah university ( reference number : tucdrec/20160107/alrahabi ) . this study enrolled 41 undergraduate dental students registered in endodontic courses in the 2015 academic year at the college of dentistry , taibah university , saudi arabia . participation was voluntary , and students were informed that they could refuse participation . a questionnaire was distributed to 19 4-year and 25 5-year students in the final month of the academic year after they had had the maximum amount of training . the questions evaluated self - confidence at performing nonsurgical root canal treatment and experiences in this discipline . the level of confidence was classified using a 5-point scale as very confident , confident , neutral , not very confident , or not at all confident . to compare results , the chi - square test and mann whitney u - test were used . statistical analyses were carried out using spss version 20.0 ( spss , chicago , il , usa ) . statistical significance was set at p < 0.05 . the overall response rate was 93% , with 18 of the 19 4-year students and 23 of the 25 final - year students returning questionnaires . table 1 shows the results for the first three questions ( first endodontic case treated , level of case difficulty , and number of endodontic treatments performed by the student ) . first endodontic case treated , level of case difficulty , and number of endodontic treatments performed self - confidence of 4th- and 5th - year dental students regarding the steps of nonsurgical root canal treatment levels of confidence in the different steps of root canal treatment confidence levels differed significantly between 4- and 5-year students in the following steps of root canal treatment : determining the working length , taking and interpreting radiographs during root canal treatment , evaluating the quality of root canal obturation , and recalling the patients periodically in the correct manner . table 3 summarizes the results of the last question regarding suggestions to improve the teaching of endodontic courses . in dentistry , evaluating competence is an important step toward validating the quality of graduating dentists , although the relationship between the self - confidence and clinical competence of medical students is not fully understood . increasing the confidence of medical students should increase their competence in clinical practice . one way to standardize education is to poll students using questionnaires to help in the assessment , evaluation , and improvement of education . this study obtained information about the confidence of undergraduate dental students at taibah university , saudi arabia regarding endodontics . it revealed that an upper incisor 78% was the most common tooth first treated by students . upper incisor root canal treatment is relatively easy , and this should encourage students . in another study , most of our students described the experience with the first case as okay 78% , while 7.4% described it as easy and 14.6% described the first case as difficult , possibly because the first case for those students involved a molar or premolar . in other studies , students considered molars to be the most difficult tooth to treat . in our study , the maximum number of teeth treated in the 4 year was four cases , by 66.7% of the students , while the maximum number of teeth treated in 5 year was 11 cases , by 8.8% of the students . the number of teeth treated by students in the 4 and 5 year did not meet the recommendations of the european society of endodontology 2001 guidelines , which advised that for adequate competency a student should complete root canal treatments in 20 teeth . although the european society of endodontology published new undergraduate curriculum guidelines for endodontology in 2013 , these focused on the quality and consistency of student performance more than simply the quantity of clinical exposure . however , one study reported that 81% of the students in 48 dental schools in the european union achieved the minimum number of root canal treatments required for their graduation : the number of treated cases ranged between 3 and 80 canals , and the average was 17 canals . in our study , we observed that confidence varied according to both the year the student was in and the practical steps of nonsurgical root canal treatment . both groups reported relatively good confidence , although there were significant differences between 4 and 5 year students regarding some steps . fourth - year students were more confident than 5-year students in the following : determining the working length , dealing with x - rays during root canal treatment , evaluating root canal obturation , and recalling patient at the correct time . this might be because there were fewer 4-year students in the sample , there are fewer requirements in 4 year , and strict supervision by supervisors helped 4-year students more than 5-year students . confidence regarding working length determination was low in both 4- and 5-year students . this might be the result of the root canal anatomy , which many dental students find difficult to learn because of its variation among individuals . the reduction in confidence regarding endodontic radiology , the evaluation of root canal obturation , and determining the correct recall period probably results from insufficient clinical exposure . murray et al . wrote that a lack of clinical exposure in the undergraduate curriculum reduces the confidence that develops with clinical practice . students ' suggestions for improving the teaching of endodontics focused on two major issues : using rotary nickel - titanium ( niti ) files during treatment and increasing credit hours for the endodontic course . introducing advances in endodontics into undergraduate training , such as niti rotary instruments , may improve the clinical experience of students and their self - confidence because it will help increase the numbers of cases treated . believe that introducing niti rotary instruments into the undergraduate dental curriculum would be safe and improve endodontics teaching because inexperienced operators can learn to use rotary instruments adequately with brief training . nevertheless , another study found that intensive preclinical training is a prerequisite for using niti rotary instruments . changing the methods of teaching endodontics so that students can complete root canal treatment more easily and quickly , with minimal procedural accidents , will improve clinical outcomes . low self - confidence can be ameliorated by increasing clinical exposure , which will help students to obtain the necessary skills through experience . fourth- and fifth - year dental students at taibah university , saudi arabia , are confident regarding root canal treatment , although they report lower confidence in some steps of the root canal treatment process . endodontics education should be improved by increasing preclinical and clinical sessions and using new teaching methods that introduce recent advances in endodontics in the undergraduate curriculum .

purpose . obesity is a serious health threat , particularly among racial / ethnic minorities and those who are uninsured , yet little is known about the implementation of nutrition or exercise counseling or the combination of both among these groups . trends in counseling by race / ethnicity and types of insurance were examined . methods . trend analyses were conducted with the california health interview surveys among those ages 1217 for the period 20032009 . results . race / ethnicity : receipt of both counseling methods declined from 20032009 for all racial / ethnic groups , except hispanics and whites , for whom increases in counseling began after 2007 . hispanics and african americans generally reported higher levels of nutrition than exercise counseling , while whites generally reported higher levels of exercise than nutrition counseling for the study period . insurance type : receipt of both counseling methods appeared to decline from 20032009 among all insurance types , although after 2007 , a slight increase was observed for the low - cost / free insurance group . those with private health insurance generally received more exercise counseling than nutrition counseling over the study period . conclusions . counseling among all racial / ethnic groups and insurance types is warranted , but particularly needed for african americans , american indian / alaska natives , and the uninsured as they are at highest risk for developing obesity . institutional and policy changes in the health care environment will be beneficial in helping to promote obesity - related counseling . objective : this study examined the endodontic experience , perceptions of endodontic practice , and self - rated confidence of dental students enrolled in taibah university , saudi arabia.materials and methods : a questionnaire was distributed to 41 undergraduate dental students registered in endodontic courses in the 2015 academic year . the questionnaire evaluated their confidence performing nonsurgical root canal treatment . the level of confidence was classified using a 5-point scale as very confident , confident , neutral , not very confident , or not at all confident . the data were analyzed using spss version 20.0 ( spss , chicago , il , usa).results : the participation rate was 93% . the maxillary incisor was the most common first tooth treated . the students were relatively confident , but their confidence levels were lower regarding endodontic radiology , evaluation of root canal obturation , and determining the correct recall period for the patient.conclusion:the confidence of undergraduates in endodontics must be enhanced to increase their clinical competence when performing root canal treatment .

a 36-year - old woman complained of an insidious onset of generalized myoclonus that first became apparent at age 27 years . she had no perinatal problems and her development was normal in childhood and juvenile periods . adult - onset myoclonus had worsened progressively from right hand to four extremities , tremulous voice and gait disturbance developed after 3 years from disease onset , and she could not continue working as a nurse . she had no history of febrile convulsions or seizure , infectious disease in the central nervous system , exposure to toxic materials , or intake of herbal drugs . her younger brother aged 40 years old also had progressive generalized myoclonus , which was detected 6 years ago at age 34 years ( figure 1 ) . the patient was alert and oriented , and her mini - mental state examination score was 30 . she did not have gaze palsy , and her vision and hearing were normal ; however , her voice was tremulous and generalized positive myoclonus was observed at four extremities and body . negative myoclonus , dystonia , tremor , and rigidity were not detected , and both motor and sensory functions were intact . there was no evidence of cerebellar dysfunction , and she did not have an ataxic or parkinsonian gait , although she staggered slightly because of myoclonus . examination of the eyes revealed cherry - red spots ( figure 2 ) , but her electroencephalogram was normal . no white matter lesion or cerebellar atrophy was detected in an mri of her brain ( figure 3 ) . neuraminidase , hexosaminidase a , and -galactosidase activities in the leukocytes and cultured fibroblasts in patient and younger brother were normal . the cherry - red spot is a pale perifoveal ring that develops when large deposits of lipid , sphingolipid , or oligosaccharide material accumulate in the ganglionic cells at the macula.2 this is a characteristic finding in storage diseases , including the sialidoses , gm1 and gm2 gangliosidoses , neuronal ceroid lipofuscinosis , niemann - pick disease ( groups a through d ) , farber s lipogranulomatosis , and metachromatic leukodystrophy . interestingly , niemann - pick disease , farber s lipogranulomatosis , and metachromatic leukodystrophy are not associated with myoclonus . moreover , the patient in this report did not have typical findings of these 3 diseases such as the organomegaly , cognitive impairment , and gaze palsy seen in niemann - pick disease4 ; the hoarseness , arthritis , and subcutaneous nodules seen in farber s lipogranulomatosis ; or the abnormal brain mri findings in metachromatic leukodystrophy . in ceroid lipofuscinosis , sialidosis , gm1 and gm2 gangliosidoses , myoclonus , and maculopathy ( e.g. a cherry - red spot ) may coexist . although they are quite similar in appearance , the macular abnormality seen in patients with neuronal ceroid lipofuscinosis ( which has been described as bulls - eye maculopathy ) can be distinguished from the cherry - red spot by color and shape , as well as by the decreased visual acuity and visual - field restriction that are common in neuronal ceroid lipofuscinosis.5 moreover , adult - onset lipofuscinosis has an autosomal dominant inheritance , rather than the autosomal recessive pattern seen in this patient.6 gm1 gangliosidosis results from a deficiency of -galatosidase ; the adult form ( type 3 ) presents as a slowly progressive dementia with prominent parkinsonian features and extra - pyramidal dysfunction , particularly dystonia.7 gm2 gangliosidosis results from a deficiency of hexosaminidase a ; the late form ( with an onset during adolescence and young adulthood ) may be characterized by cognitive dysfunction , cerebellar dysfunction , upper and lower motor neuron involvement , and extrapyramidal dysfunction.8 because -galatosidase and hexosaminidase activity was normal in this patient , because she has a normal level of intelligence , and because no other prominent pyramidal or extrapyramidal dysfunction was detected , we might not diagnose this patient as gm1 and gm2 gangliosidoses . sialidosis is an inherited , autosomal recessive disease associated with a neuraminidase deficiency.9 it has 2 major clinical manifestations : type i ( late , adult onset ) and type ii ( early , infantile onset ) . type i sialidosis is typically found in patients aged 8 to 25 years and is characterized by cherry - red spot myoclonus , seizure , neuropathy , corneal clouding , and difficulty walking , but with normal vision and intelligence ( obrien , 1978 ) . type ii sialidosis is characterized by dysmorphism , myoclonus , mental retardation , ocular cherry - red spots , and hepatosplenomegaly . the patient in this report may present clinical evidence of type i sialidosis , but her laboratory findings do not support this diagnosis ; activities of the neuraminidase were normal . a similar case of progressive myoclonic epilepsy has been reported.10 differences from the previous report which described a patient with progressive myoclonic epilepsy , cherry - red spots and negative enzyme deficiency were theses ; 1 ) the onset - age was older than the previous report ( 27 versus 13 years , adult versus juvenile - onset ) ; 2 ) the patient had a sibling with same disease , which indicated she had inherited disease , but the patient in previous report did not have familial history ; 3 ) the patient in our report did not have a history of seizure , and it is different from the patient with myoclonic epilepsy . although the cause of cherry - red spot myoclonus is not clear , to our knowledge , this is the first report of adult - onset familial cherry - red spot myoclonus caused by an unknown type of lysosomal storage disease in korea . with the worldwide spread of mycobacterium tuberculosis ( mtb ) strains resistant to both isoniazid and rifampicin ( rif ) , multidrug - resistant ( mdr ) tuberculosis ( tb ) has become a major public health problem posing formidable challenges due to its complex diagnostic and treatment requirements . this highlights the need for having rapid molecular diagnostic techniques which could help facilitate early diagnosis and appropriate delivery of anti - tubercular therapy . xpert mtb / rif assay ( cepheid ) , a real - time automated nucleic acid amplification system is one such technique which detects mtb as well as mutations that confer resistance to rif in > 2 h. it was installed in our institute ( guru gobind singh medical college and hospital , faridkot ) in october 2013 under revised national tuberculosis control programme ( rntcp ) tb xpert project supported by who - stop tb partnership - unitaid . rif is a principle first line anti - tb drug and rif resistance is a surrogate marker for mdr - tb . the genetic basis of rif resistance ( in approximately 95% cases ) is the presence of mutations in 81 bp rif resistance determining region ( rrdr ) of the rpob gene , corresponding to codons 507533 ( escherichia coli numbering system ) , which codes for a beta subunit of rna polymerase of mtb . studies conducted in diverse geographical areas have shown that the burden of mdr - tb and the mutations responsible for drug resistance vary from country to country and region to region . however , there is not enough of information about this important aspect from north india and the data from malwa region of punjab is almost lacking . therefore , this retrospective study was conducted to determine the frequency of rif resistance and rpob gene mutations among the suspected tb / mdr cases of malwa region of punjab ( districts : faridkot , fazilka , firozpur , moga , bathinda , and muktsar - area approximately 14,981 km ) , using xpert mtb / rif assay . knowledge of the pattern of mutations present in rif - resistant isolates could provide insight into the epidemiology of rif - resistant mtb isolates of this particular area . a total of 1612 sputum specimens which originated from patients with suspected mdr pulmonary tb of malwa region of punjab were received in microbiology laboratory , under rntcp for xpert mtb / rif assay between october 2013 and february 2015 . the samples were subjected to ziehl - neelsen ( zn ) staining and xpert mtb / rif assay . the xpert mtb / rif assay was performed directly on sputum specimens using the xpert mtb / rif assay g4 version 5 and the software version 4.4a as per the manufacturer 's instruction ( cepheid , sunnyvale , ca , usa ) . briefly , it consisted of inactivation of the sputum specimen with naoh and isopropanol ( sample reagent ) used in 1:2 ratio for 15 min . the mixture was then introduced into the xpert mtb / rif cartridge and loaded into the xpert mtb / rif instrument for dna extraction and amplification of 192 bp segments of the rpob gene . the detection consists of hybridization of the amplicon with five overlapping probes complementary to the rpob core the reports were communicated electronically to the concerned district tb officer and drug - resistant tb center . chi - square test of proportions was used to identify a significant difference between two or more groups , and p < 0.05 was considered as statistically significant . to determine the association of various epidemiological characteristics of the patients to rif - resistant tb , odds ratio ( or ) and 95% confidence interval ( ci ) were calculated using spss statistical software version 20.0.0 ( spss inc . , the relationship of smear positivity grade with the relative bacterial burden and xpert mtb / rif ct value ( given in genexpert operator 's manual by cepheid ) was also analyzed . chi - square test of proportions was used to identify a significant difference between two or more groups , and p < 0.05 was considered as statistically significant . to determine the association of various epidemiological characteristics of the patients to rif - resistant tb , odds ratio ( or ) and 95% confidence interval ( ci ) were calculated using spss statistical software version 20.0.0 ( spss inc . , the relationship of smear positivity grade with the relative bacterial burden and xpert mtb / rif ct value ( given in genexpert operator 's manual by cepheid ) was also analyzed . out of 1612 specimens tested , 1308 were positive for mtb by xpert mtb / rif assay . there were 34 errors and 18 specimens showed no results . all these specimens were retested , and valid results were obtained except in two ( one showed error in the second sample also and another could not be processed due to insufficient material in the second specimen ) . zn staining of the 1308 specimens positive for mtb by xpert mtb / rif assay showed that 1240 were positive for acid fast - bacilli ( afb ) with 272 ( 21.9% ) being grade 3 positive . correlation of smear positivity grade with relative bacterial burden and ct value showed that out of total 272 grade 3 positive specimens , 69.4% , 22.5% , 6.5% , and 1.6% had high ( ct < 16 ) , medium ( ct 1622 ) , low ( ct 2028 ) , and very low ( ct > 28 ) bacterial load , respectively . of the specimens showing grade 2 , grade 1 scanty positivity , the maximum belonged to high and medium , high and medium , and medium bacterial load groups , respectively . in sputum negative specimens ( 68/1308 ) the maximum number belonged to low bacterial load group . of 1308 mtb - positive samples by xpert tb / rif assay , 130 ( 9.9% ) demonstrated rif resistance . results were obtained in 4 specimens and on retesting 2 came out to be rif sensitive . all these 4 specimens were smear - positive and had ct value was > 28 . the study of the association of epidemiological characteristics of the patients to the rif - resistant tb showed that rif resistance was present in statistically significant higher number in previously treated patients in comparison to the new patients ( 95% ci 1.1487.135 , p = 0.006 and or = 2.86 ) and in patients with afb - positive sputum smears to afb - negative smears ( 95% ci 0.91115.5 , p = 0.048 and or 3.7 ) . however , the difference was found to be statistically insignificant between the different age groups of patients ( p = 0.053 ) , male and female patients ( 95% ci 0.7061.57 , p = 0.798 and or 1.054 ) , patients from rural and urban background ( 95% ci 0.691.49 , p = 0.918 and or 1.02 ) and hiv - positive and hiv - negative patients ( 95% ci 0.2194.13 , p = 0.949 and or 0.953 ) . the study of mutation pattern of rif resistance in 130 specimens revealed that in 129 , rpob gene mutations in 81 bp rrdr of mtb were detected by one of the 5 different probes ( a , b , c , d , e ) , while in one specimen , mutation combination , i.e. mutations associated with more than one probe ( a and b both ) was present . the probe frequencies associated with the observed rif resistance were as follow : e 73/130 ( 56% ) , b 28/130 ( 21.5% ) , d 18/130 ( 13.8% ) , a 11/130 ( 8.4% ) , and c 1/130 ( 0.7% ) [ table 1 ] . accordingly , the frequencies of mutations at 5 different rpob gene regions were 529533 ( 56% ) , 512518 ( 21.5% ) , 523529 ( 13.8% ) , 507511 ( 8.4% ) , and 518523 ( 0.7% ) . district - wise pattern of mutations seen in 130 rifampicin resistant tuberculosis specimens correlation of mutations with the rif - resistant sputum specimens obtained from different districts [ table 1 ] revealed that probe e which represented mutation in region 529533 was the most common in all the six districts of the study . only one specimen revealed rif resistance by probe c depicting mutation in codon region 518523 . out of 1612 specimens tested , 1308 were positive for mtb by xpert mtb / rif assay . there were 34 errors and 18 specimens showed no results . all these specimens were retested , and valid results were obtained except in two ( one showed error in the second sample also and another could not be processed due to insufficient material in the second specimen ) . zn staining of the 1308 specimens positive for mtb by xpert mtb / rif assay showed that 1240 were positive for acid fast - bacilli ( afb ) with 272 ( 21.9% ) being grade 3 positive . correlation of smear positivity grade with relative bacterial burden and ct value showed that out of total 272 grade 3 positive specimens , 69.4% , 22.5% , 6.5% , and 1.6% had high ( ct < 16 ) , medium ( ct 1622 ) , low ( ct 2028 ) , and very low ( ct > 28 ) bacterial load , respectively . of the specimens showing grade 2 , grade 1 scanty positivity , the maximum belonged to high and medium , high and medium , and medium bacterial load groups , respectively . in sputum negative specimens ( 68/1308 ) the maximum number belonged to low bacterial load group . of 1308 mtb - positive samples by xpert tb / rif assay , 130 ( 9.9% ) demonstrated rif resistance . results were obtained in 4 specimens and on retesting 2 came out to be rif sensitive . all these 4 specimens were smear - positive and had ct value was > 28 . the study of the association of epidemiological characteristics of the patients to the rif - resistant tb showed that rif resistance was present in statistically significant higher number in previously treated patients in comparison to the new patients ( 95% ci 1.1487.135 , p = 0.006 and or = 2.86 ) and in patients with afb - positive sputum smears to afb - negative smears ( 95% ci 0.91115.5 , p = 0.048 and or 3.7 ) . however , the difference was found to be statistically insignificant between the different age groups of patients ( p = 0.053 ) , male and female patients ( 95% ci 0.7061.57 , p = 0.798 and or 1.054 ) , patients from rural and urban background ( 95% ci 0.691.49 , p = 0.918 and or 1.02 ) and hiv - positive and hiv - negative patients ( 95% ci 0.2194.13 , p = 0.949 and or 0.953 ) . the study of mutation pattern of rif resistance in 130 specimens revealed that in 129 , rpob gene mutations in 81 bp rrdr of mtb were detected by one of the 5 different probes ( a , b , c , d , e ) , while in one specimen , mutation combination , i.e. mutations associated with more than one probe ( a and b both ) was present . the probe frequencies associated with the observed rif resistance were as follow : e 73/130 ( 56% ) , b 28/130 ( 21.5% ) , d 18/130 ( 13.8% ) , a 11/130 ( 8.4% ) , and c 1/130 ( 0.7% ) [ table 1 ] . accordingly , the frequencies of mutations at 5 different rpob gene regions were 529533 ( 56% ) , 512518 ( 21.5% ) , 523529 ( 13.8% ) , 507511 ( 8.4% ) , and 518523 ( 0.7% ) . district - wise pattern of mutations seen in 130 rifampicin resistant tuberculosis specimens correlation of mutations with the rif - resistant sputum specimens obtained from different districts [ table 1 ] revealed that probe e which represented mutation in region 529533 was the most common in all the six districts of the study . only one specimen revealed rif resistance by probe c depicting mutation in codon region 518523 . although , india ranks first out of the 22 countries with the highest burden of tb , but the reliable information on the magnitude of mdr - tb in the country is largely unavailable . by using xpert mtb / rif assay , we observed rif resistance which is a surrogate marker of mdr - tb in 9.9% of suspected cases of mdr - tb of malwa region of punjab . however , higher prevalence of mdr - tb has been reported in other indian studies ( lucknow 27.8% , new delhi 17.9% , and central india 17% ) . globally , 3.7% of new cases and 20% of previously treated cases are estimated to have mdr - tb . in india , the estimated figure in new cases with mdr - tb is 2.1% with ci 1.52.7 and estimated percentage in previously treated cases is 15% with ci of 1317 , respectively . rntcp carried out drug - resistant surveys in accordance with global guidelines and indicated a low prevalence of mdr - tb , i.e. , > 3% among new cases and 1217% in previously treated cases in gujarat , and maharashtra and andhra pradesh . in the present study , the figure was 3.9% ( 5/126 ) in the new and 10.6% ( 125/1182 ) in the retreatment cases , and the difference between the two was statistically significant ( p = 0.006 ) . gupta et al . had also observed that mdr - tb was significantly higher in previously treated cases compared to new cases and concluded that the previous exposure to anti - tb agents was the most common cause of developing mdr . in the present study , another statistically significant association was seen between smear - positive mdr cases in comparison to smear - negative mdr cases ( p = 0.048 ) . gupta et al . had also observed a higher but statistically insignificant association of mdr - tb with smear positivity . this could be because all the mdr - tb patients of our study had pulmonary tb and these patients are more likely to have cavitary lesions and positive sputum smear results . our study population showed slightly higher prevalence of rif resistance in hiv - negative patients than hiv - positive patients but statistically the difference was insignificant ( p = 0.949 ) . this is in contrast to the study of gupta et al . who observed a statistically significant difference in mdr - tb in hiv - negative in comparison to a hiv - positive group of patients . quy et al . reported that the failure of treatment of mdr - tb was associated with mdr and not with hiv infection . although statistically there were insignificant differences between different age groups showing mdr - tb ( p = 0.053 ) , a maximum number of cases in the present study were in the age group of 2140 years . other indian studies have also reported the predominance of younger age group with mdr - tb . there were also a higher number of male patients and patients from a rural background with mdr - tb in our study . as young adult males are economically productive segment of our society , high mdr - tb in this group has several socioeconomic implications . while detecting tb / mdr cases , xpert mtb / rif assay showed 2.1% ( 34/1612 ) errors in the present study . mboowa et al . have reported 12% errors using xpert mtb / rif assay g3 version 3 and g4 version 5 , with more than half being contributed by the former version . the lower percentage of errors in the present study could be because that we used the upgraded g4 version 5 of xpert mtb / rif assay . similar to our study , rufai et al . have reported 1.8% errors using xpert mtb / rif assay g4 . nwokoye et al . have stated that low bacterial load limits the ability of the xpert mtb / rif assay to correctly identify mutated and wildtype sequences in the core region of the rpob gene . the indeterminate results of rif resistance detected in four samples of the present study could be because of their high ct value ( > 28 ) corresponding to the very low bacterial burden . while using xpert mtb / rif assay for the detection of mtb and rif resistance in mdr suspected cases , we could collect additional information on mutations associated with rif resistance . it was observed that the pattern of mutations in the 81 bp rrdr of mtb isolates of the present study was almost similar to that reported from other parts of india . the most common rrdr rpob gene mutations were in the gene region 529533 ( 56% ; table 1 ) in all the six districts of malwa region and were recognized by probe e. mboowa et al . too used xpert mtb / rif assay and the most common gene mutations observed were in codon 531 ( 58% ) followed by 513 ( 25% ) , 526 ( 8% ) , and 511 ( 8% ) designed by probes e , b , d , and a. singhal et al . used genotype mtbdr plus assay and reported 531 as the most commonly mutated codon in 59.0% . similarly , mani et al . reported that the codons most commonly involved in these mutations were 531 ( 53% ) and 526 ( 19% ) in a study from south india . the resistant mutants isolated more frequently in clinical practice have higher mean relative fitness and their prevalence depend on their ability to survive . this might be the reason for the higher occurrence of mutations in codon 531533 . in our study , we observed rif resistance by probe c ( 518523 ) in one of the 130 rif resistant sputum samples . this is similar to the findings of gupta et al . who used dna sequencing combined with ms - pcr assay to observe mutation in this rpob region . in contrast , no rif resistance was found to be associated with probe c in the study of mboowa et al . this could probably be because of lesser susceptibility of this genetic region to mutations or because the selection pressure shaping ( producing ) probe c associated rif resistance is less in malwa region of punjab ( north india ) . mutation combination ( probe a and b ) was observed in one ( 1/130 ) of the rif - resistant specimens in the present study . while , singhal et al . found 6 strains ( 6/366 ) with more than one mutations and mboowa et al . reported no specimen with more than one probe failure ( mutation combination ) while employing xpert mtb / rif assay in their study . probably , mutations continue to arise due to the ability of mtb to adapt to drug exposure . the limitation of the study was that no gold standard was used for the comparison of xpert mtb / rif assay results . xpert mtb / rif is a better screening tool for detection of mtb and rif resistance in a shorter period of time , and this could help improve early recognization of mdr - tb and prevention of its further transmission in malwa region of punjab . this assay also appears to be a useful technique to have simultaneous preliminary information regarding the mutation pattern of rif resistance in mtb isolates which could be helpful in understating the epidemiology of the disease and identification of hot spots for implementation of tb control program . however , for the confirmation and the detailed study on these mutations , dna sequencing remains indispensable .

we report a case of a 36-year - old woman with progressive generalized myoclonus that first became apparent 9 years ago . her younger brother had similar problems . examination of her eyes revealed cherry - red spots . hexosaminidase a , -galactosidase and neuraminidase activity were normal . although the laboratory findings were negative , cherry - red spots , progressive myoclonus and autosomal recessive inheritance pattern suggested that she had an unknown type of lysosomal storage disease . background : xpert mtb / rif assay has revolutionized the diagnosis of tuberculosis ( tb ) by simultaneously detecting the bacteria and resistance to rifampicin ( rif ) , a surrogate marker for multidrug - resistant tb ( mdr - tb ) in < 2 h. the rif resistance pattern in malwa region of punjab , india , is not documented . here , we report the epidemiology of rif - resistant tb and mutations in rpob gene of mycobacterium tuberculosis ( mtb).materials and methods : a total of 1612 specimens received between october 2013 and february 2015 were tested by xpert mtb / rif assay following manufacturer 's instructions . the results thus obtained were analyzed using spss version 20.0.0 ( spss inc . , chicago , il , usa ) statistical software.result:rif resistance was statistically higher in previously treated patients in comparison to the new patients ( p = 0.006 ) and in patients with acid fast - bacilli ( afb ) positive smears to afb - negative smears ( p = 0.048 ) . rif resistance mutations in 130 specimens revealed frequency of e 73/130 ( 56% ) , b 28/130 ( 21.5% ) , d 18/130 ( 13.8% ) , a 11/130 ( 8.4% ) , and c 1/130 ( 0.7% ) while in one specimen , mutation combination , i.e. , mutations associated with more than one probe ( a and b both ) was present.conclusion:xpert mtb / rif assay is a user - friendly screening tool for detection of mtb and rif resistance from suspected tb / mdr cases in a shorter period of time . it could also serve as a useful technique to have simultaneous preliminary information regarding the mutation pattern of rif resistance in mtb isolates .

the hallmark of autoimmune diseases is the pathologic activity of the immune system of an organism directed against its own cells and tissues . the disease is a direct consequence of tissue and/or organ damage as mediated by autoreactive components of the immune system , that is , autoreactive t - lymphocytes and/or autoantibodies . for diagnostic purposes , autoantibodies are the most important analytes . within the systemic autoimmune rheumatic diseases ( sard ) , anti - nuclear antibodies ( ana ) , directed against various cellular components , and associated autoantibodies , such as antibodies reactive with dsdna and extractable nuclear antigens ( ena ) , are fundamental for diagnosis [ 13 ] . traditionally , ana are detected by indirect immunofluorescence ( iif ) performed on human epithelial cells ( hep-2 ) . this technique requires a multistage process consistent with visual determination of the staining pattern , serial titrations of positive sera , followed by a second test in which autoantigen specificity is determined [ 2 , 4 ] . recently , the american college of rheumatology ( acr ) stated that ana detection by iif is still considered the gold standard . this was primarily based on the high sensitivity of the iif assay and the inclusion of ana detection by iif assay in diagnostic criteria of systemic lupus erythematosus ( sle ) and autoimmune hepatitis ( aih ) [ 68 ] . in addition , ana can also be considered as a screening test for samples that require further testing for autoantigen specificity , that is , dsdna and ena [ 2 , 9 ] . the specificity of ana detection by iif , however , is relatively poor , especially when low titres are used for screening . indeed , at a 1 : 40 serum dilution , 2530% of healthy individuals may test positive for ana and this increases even further upon ageing [ 1 , 10 ] . overall , it is recommended that the serum dilution that gives a specificity of 95% in healthy individuals should be used as cut - off . moreover , the clinical significance rises with increasing titres [ 11 , 12 ] , as well as with the identification of the responsible autoantigen [ 1 , 9 ] . obviously , a positive ana test must always be interpreted cautiously and only within the clinical context of the patient . in a clinical setting where the pretest probability of sard is generally low , as in primary care , the added value of a positive ana test is lower as compared to secondary and tertiary care situations where pretest probabilities of sard are often higher . in the current study , we determined the prevalence of ana in primary ( general practices ) , secondary ( regional hospital ) , and tertiary care ( university hospital ) . besides data on ana prevalence , also ana titres and anti - ena and anti - dsdna antibodies were included in our analyses . we hypothesize that ana prevalence , ana titre , and anti - ena / dsdna reactivity increase from primary to tertiary care as these situations are expected to be also associated with an increasing pretest probability of sard . in the present study , three different patient populations from the southern part of the netherlands were evaluated and compared with each other . these three populations consisted of patients who were tested for ana between november 2011 and august 2012 in suspicion of an autoimmune disease . all ana requests were considered to involve the diagnostic workup since none of the patients had requests for ana ( and/or anti - ena / dsdna ) at least 4 years prior to the study period . in the first patient population ( n = 1453 ) ana were requested by general practitioners ( primary care ) . the second population ( n = 1621 ) had an ana request in a regional hospital ( secondary care ) , while the third population ( n = 1168 ) had an ana request in a university hospital ( tertiary care ) . testing for ana in the first and second cohorts was performed in the atrium mc ( heerlen , the netherlands ) , while the ana tests in the third cohort were performed in the maastricht university medical centre ( mumc , maastricht , the netherlands ) . furthermore , in both regional and university hospitals , the origin , that is , hospital department , of ana requests was documented . ana detection by iif was performed on hep-2000 cells according to the instructions provided by the manufacturer ( immuno concepts , sacramento , ca ) . hep-2000 cells are transfected with the gene for ssa-60 , which makes these cells more sensitive for ssa - antibody detection [ 14 , 15 ] . fitc - conjugated goat anti - human igg antibody was used for detection of ana . five staining patterns were considered ana positive : homogenous , atypical speckled , speckled , centromere , and nucleolar . in case of mixed - patterns , slides were evaluated with a fluorescent microscope ( axioskop , carl zeiss microscopy gmbh , jena , germany ) with led light source . all slides were evaluated by two independent observers ; in case of a difference in opinion , a third observer was decisive . the presence of anti - ena antibodies was screened by a commercially available line immunoassay ( ana 3 profile euroline , euroimmun , lbeck , germany ) . after the first incubation with diluted serum , a second incubation was performed with goat anti - human igg linked to alkaline - phosphatase . finally , a third incubation took place with bromochloroindolyl phosphate and nitro blue tetrazolium chloride ( bcip / nbt ) as substrate to detect anti - ena antibodies . although the ana 3 profile euroline kit enables detection of 15 different antigens , only eight were evaluated in the current study : ro52 , ss - a / ro60 , ss - b / la , nrnp / sm , sm , scl-70 ( topoisomerase 1 ) , jo-1 , and cenp - b . reading of the results was automated and the colour intensities of the reactions were evaluated by the eurolinescan program ( euroimmun ) to enable semiquantitative determination , that is , equivocal , 1 + , 2 + , and 3 + . results were considered positive for sm , scl-70 , and jo-1 if the intensity was at least equivocal , while for ss - a / ro60 , ss - b / la , nrnp / sm , and cenp - b an intensity of at least 1 + was required . finally , the cutoff for ro52 was 2 + . positive lia results , as defined above , were confirmed with a commercially available feia ( elia , immunodiagnostics , thermo fisher scientific , freiburg , germany ) . this method uses highly purified ( smd ) or recombinant ( ss - a / ro60 , ro52 , ss - b / la , cenp - b , scl-70 , jo-1 , rnp70 , and u1rnp ) human antigens that are coated on irradiated polystyrene cups . the assay was performed according to the manufacturer 's instructions . the sera were diluted 1 : 50 with dilution buffer . after binding of anti - ena antibodies , the cups were washed and subsequently incubated with mouse anti - human igg ( heavy chain specific ) conjugated to -galactosidase . in case of antibody association , binding was detected fluorometrically using 4-methylumbellifery--d - galactoside ( 0.01% ) as substrate . for all antigens , values above 10 u / ml were considered positive . in primary and secondary care , anti - dsdna antibodies were detected with a commercially available feia ( elia , immunodiagnostics , thermo fisher scientific ) . this method uses a circular plasmid dsdna , purified from escherichia coli , as antigen . the assay was performed according to the manufacturer 's instructions and as described for the anti - ena antibodies . the sera were diluted 1 : 10 in dilution buffer and values above 15 u / ml were considered positive . in tertiary care , anti - dsdna antibodies were detected by the crithidia luciliae immunofluorescence test ( clift ; immuno concepts ) . fitc - conjugated goat anti - human igg antibody was used for detection of anti - dsdna antibodies . slides were evaluated with a fluorescent microscope ( axioskop , carl zeiss microscopy gmbh ) with led light source . all slides were evaluated by two independent observers ; in case of a difference in opinion , a third observer was decisive . all samples were tested first by ana iif . if the result was negative , no further testing was performed , unless specifically requested . however , the results of these additional tests were not included in the present study . if the ana iif was positive , irrespective of the staining pattern , titration was performed ( 1 : 320 and 1 : 1280 ) . if a homogenous ana pattern was detected , testing for anti - dsdna antibodies was performed by feia ( primary and secondary care ) or clift ( tertiary care ) . additionally , in case of a homogenous , ( atypical ) speckled , or centromere pattern , typing for anti - ena antibodies was performed by lia . because of the high correlation between the atypical speckled ana pattern and ss - a / ro60 antibodies , these antibodies were considered positive if the atypical speckled ana pattern was observed in combination with either a positive lia or a positive feia . similarly , anti - cenp - b antibodies were considered positive when a centromere ana pattern was observed in combination with a positive anti - cenp - b result in at least one of both anti - ena antibody test systems . since the lia does not enable specific distinction of anti - rnp antibodies , positivity for the nrnp / sm complex was followed by testing for antibodies against rnp70 and u - rnp by feia . chicago , il ) or graphpad prism version 6 ( graphpad software inc . , la jolla , ca ) . the kolmogorov - smirnov analysis was performed to determine whether the age distributions of the three study populations were normally distributed . furthermore , the chi square test , with yates ' correction if appropriate , was performed when comparing proportions of groups . in the case of small samples , of the three patient populations included , 90 ( 6.2% ) , 175 ( 10.8% ) , and 187 ( 16.0% ) patients from primary ( n = 1453 ) , secondary ( n = 1621 ) , and tertiary care ( n = 1168 ) , respectively , were tested positive for ana and were therefore eligible for the current study . gender ( f / m ) and age ( median and range ) distribution were as follows : 78/12 and 57.2 ( 1595 ) for primary care , 129/46 and 57.0 ( 1793 ) for secondary care , and 130/57 and 57.3 ( 384 ) for tertiary care . the gender distribution differed significantly ( p = 0.009 ) due to a strong female preponderance in primary care . the age distribution differed significantly ( p = 0.005 ) due to the fact that in tertiary care age distribution was skewed negatively . evaluation of the origin , that is , hospital departments , of the ana requests in secondary and tertiary care revealed four departments , that is , rheumatology , dermatology , internal medicine , and neurology , which requested the majority of the ana screening tests ( figure 1(a ) ) , that is , 86% and 68% , respectively . the prevalence of ana in the 3 distinct clinical settings is depicted in figure 2(a ) . the higher relative ana prevalence in tertiary care ( 16.0% ) versus secondary care ( 10.8% ) was apparent in all 4 clinical disciplines that requested the majority of ana screening test ( figure 1(a ) ) ; this also holds for the other clinical disciplines ( data only shown as pooled results ) . typically , in secondary care 39.5% of overall ana requests came from the rheumatology department , while in tertiary care this was 15.4% . within the positive ana cohorts , patients from primary care had relatively low titres out of 90 positive ana tests 63 sera ( 70% ) revealed an ana titre of 1 : 80 . in secondary and tertiary care , a titre of 1 : consequently , higher titres were observed more frequently in secondary and tertiary care than in primary care . the distribution in titres between secondary and tertiary care was not different . at first glance , there is no apparent difference in the distribution of ana patterns between the three health care levels ( figure 2(c ) ) . also , comparison of the distribution of ana patterns with a titre of 1 : 1280 , considered to have the highest positive likelihood ratio , showed no significant differences ( data not shown ) . the prevalence of anti - ena and anti - dsdna antibodies , as defined by the algorithm described above , is presented in figure 3(a ) . in the primary care , 19 ( 21.1% ) patients with positive ana ( n = 90 ) were tested positive for anti - ena and/or anti - dsdna antibodies , that is , 1.3% of the total cohort . in the secondary and tertiary care , 68 of 175 ( 38.9% ) and 40 of 187 ( 21.4% ) ana positive patients revealed anti - ena and/or anti - dsdna reactivity , respectively . this is 4.2% and 3.4% of the total secondary and tertiary care cohorts , respectively ( p = 0.367 ) . significantly more positive anti - ena and/or anti - dsdna results were found in the total secondary and tertiary care cohorts than in the primary care cohort ( p < 0.0001 and p = 0.0006 , resp . ) . in secondary care , the prevalence of anti - ena and/or anti - dsdna reactivity within the positive ana samples was the highest in the requests from rheumatology ( figure 1(b ) ) . in tertiary care , however , departments of rheumatology and neurology had , on average , reduced prevalence of anti - ena and/or anti - dsdna reactivity as compared to the departments of dermatology and internal medicine ( figure 1(b ) ) . since the relevance of anti - ena and anti - dsdna antibodies is considered to increase when combined reactivity is observed , we analysed the prevalence of anti - ena and/or anti - dsdna antibodies in relation to combined reactivity ( figure 3(b ) ) . in the primary care setting , single positivity appeared to be more abundant than in secondary and tertiary care , but this difference was statistically not significant . no differences were observed between secondary and tertiary care settings either . with respect to the antigens recognized by the specific antibodies , no apparent differences were observed in the prevalence of antibodies reactive to rnp , ssa60 , ro52 , ssb , cenp - b , and dsdna ( figure 3(c ) ) . anti - scl70 antibodies were only detected in samples of patients from secondary and tertiary care . however , the absolute number , one in each cohort , was low in both of these settings . in the present study on the analyses of ana prevalence , ana titre and anti - ena specificity in the primary , secondary , and tertiary care , our results indicate that ( i ) ana prevalence significantly increases from primary to tertiary care , ( ii ) low titres ( 1 : 80 ) are more frequently observed in the primary care , and ( iii ) anti - ena and anti - dsdna specificities are significantly more prevalent in the secondary care than in the primary care . interpretation of the data obtained in the current study is highly dependent on the viewpoint on clinical utility of an ana test result . this result may help the clinician to identify a patient with sard , but especially in situations with low pretest probabilities of such diseases , the risk of false interpretation of a positive ana is high . this risk of false positive interpretation will decrease if the positive ana is characterized by high titre and includes ( multi)-reactivity for ena and/or dsdna , since these characteristics are associated with higher positive likelihood ratios [ 11 , 12 , 17 ] . next , one has to realize that ana testing is performed in the context of multiple diseases , varying from distinct sard to autoimmune liver diseases . interpretation of a positive ana test may be different for each distinct disease : patients with , for instance , aih or systemic sclerosis often have a positive ana with no ena reactivity , while a positive ana test as such is part of the classification criteria for sle , as well as aih [ 68 ] . a negative ana test result , on the other hand , may also be very useful to exclude a specific set of diseases and may drive attention to other diseases . again , this differs for the distinct sard : a negative ana test has a high negative predictive value for sle and systemic sclerosis but is less helpful to exclude sjgren 's syndrome or myositis [ 2 , 3 ] . obviously , definite interpretation of our dataset is hampered by the lack of clinical data from the patients of the three cohorts . the assumption of the primary care having a relatively low pretest probability was based on previous studies [ 13 , 18 , 19 ] . for future studies , we recommend the inclusion of clinical data in order to be able to thoroughly assess pretest probabilities and strengthen our assumptions . this study , however , also has noteworthy strengths , that is , only data obtained during the diagnostic workup of patients ( not follow - up samples ) were included and the same testing algorithm and reagents ( except for detection of anti - dsdna antibodies ) were used for all three patient cohorts . as expected , we observed a gradual increase in the prevalence of ana from primary to tertiary care . in our present study ana was detected by iif in a screening dilution of 1 : 80 . in several studies it has been reported that 1015% of healthy controls are ana positive in this serum dilution [ 1 , 10 , 20 ] . obviously , a positive ana test result is not only dependent on the dilution factor but also on the quality of other reagents , that is , fluorescent conjugate , cell substrate , and the fluorescent microscope . for standardization purposes , it has , therefore , been recommended to screen for ana with a specificity of 95% . taking this into account , the mere presence of ana in our primary care cohort ( prevalence 6.2% ) lacks discriminating power to identify patients with sard . on the other hand , due to the high negative predictive value of a negative ana result furthermore , since both high titre and anti - ena and/or anti - dsdna ( multi-)reactivity may increase the likelihood of identifying a patient with sard [ 11 , 12 , 17 ] , it is apparent in our primary care cohort that 30% of ana positive sera are of medium to high titre ( n = 27 ) , 21% contain anti - ena and/or anti - dsdna reactivity ( n = 19 ) , and 37% of the latter reveal multireactivity ( n = 7 ) . in our secondary and tertiary cohorts , the ana prevalence is also relatively low ( 11 and 16% , resp . ) , but in both clinical settings about half of the ana positive sera are of high titre ( 48 - 49% ) . as expected , the observed anti - ena and/or anti - dsdna reactivity of ana positive sera in the secondary care ( 39% ) is higher than in the primary care . surprisingly , anti - ena and/or anti - dsdna reactivity in the tertiary care ( 21% ) is lower than in the secondary care and similar to the primary care . this might be related to the spectrum of diseases investigated in the tertiary versus the secondary centres . also the higher number of ana requests by tertiary care departments not typically involved in the diagnosis of sard might be the result of academic profiling of the respective departments . for instance , the cardiology department of the mumc is specialized in inflammatory cardiomyopathies and a possible autoimmune aetiology of these diseases . cardiology requested 108 ana tests ( 9.2% ) , revealing 13% ana positive results of which 20% was anti - ena and/or anti - dsdna antibody positive . a second important difference is the presence of a division of clinical immunology within the department of internal medicine of the tertiary care hospital , implying that many patients with sard are evaluated by clinical immunologists instead of rheumatologists . the latter difference might at least explain the lower relative prevalence of anti - ena and anti - dsdna antibodies in the rheumatology department of the tertiary care hospital . our study shows that in secondary care the majority of positive ana results ( 92.6% ) , as well as the positive anti - ena and anti - dsdna results ( 95.6% ) , are linked to the departments of rheumatology , dermatology , internal medicine , and neurology . obviously , patients with the highest pretest probabilities for sard , as associated with the initial clinical presentation , are most likely referred to these clinical departments . in clinical settings with lower pretest probabilities , ana positive sera are more likely to be of no clinical significance [ 13 , 18 , 19 ] . it might therefore be an option , in particular in primary care settings , to move away from the traditional ana screening test . in this context , the recent discovery of the dense fine speckled ( dfs70 ) antigen is promising and could offer a possible solution for the identification and exclusion of positive sera with no clinical relevance [ 21 , 22 ] . the typical ana dense fine speckled pattern ( dfs ) , known to be associated with the dfs70 antigen , has been found to be commonly prevalent in healthy individuals with ana positive sera ( 33.1% ) , whereas in sard patients 0.0% of the sera revealed a dfs pattern . another study revealed that 2 - 3% of sard patients had antibodies directed to the dfs70 antigen . obviously , in sard patients other anti - ena antibodies might be present that hamper correct recognition of the dfs iif pattern indeed , the identification and correct interpretation of the dfs pattern might prove to be challenging for diagnostic laboratories and would require additional training . moreover , the majority of patterns recognized do not seem to be compatible with a dfs pattern , but this might be the consequence of only interpreting the strongest pattern in the current study . since the identification of the dfs pattern might be challenging for routine diagnostic laboratories and inaccurate interpretation can have significant consequences , an immunoadsorption protocol to diminish anti - dfs70 antibody reactivity to hep-2000 cells could be implemented in the current iif assay in order to significantly improve the performance characteristics of the ana iif test [ 21 , 25 ] . this approach sustains recognition of sard - related autoantibodies in sera with combined reactivities , that is , anti - dfs70 and other anti - ena antibodies . another alternative testing algorithm could , instead of ana iif , include solid phase assays including multiplex and screening assays for well - defined anti - ena and anti - dsdna reactive antibodies [ 11 , 12 , 26 , 27 ] . both approaches enable to distinguish or reduce the number of positive ana results lacking clinical relevance . in addition , multiplex anti - ena screening assays are considered to better recognize particular antigens , for example , ssa and jo-1 , as compared to ana iif testing [ 3 , 11 , 12 , 28 ] . altogether , the results indicate that in the primary care the usage of traditional ana screening tests is more prone to false - interpretation of positive ana results . rather , an alternative testing algorithm for detection of patients with sard might be more appropriate . this might either be achieved by immunoadsorption of anti - dfs70 antibodies or direct screening for anti - ena antibodies . obviously , a patient with severe clinical manifestations typical for sard presenting in a primary care setting should be referred directly , that is , without any laboratory testing , to the rheumatologist or clinical immunologist . this recommendation may not only apply for general practitioners but may also hold for clinical departments that are less likely to encounter patients suspected of sard . in the last 2 decades , cross - sectional imaging , in particular multidetector computed tomography ( mdct ) , has become the main investigation of choice in the locoregional and distant staging of many tumours within the abdomen and pelvis . mdct benefits from having a high spatial resolution and fast acquisition time , making it an ideal tool to stage and restage tumours . the routine use of intravenous ( and oral contrast ) media , unless contraindicated , also helps to delineate sites of disease , especially within solid organs . the identification of nodal involvement on ct is based on nodal size , typically using a minimum cutoff of 1 cm in short axis diameter , although this can lead to both under- and over - staging of metastatic nodal involvement . the purpose of this pictorial review is to discuss the imaging appearances on ct of some of the more common cancers arising within the abdomen and pelvis and to describe their typical sites of local , nodal and haematogenous tumour spread . cancers arising from the stomach , pancreas , colon , kidney , ovary and prostate will be reviewed , with summary tables provided . ninety percent of tumours are adenocarcinomas , which are subdivided into two main histological types : ( 1 ) the well - differentiated ( intestinal ) type , which is associated with atrophic gastritis , occurs in older patients and has a better survival rate and ( 2 ) the undifferentiated ( diffuse / signet ring ) type , which is more common , occurs more frequently in women and has a poorer prognosis . gastric adenocarcinoma occurs either proximally ( cardia ) or distally ( non - cardia ) , with the former increasing in incidence whilst the latter is declining ( see table 1 ) . table 1summary table of local , nodal and haematogenous spread in gastric cancerlocal spreadlocal spread into adjacent structures ( e.g. pancreas , colon , spleen)lymph node spreadperigastric : pericardial ; lesser curvature ; greater curvature ; suprapyloricextraperigastric : left gastric ; common hepatic ; coeliac ; splenic hilum and artery ; hepatic pedicle ; retropancreatic ; mesenteric root ; middle colic ; para - aortichaematogenous spreadmost commonly the liver ( 25% at presentation ) and peritoneumnotestranscoelomic spread can occur through the peritoneum ( e.g. kruckenberg tumours)retropancreatic , para - aortic and mesenteric nodes are classified as m1 metastatic disease nodal disease in gist tumours is extremely rare summary table of local , nodal and haematogenous spread in gastric cancer on ct , the use of oral contrast agents to distend the stomach is essential , preferably with water rather than dilute gastrograffin to avoid beam - hardening artefacts . involvement of the stomach wall is seen as thickening or irregularity of the inner / middle layer of the gastric wall ( t1 tumours ) or transmural thickening with abnormal contrast enhancement ( t2 tumours ) . wall thickness has been reported to be greater and of higher attenuation in the diffuse histological type compared to the intestinal type . gastric cancer spreads in a predictable fashion through the gastric wall with the presence of perigastric fat stranding and nodular outline suggestive of serosal involvement ( t3 tumours ) ( fig . 1 ) . 1gastric adenocarcinoma : axial contrast - enhanced ct ( cect ) showing tumour arising from the lesser curvature of the stomach ( asterisk ) associated with enlarged regional gastrohepatic nodes ( arrows ) gastric adenocarcinoma : axial contrast - enhanced ct ( cect ) showing tumour arising from the lesser curvature of the stomach ( asterisk ) associated with enlarged regional gastrohepatic nodes ( arrows ) spread to perigastric and less frequently other intra - abdominal nodes may occur ( table 1 ; fig . 1 ) . sign describes the clinical finding of an enlarged left supraclavicular lymph node due to metastatic involvement . abdominal cancers may metastasise to this site via the thoracic duct , and gastric malignancy is classically described as the commonest primary tumour to do this . obstructive jaundice with associated bile duct dilatation may occur , due to enlarged porta hepatis nodes or due to the spread of gastric tumour via the gastrohepatic ligament directly into the liver . haematogeneous spread via the portal vein to the liver occurs in 25% of patients at presentation , with liver metastases typically appearing as rim - enhancing low - attenuation lesions during the portal venous phase . peritoneal disease secondary to gastric adenocarcinoma may mimic the appearance of peritoneal disease secondary to metastatic ovarian carcinoma , with omental cake and/or discrete peritoneal deposits noted within the abdominal cavity . trans - coelomic spread is also characteristic of gastric cancer , with spread to the ovary resulting in a krukenberg tumour that typically appears as a mixed solid / cystic adnexal mass . involvement of the peritoneal reflection within the pelvis can result in a positive blumer s shelf finding , with tumour found high on the anterior rectal wall . it should be noted that other histological tumour types arising from the stomach have different patterns of tumour spread . gastrointestinal stromal tumours ( gists ) are the most common primary mesenchymal neoplasms of the gastrointestinal tract , distinct from true smooth muscle and neural tumours , and are derived from the interstitial cells of cajal . they are most frequently located within the stomach ( 6070% ) and should be regarded as having malignant potential . gists typically involve the outer muscular layer of the gi tract , and thus often demonstrate exophytic growth ( fig . 2 ) . most appear as well - defined extraluminal or combined intraluminal / extraluminal masses on ct . if small , they tend to be of homogeneous attenuation ; if larger , they are more heterogeneous on both the unenhanced and enhanced scans because of necrosis or haemorrhage . the central areas of necrosis may communicate with the gastric lumen . if arising from the stomach , the tumour may extend into the gastrohepatic ligament , the gastrosplenic ligament and the lesser sac . nodal spread is extremely rare , but haematogenous spread can occur , often to the liver with nearly 50% of patients with gists presenting with metastases ( fig . 2 ) . 2gastrointestinal stromal tumour ( gist ) : axial cect showing typical large low attenuation exophytic tumour ( asterisk ) arising from the greater curvature of the stomach associated with liver metastasis ( arrow ) . note there are no significantly enlarged regional nodes gastrointestinal stromal tumour ( gist ) : axial cect showing typical large low attenuation exophytic tumour ( asterisk ) arising from the greater curvature of the stomach associated with liver metastasis ( arrow ) . note there are no significantly enlarged regional nodes pancreatic ductal adenocarcinoma accounts for more than 80% of malignant tumours of the pancreas , and ct is the initial imaging modality used for staging of suspected pancreatic cancer with ct ( along with mri and endoscopic sonography ) used to distinguish between potentially resectable and non - resectable patients . for cancer at the head of the pancreas , contraindications to surgery include distant metastases , circumferential involvement of the superior mesenteric vein - portal vein segment more than 2 cm long , thrombus in the vein , and occlusion or circumferential invasion of the celiac , hepatic or superior mesenteric arteries ( see table 2 ) . table 2summary table of local , nodal and haematogenous spread in pancreatic cancerlocal spreadtumour spreads by direct perivascular and perineural invasion;local invasion can involve the stomach , duodenum and retroperitoneum;head / uncinate process tumours : these usually extend along the sma and mesenteric root;body / tail tumours : these usually infiltrate the celiac , hepatic or splenic arterieslymph node spreadprimary drainage : superior , inferior , anterior , posterior and splenic lymph nodes;secondary drainage : porta hepatis , common hepatic , coeliac , mesenteric root lymph nodes;tertiary drainage : peri - aortic and distal superior mesenteric lymph nodeshaematogenous spreadthese usually involve the liver and peritoneal surfacesnoteearly lymphatic and haematogenous micrometastases at presentation are common summary table of local , nodal and haematogenous spread in pancreatic cancer when performing a staging ct , ideally at least two contrast - enhanced acquisitions ( during the late arterial and venous phases ) are advised . the pancreas is a retroperitoneal organ and has a close anatomic relationship with the abdominal peritoneal reflections . the pancreatic head is connected to the liver and lesser curvature of the stomach via the hepatoduodenal ligament and gastrohepatic ligaments , respectively . the tail of the pancreas is in continuity with the hilum of the spleen and the greater curvature of the stomach via the splenorenal and gastrosplenic ligaments . pancreatic cancer can invade these adjacent peritoneal ligaments ( as well as the transverse mesocolon ) to involve the stomach , duodenum ( fig . tumour tends to spread by direct perivascular and perineural invasion within the subperitoneal space . if arising from the pancreatic head or uncinate process , tumours usually extend along the superior mesenteric artery and root of the mesentery . biliary and pancreatic duct dilatation 3pancreatic adenocarcinoma : a axial cect showing an example of a tumour arising in the head of the pancreas ( asterisk ) with invasion into the duodenum . there is a focal thrombus in the portal vein ( arrow ) ; b axial cect in same patient as ( a ) ; there is associated intra- ( arrows ) and extrahepatic ( not shown ) bile duct dilatation ; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas ( asterisk ) encasing the origin of the celiac axis pancreatic adenocarcinoma : a axial cect showing an example of a tumour arising in the head of the pancreas ( asterisk ) with invasion into the duodenum . there is a focal thrombus in the portal vein ( arrow ) ; b axial cect in same patient as ( a ) ; there is associated intra- ( arrows ) and extrahepatic ( not shown ) bile duct dilatation ; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas ( asterisk ) encasing the origin of the celiac axis spread to regional nodes occurs , involving either peripancreatic nodes , nodes at the celiac axis and porta hepatis , or sometimes nodes further afield ( table 2 ) . most colorectal cancers arise from adenomatous polyps , with polyps greater than 2 cm in size associated with a greater than 40% risk of malignancy . for local staging of rectal cancer , mri is now the standard imaging modality because of its increased soft tissue resolution and multiplanar capabilities . colorectal tumours may appear as polypoid or infiltrative lesions of the bowel wall ( fig . early colonic tumours are better detected when endoluminal distension is given and the colon has been prepared , as in ct colonography . tumour spread through the serosa appears as pericolonic fat stranding , but may be confused with an associated desmoplastic reaction ( possibly mimicking diverticulitis within the sigmoid colon ) . the presence of fluid in the root of the sigmoid mesentery and engorgement of the adjacent sigmoid mesenteric vasculature favours diverticulitis , whilst the presence of pericolic lymph nodes is suspicious for colon cancer . tumour spread to adjacent organs is suggested by loss of the fat planes and direct invasion ( see table 3 ) . table 3summary table of local , nodal and haematogenous spread in colorectal cancerlocal spreadinvasion through the bowel wall into the peri - colonic fat and adjacent structureslymph node spreadfollows the vascular distribution of vessels in mesocolon;ascending mesocolon : nodes along the ileocolic vessels and right colic vessel;transverse mesocolon : nodes along the middle colic vessels;sigmoid and descending mesocolon : nodes along the inferior mesenteric vein;regional lymph nodes for rectal cancers include : mesorectal , sigmoid mesenteric , inferior mesenteric , lateral sacral , presacral , internal iliac , sacral promontory , superior rectal , middle rectal and inferior rectalhaematogenous spreadliver ( via the portal vein ) > lung , adrenal glands , bonesnotesin rectal cancers that have perforated the peritoneum , transcoelomic spread favours the lower right small bowel mesentery and the pouch of douglasfig . 4colonic adenocarcinoma : a axial non - contrast - enhanced ct showing tumour ( asterisk ) in the ascending colon with enlarged ileocolic nodes ( arrows ) ; b axial contrast - enhanced ct shows a large liver metastasis ( asterisk ) summary table of local , nodal and haematogenous spread in colorectal cancer colonic adenocarcinoma : a axial non - contrast - enhanced ct showing tumour ( asterisk ) in the ascending colon with enlarged ileocolic nodes ( arrows ) ; b axial contrast - enhanced ct shows a large liver metastasis ( asterisk ) nodal spread depends on the site of the primary tumour and follows the vascular distribution of the vessels within the mesocolon [ 11 , 12 ] . these vessels include the ileocolic vessels and right colic vessel for the ascending mesocolon , the middle colic vessels for the transverse mesocolon , and the inferior mesenteric vein for the sigmoid and descending mesocolon . for rectal tumours , drainage is usually cranial within the mesorectum to involve the regional lymph nodes ( table 3 ) . 5 ) is less common , with involvement of the inguinal nodes only seen with lower rectal tumours when there is proximal lymphatic blockage ( e.g. extensive adenopathy ) . 5rectal adenocarcinoma : coronal reformat cect showing a bulky rectal tumour ( asterisk ) with enlarged right internal iliac nodes ( double asterisk ) and left paraaortic node ( arrow ) rectal adenocarcinoma : coronal reformat cect showing a bulky rectal tumour ( asterisk ) with enlarged right internal iliac nodes ( double asterisk ) and left paraaortic node ( arrow ) metastatic disease at presentation is uncommon ( < 1015% of cases ) . haematogeneous spread is predominantly seen within the liver , with metastases deriving their blood supply from the hepatic artery ( compared to normal liver parenchyma , which is primarily supplied by the portal vein ) . as a result , liver metastases imaged during the portal venous phase are seen as heterogeneous ring - enhancing metastases that are hypodense to the surrounding liver parenchyma ( fig . for example , the venous drainage of the colon and upper rectum is via the portal vein , and thus the liver is a common site of spread . however , the lower rectum has a dual drainage , with the superior haemorrhoidal vein draining into the inferior mesenteric vein and then into the portal vein , and the middle and inferior haemorrhoidal veins draining into the pelvic veins and then directly into the inferior vena cava . this explains why distal rectal cancers can result in isolated pulmonary metastases without hepatic metastases . if rectal tumours perforate the peritoneal membrane , transcoelomic spread may occur , favouring the lower right small bowel mesentery and pouch of douglas . if colorectal tumours are of mucinous histology , widespread intraperitoneal mucinous metastases may occur with characteristic scalloping of adjacent viscera ( pseudomyxoma peritoneii ) ( fig . 6pseudomyxoma peritoneii : axial cect showing mucinous ascites exerting mass effect ( scalloping ) on the solid organs ; primary was of gastrointestinal tract origin pseudomyxoma peritoneii : axial cect showing mucinous ascites exerting mass effect ( scalloping ) on the solid organs ; primary was of gastrointestinal tract origin on ct , renal tumours appear as hypervascular masses , with larger lesions of heterogenous attenuation . with increasing size , they spread through the renal capsule into the perinephric fat to involve gerota s fascia . early extension through the renal capsule is recognized by an indistinct tumour margin , thickened perirenal fascia and perinephric fat stranding . extension into the renal vein occurs in 20% of patients at presentation with involvement of the inferior vena cava ( ivc ) in 510% of patients ( fig . tumour thrombus is seen as a filling defect within the vein , which should not be confused with streaming artefact from unopacified blood . the presence of venous distension on its own may be misleading as rccs are hypervascular and hence tend to have increased blood flow and venous drainage . distinction between tumour thrombus and bland thrombus within the ivc may be difficult , although the former may have enhancing tumour vessels within it . in advanced tumours , direct invasion into adjacent structures such as the diaphragm , and posterior abdominal wall muscles may be seen ( see table 4 ) . table 4summary table of local , nodal and haematogenous spread in renal cell carcinomalocal spreadperinephric fat ; ipsilateral adrenal ; adjacent viscera ( including muscles);renal vein invasion ( ivc)lymph node spreadvia lymphatics following the renal vessels to the ipsilateral para - aortic nodes ; direct connections with the thoracic duct and mediastinum also existhaematogenous spreadcommon sites : lungs > bones , cns , adrenalsnoteextension into renal vein occurs in 20% of patients at presentation ; ivc involvement in 510%fig . 7metastatic renal cell carcinoma : a coronal reformat cect showing the inferior vena cava ( ivc ) greatly expanded by tumour thrombus ( arrows ) with associated enlarged mediastinal nodes ( asterisks ) . the primary renal tumour has been excised ; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient , which increased in size on interval ct studies , in keeping with a metastatic deposit ; note the numerous venous collaterals in the anterior abdomen ( short arrows ) due to the ivc thrombus ; c axial cect shows a large lytic deposit in the lumbar spine ( asterisk ) in the same patient causing compromise of the spinal canal and cord ( arrows ) summary table of local , nodal and haematogenous spread in renal cell carcinoma metastatic renal cell carcinoma : a coronal reformat cect showing the inferior vena cava ( ivc ) greatly expanded by tumour thrombus ( arrows ) with associated enlarged mediastinal nodes ( asterisks ) . the primary renal tumour has been excised ; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient , which increased in size on interval ct studies , in keeping with a metastatic deposit ; note the numerous venous collaterals in the anterior abdomen ( short arrows ) due to the ivc thrombus ; c axial cect shows a large lytic deposit in the lumbar spine ( asterisk ) in the same patient causing compromise of the spinal canal and cord ( arrows ) lymphatic spread from rcc tends to follow the renal veins to involve the ipsilateral para - aortic nodes . there are also direct connections with the thoracic duct and mediastinum , which can account for the rare presence of mediastinal and hilar node involvement ( fig . metastatic involvement in renal cell carcinoma is seen ( in order of decreasing frequency ) in : the lung ( 5060% ) , bone ( 3040% ) , liver ( 3040% ) , adrenal gland ( 5% ) , contralateral kidney ( 5% ) , retoperitoneum ( 5% ) and brain ( 5% ) . more unusual sites that have been reported include the pancreas , peritoneum , bowel , thyroid and muscle ( fig . 7c ) . cross - sectional imaging ( ct or mri ) is used for preoperative tumour assessment , with ct being the imaging modality of choice for assessing metastatic disease . bowel opacification is usually achieved with a positive oral contrast medium ( e.g. dilute gastrograffin ) , which is essential for the detection of peritoneal deposits . intravenous contrast is also routinely administered unless contraindicated , with imaging performed in the portal venous phase . conversely , local pelvic spread is better assessed with pelvic mri than ct ( see table 5 ) . table 5summary table of local , nodal and haematogenous spread in ovarian carcinomalocal spreaduterus and broad ligament ( via the fallopian tube);direct invasion of the rectum , colon , bladder and pelvic side walllymph node spreadvia lymphatics travelling along with the ovarian vessels to terminate in common iliac and para - aortic nodes;via the broad ligament to terminate in the internal iliac and obturator nodes;via the round ligament to terminate in the external iliac and inguinal nodestrans - coelomic spreadcommon sites : undersurface of the diaphragm , liver surface , pouch of douglas , omentum , serosal bowel surfaceshaematogenous spreadthis occurs late during the disease;liver > lungs , kidney , bonenoteascites arise from increased production from tumour surfaces and/or occlusion of retroperitoneal lymph nodes summary table of local , nodal and haematogenous spread in ovarian carcinoma primary ovarian cancer has variable appearances , presenting either as a solid mass or mixed solid / cystic lesion ( fig . ovarian cancer spreads locally to adnexal structures , the uterus and the contralateral ovary , although bilateral ovarian cancer may occur in 1150% of cases . invasion of the pelvic side wall is indicated when the tumour is seen encasing the iliac vessels or is within 3 mm of the pelvic sidewall . 8ovarian carcinoma : coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis ( asterisk ) , with large - volume para - aortic adenopathy ( double asterisk ) , ascites and small peritoneal deposits ( arrows ) . although para - aortic nodal involvement is seen in metastatic ovarian carcinoma , the large volume is rather unusual ; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient ( arrows ) ; c axial cect in the same patient as ( b ) showing a thick omental cake ( arrows ) ; d axial cect in the same patient as ( b ) showing multiple enlarged ( short axis diameter > 5 mm ) cardiophrenic nodes ( arrows ) ovarian carcinoma : coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis ( asterisk ) , with large - volume para - aortic adenopathy ( double asterisk ) , ascites and small peritoneal deposits ( arrows ) . although para - aortic nodal involvement is seen in metastatic ovarian carcinoma , the large volume is rather unusual ; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient ( arrows ) ; c axial cect in the same patient as ( b ) showing a thick omental cake ( arrows ) ; d axial cect in the same patient as ( b ) showing multiple enlarged ( short axis diameter > 5 mm ) cardiophrenic nodes ( arrows ) peritoneal dissemination is the commonest mode of spread , being found in approximately 70% of patients at presentation . common sites include the greater omentum , paracolic gutters , the pouch of douglas , the liver capsule ( fig . less commonly , implants may be seen within the mesentery , along the porta hepatis , lesser sac , splenic surface and gastrosplenic ligament [ 1618 ] . ascites are often present , due to either increased production from tumour surfaces or reduced peritoneal resorption from invasion of lymphatics by tumour cells . peritoneal deposits on ct have a variety of appearances including enhancing nodular soft tissue lesions , linear / plaque - like thickening of peritoneal reflections ( fig . 8c ) , tiny calcifications or mixed solid and cystic or purely cystic lesions [ 19 , 20 ] . the involvement of bowel serosa can lead to ( asymmetric ) bowel wall thickening , tethering of bowel loops and bowel obstruction . liver surface implants ( subcapsular deposits ) may be seen , with deposits causing characteristic scalloping of the liver capsule . metastatic mucinous tumours of the ovary can lead to pseudomyxoma peritoneii , although in many of these cases , the primary mucinous tumour actually arises from the gastrointestinal tract with secondary involvement of the ovary . lymphatic spread occurs via three routes and is usually seen in conjunction with peritoneal spread . routes include : ( 1 ) along the ovarian vessels to the common iliac and para - aortic nodes ( fig . 8a ) ; ( 2 ) via the broad ligament and parametria to the internal iliac and obturator nodes ; and ( 3 ) rarely via the round ligaments to the external iliac and inguinal nodes . cardiophrenic nodes ( defined as > 5 mm short axis diameter on ct ) are characteristically involved as they are the main lymphatic drainage route from the peritoneal cavity ( fig . haematogenous dissemination is rare in ovarian carcinoma with a malignant pleural effusion being the most common manifestation . at presentation , other sides of spread include the spleen , kidneys , adrenals , lungs , brain and bone . mri is traditionally used for local staging of prostate cancer because of its high contrast resolution ( as in rectal cancer ) . it is able to differentiate organ - confined disease from tumour that demonstrates extracapsular spread . direct involvement of the seminal vesicles or adjacent organs is also well seen on mri . ct is used for nodal and distant staging of prostate cancer ( see table 6 ) . table 6summary table of local , nodal and haematogenous spread in prostate cancerlocal spreaddirect extension through the prostate capsule into the seminal vesicles and bladder baselymph node spreadorder of nodal involvement : obturator , presacral , internal iliac , common iliachaematogenous spreadbone > > lung and liver;spinal bone metastases are the commonest site ( due to the direct communication between the presacral and periprostatic veins)notesdenonvillier s fascia forms a relative natural barrier to rectal spread;tumours at the apex of the prostate are more likely to demonstrate extracapsular extension because of relatively little capsule at this level summary table of local , nodal and haematogenous spread in prostate cancer classically , two lymph node metastatic patterns have been reported . 9a ) and para - aortic lymph nodes , and this pelvic nodal involvement is sequential with spread to the obturator nodes , presacral nodes , internal iliac and common iliac nodes . pattern 2 involves para - aortic nodes only , with this second pattern of nodal spread felt to be due to haematogenous dissemination . 9prostate carcinoma : a coronal reformat cect showing irregular enlarged prostate tumour ( white arrow ) extending into the bladder with enlarged left pelvic side wall node ( black arrow ) , liver metastases ( double arrows ) and lesion in the left pubic ramus with large soft tissue component ( * ) . note the bones are sclerotic in keeping with diffuse bony metastases ; b sagittal reformat cect in a different patient on bone windows , showing multiple sclerotic metastases in the thoracolumbar spine prostate carcinoma : a coronal reformat cect showing irregular enlarged prostate tumour ( white arrow ) extending into the bladder with enlarged left pelvic side wall node ( black arrow ) , liver metastases ( double arrows ) and lesion in the left pubic ramus with large soft tissue component ( * ) . note the bones are sclerotic in keeping with diffuse bony metastases ; b sagittal reformat cect in a different patient on bone windows , showing multiple sclerotic metastases in the thoracolumbar spine distant spread occurs in one third of patients with involved sites including bone ( 90% ) , lung ( 50% ) , liver ( 25% ) ( fig . osteoblastic sclerotic metastases rather than lytic bone lesions are usually seen on ct . in order of decreasing frequency , skeletal metastases are seen within the vertebrae ( fig . spinal involvement is initially more common within the lumbar region and less likely within the cervical spine ( 97% cf . pulmonary metastases occur in between 527% of patients at presentation and are more often seen in association with the second pattern of nodal involvement . pulmonary involvement can either appear as lymphangitis carcinomatosa ( due to direct invasion of the pulmonary lymphatics ) or as pulmonary nodules ( due to haematogenous spread ) [ 22 , 24 ] . ninety percent of tumours are adenocarcinomas , which are subdivided into two main histological types : ( 1 ) the well - differentiated ( intestinal ) type , which is associated with atrophic gastritis , occurs in older patients and has a better survival rate and ( 2 ) the undifferentiated ( diffuse / signet ring ) type , which is more common , occurs more frequently in women and has a poorer prognosis . gastric adenocarcinoma occurs either proximally ( cardia ) or distally ( non - cardia ) , with the former increasing in incidence whilst the latter is declining ( see table 1 ) . table 1summary table of local , nodal and haematogenous spread in gastric cancerlocal spreadlocal spread into adjacent structures ( e.g. pancreas , colon , spleen)lymph node spreadperigastric : pericardial ; lesser curvature ; greater curvature ; suprapyloricextraperigastric : left gastric ; common hepatic ; coeliac ; splenic hilum and artery ; hepatic pedicle ; retropancreatic ; mesenteric root ; middle colic ; para - aortichaematogenous spreadmost commonly the liver ( 25% at presentation ) and peritoneumnotestranscoelomic spread can occur through the peritoneum ( e.g. kruckenberg tumours)retropancreatic , para - aortic and mesenteric nodes are classified as m1 metastatic disease nodal disease in gist tumours is extremely rare summary table of local , nodal and haematogenous spread in gastric cancer on ct , the use of oral contrast agents to distend the stomach is essential , preferably with water rather than dilute gastrograffin to avoid beam - hardening artefacts . involvement of the stomach wall is seen as thickening or irregularity of the inner / middle layer of the gastric wall ( t1 tumours ) or transmural thickening with abnormal contrast enhancement ( t2 tumours ) . wall thickness has been reported to be greater and of higher attenuation in the diffuse histological type compared to the intestinal type . gastric cancer spreads in a predictable fashion through the gastric wall with the presence of perigastric fat stranding and nodular outline suggestive of serosal involvement ( t3 tumours ) ( fig . 1 ) . 1gastric adenocarcinoma : axial contrast - enhanced ct ( cect ) showing tumour arising from the lesser curvature of the stomach ( asterisk ) associated with enlarged regional gastrohepatic nodes ( arrows ) gastric adenocarcinoma : axial contrast - enhanced ct ( cect ) showing tumour arising from the lesser curvature of the stomach ( asterisk ) associated with enlarged regional gastrohepatic nodes ( arrows ) spread to perigastric and less frequently other intra - abdominal nodes may occur ( table 1 ; fig . 1 ) . sign describes the clinical finding of an enlarged left supraclavicular lymph node due to metastatic involvement . abdominal cancers may metastasise to this site via the thoracic duct , and gastric malignancy is classically described as the commonest primary tumour to do this . obstructive jaundice with associated bile duct dilatation may occur , due to enlarged porta hepatis nodes or due to the spread of gastric tumour via the gastrohepatic ligament directly into the liver . haematogeneous spread via the portal vein to the liver occurs in 25% of patients at presentation , with liver metastases typically appearing as rim - enhancing low - attenuation lesions during the portal venous phase . peritoneal disease secondary to gastric adenocarcinoma may mimic the appearance of peritoneal disease secondary to metastatic ovarian carcinoma , with omental cake and/or discrete peritoneal deposits noted within the abdominal cavity . trans - coelomic spread is also characteristic of gastric cancer , with spread to the ovary resulting in a krukenberg tumour that typically appears as a mixed solid / cystic adnexal mass . involvement of the peritoneal reflection within the pelvis can result in a positive blumer s shelf finding , with tumour found high on the anterior rectal wall . it should be noted that other histological tumour types arising from the stomach have different patterns of tumour spread . gastrointestinal stromal tumours ( gists ) are the most common primary mesenchymal neoplasms of the gastrointestinal tract , distinct from true smooth muscle and neural tumours , and are derived from the interstitial cells of cajal . they are most frequently located within the stomach ( 6070% ) and should be regarded as having malignant potential . gists typically involve the outer muscular layer of the gi tract , and thus often demonstrate exophytic growth ( fig . 2 ) . most appear as well - defined extraluminal or combined intraluminal / extraluminal masses on ct . if small , they tend to be of homogeneous attenuation ; if larger , they are more heterogeneous on both the unenhanced and enhanced scans because of necrosis or haemorrhage . the central areas of necrosis may communicate with the gastric lumen . if arising from the stomach , the tumour may extend into the gastrohepatic ligament , the gastrosplenic ligament and the lesser sac . nodal spread is extremely rare , but haematogenous spread can occur , often to the liver with nearly 50% of patients with gists presenting with metastases ( fig . 2 ) . 2gastrointestinal stromal tumour ( gist ) : axial cect showing typical large low attenuation exophytic tumour ( asterisk ) arising from the greater curvature of the stomach associated with liver metastasis ( arrow ) . note there are no significantly enlarged regional nodes gastrointestinal stromal tumour ( gist ) : axial cect showing typical large low attenuation exophytic tumour ( asterisk ) arising from the greater curvature of the stomach associated with liver metastasis ( arrow ) . pancreatic ductal adenocarcinoma accounts for more than 80% of malignant tumours of the pancreas , and 70% of tumours arise within the pancreatic head . ct is the initial imaging modality used for staging of suspected pancreatic cancer with ct ( along with mri and endoscopic sonography ) used to distinguish between potentially resectable and non - resectable patients . for cancer at the head of the pancreas , contraindications to surgery include distant metastases , circumferential involvement of the superior mesenteric vein - portal vein segment more than 2 cm long , thrombus in the vein , and occlusion or circumferential invasion of the celiac , hepatic or superior mesenteric arteries ( see table 2 ) . table 2summary table of local , nodal and haematogenous spread in pancreatic cancerlocal spreadtumour spreads by direct perivascular and perineural invasion;local invasion can involve the stomach , duodenum and retroperitoneum;head / uncinate process tumours : these usually extend along the sma and mesenteric root;body / tail tumours : these usually infiltrate the celiac , hepatic or splenic arterieslymph node spreadprimary drainage : superior , inferior , anterior , posterior and splenic lymph nodes;secondary drainage : porta hepatis , common hepatic , coeliac , mesenteric root lymph nodes;tertiary drainage : peri - aortic and distal superior mesenteric lymph nodeshaematogenous spreadthese usually involve the liver and peritoneal surfacesnoteearly lymphatic and haematogenous micrometastases at presentation are common summary table of local , nodal and haematogenous spread in pancreatic cancer when performing a staging ct , ideally at least two contrast - enhanced acquisitions ( during the late arterial and venous phases ) are advised . the pancreas is a retroperitoneal organ and has a close anatomic relationship with the abdominal peritoneal reflections . the pancreatic head is connected to the liver and lesser curvature of the stomach via the hepatoduodenal ligament and gastrohepatic ligaments , respectively . the tail of the pancreas is in continuity with the hilum of the spleen and the greater curvature of the stomach via the splenorenal and gastrosplenic ligaments . pancreatic cancer can invade these adjacent peritoneal ligaments ( as well as the transverse mesocolon ) to involve the stomach , duodenum ( fig . tumour tends to spread by direct perivascular and perineural invasion within the subperitoneal space . if arising from the pancreatic head or uncinate process , tumours usually extend along the superior mesenteric artery and root of the mesentery . biliary and pancreatic duct dilatation 3pancreatic adenocarcinoma : a axial cect showing an example of a tumour arising in the head of the pancreas ( asterisk ) with invasion into the duodenum . there is a focal thrombus in the portal vein ( arrow ) ; b axial cect in same patient as ( a ) ; there is associated intra- ( arrows ) and extrahepatic ( not shown ) bile duct dilatation ; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas ( asterisk ) encasing the origin of the celiac axis pancreatic adenocarcinoma : a axial cect showing an example of a tumour arising in the head of the pancreas ( asterisk ) with invasion into the duodenum . there is a focal thrombus in the portal vein ( arrow ) ; b axial cect in same patient as ( a ) ; there is associated intra- ( arrows ) and extrahepatic ( not shown ) bile duct dilatation ; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas ( asterisk ) encasing the origin of the celiac axis spread to regional nodes occurs , involving either peripancreatic nodes , nodes at the celiac axis and porta hepatis , or sometimes nodes further afield ( table 2 ) . most colorectal cancers arise from adenomatous polyps , with polyps greater than 2 cm in size associated with a greater than 40% risk of malignancy . for local staging of rectal cancer , mri is now the standard imaging modality because of its increased soft tissue resolution and multiplanar capabilities . colorectal tumours may appear as polypoid or infiltrative lesions of the bowel wall ( fig . early colonic tumours are better detected when endoluminal distension is given and the colon has been prepared , as in ct colonography . tumour spread through the serosa appears as pericolonic fat stranding , but may be confused with an associated desmoplastic reaction ( possibly mimicking diverticulitis within the sigmoid colon ) . the presence of fluid in the root of the sigmoid mesentery and engorgement of the adjacent sigmoid mesenteric vasculature favours diverticulitis , whilst the presence of pericolic lymph nodes is suspicious for colon cancer . tumour spread to adjacent organs is suggested by loss of the fat planes and direct invasion ( see table 3 ) . table 3summary table of local , nodal and haematogenous spread in colorectal cancerlocal spreadinvasion through the bowel wall into the peri - colonic fat and adjacent structureslymph node spreadfollows the vascular distribution of vessels in mesocolon;ascending mesocolon : nodes along the ileocolic vessels and right colic vessel;transverse mesocolon : nodes along the middle colic vessels;sigmoid and descending mesocolon : nodes along the inferior mesenteric vein;regional lymph nodes for rectal cancers include : mesorectal , sigmoid mesenteric , inferior mesenteric , lateral sacral , presacral , internal iliac , sacral promontory , superior rectal , middle rectal and inferior rectalhaematogenous spreadliver ( via the portal vein ) > lung , adrenal glands , bonesnotesin rectal cancers that have perforated the peritoneum , transcoelomic spread favours the lower right small bowel mesentery and the pouch of douglasfig . 4colonic adenocarcinoma : a axial non - contrast - enhanced ct showing tumour ( asterisk ) in the ascending colon with enlarged ileocolic nodes ( arrows ) ; b axial contrast - enhanced ct shows a large liver metastasis ( asterisk ) summary table of local , nodal and haematogenous spread in colorectal cancer colonic adenocarcinoma : a axial non - contrast - enhanced ct showing tumour ( asterisk ) in the ascending colon with enlarged ileocolic nodes ( arrows ) ; b axial contrast - enhanced ct shows a large liver metastasis ( asterisk ) nodal spread depends on the site of the primary tumour and follows the vascular distribution of the vessels within the mesocolon [ 11 , 12 ] . these vessels include the ileocolic vessels and right colic vessel for the ascending mesocolon , the middle colic vessels for the transverse mesocolon , and the inferior mesenteric vein for the sigmoid and descending mesocolon . for rectal tumours , drainage is usually cranial within the mesorectum to involve the regional lymph nodes ( table 3 ) . 5 ) is less common , with involvement of the inguinal nodes only seen with lower rectal tumours when there is proximal lymphatic blockage ( e.g. extensive adenopathy ) . 5rectal adenocarcinoma : coronal reformat cect showing a bulky rectal tumour ( asterisk ) with enlarged right internal iliac nodes ( double asterisk ) and left paraaortic node ( arrow ) rectal adenocarcinoma : coronal reformat cect showing a bulky rectal tumour ( asterisk ) with enlarged right internal iliac nodes ( double asterisk ) and left paraaortic node ( arrow ) metastatic disease at presentation is uncommon ( < 1015% of cases ) . haematogeneous spread is predominantly seen within the liver , with metastases deriving their blood supply from the hepatic artery ( compared to normal liver parenchyma , which is primarily supplied by the portal vein ) . as a result , liver metastases imaged during the portal venous phase are seen as heterogeneous ring - enhancing metastases that are hypodense to the surrounding liver parenchyma ( fig . if the primary cancer is mucinous , liver metastases may be cystic or calcified . sites of distant metastases are determined by the venous drainage of the primary site . for example , the venous drainage of the colon and upper rectum is via the portal vein , and thus the liver is a common site of spread . however , the lower rectum has a dual drainage , with the superior haemorrhoidal vein draining into the inferior mesenteric vein and then into the portal vein , and the middle and inferior haemorrhoidal veins draining into the pelvic veins and then directly into the inferior vena cava . this explains why distal rectal cancers can result in isolated pulmonary metastases without hepatic metastases . if rectal tumours perforate the peritoneal membrane , transcoelomic spread may occur , favouring the lower right small bowel mesentery and pouch of douglas . if colorectal tumours are of mucinous histology , widespread intraperitoneal mucinous metastases may occur with characteristic scalloping of adjacent viscera ( pseudomyxoma peritoneii ) ( fig . 6pseudomyxoma peritoneii : axial cect showing mucinous ascites exerting mass effect ( scalloping ) on the solid organs ; primary was of gastrointestinal tract origin pseudomyxoma peritoneii : axial cect showing mucinous ascites exerting mass effect ( scalloping ) on the solid organs ; primary was of gastrointestinal tract origin on ct , renal tumours appear as hypervascular masses , with larger lesions of heterogenous attenuation . with increasing size , they spread through the renal capsule into the perinephric fat to involve gerota s fascia . early extension through the renal capsule is recognized by an indistinct tumour margin , thickened perirenal fascia and perinephric fat stranding . extension into the renal vein occurs in 20% of patients at presentation with involvement of the inferior vena cava ( ivc ) in 510% of patients ( fig . tumour thrombus is seen as a filling defect within the vein , which should not be confused with streaming artefact from unopacified blood . the presence of venous distension on its own may be misleading as rccs are hypervascular and hence tend to have increased blood flow and venous drainage . distinction between tumour thrombus and bland thrombus within the ivc may be difficult , although the former may have enhancing tumour vessels within it . in advanced tumours , direct invasion into adjacent structures such as the diaphragm , and posterior abdominal wall muscles may be seen ( see table 4 ) . table 4summary table of local , nodal and haematogenous spread in renal cell carcinomalocal spreadperinephric fat ; ipsilateral adrenal ; adjacent viscera ( including muscles);renal vein invasion ( ivc)lymph node spreadvia lymphatics following the renal vessels to the ipsilateral para - aortic nodes ; direct connections with the thoracic duct and mediastinum also existhaematogenous spreadcommon sites : lungs > bones , cns , adrenalsnoteextension into renal vein occurs in 20% of patients at presentation ; ivc involvement in 510%fig . 7metastatic renal cell carcinoma : a coronal reformat cect showing the inferior vena cava ( ivc ) greatly expanded by tumour thrombus ( arrows ) with associated enlarged mediastinal nodes ( asterisks ) . the primary renal tumour has been excised ; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient , which increased in size on interval ct studies , in keeping with a metastatic deposit ; note the numerous venous collaterals in the anterior abdomen ( short arrows ) due to the ivc thrombus ; c axial cect shows a large lytic deposit in the lumbar spine ( asterisk ) in the same patient causing compromise of the spinal canal and cord ( arrows ) summary table of local , nodal and haematogenous spread in renal cell carcinoma metastatic renal cell carcinoma : a coronal reformat cect showing the inferior vena cava ( ivc ) greatly expanded by tumour thrombus ( arrows ) with associated enlarged mediastinal nodes ( asterisks ) . the primary renal tumour has been excised ; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient , which increased in size on interval ct studies , in keeping with a metastatic deposit ; note the numerous venous collaterals in the anterior abdomen ( short arrows ) due to the ivc thrombus ; c axial cect shows a large lytic deposit in the lumbar spine ( asterisk ) in the same patient causing compromise of the spinal canal and cord ( arrows ) lymphatic spread from rcc tends to follow the renal veins to involve the ipsilateral para - aortic nodes . there are also direct connections with the thoracic duct and mediastinum , which can account for the rare presence of mediastinal and hilar node involvement ( fig . metastatic involvement in renal cell carcinoma is seen ( in order of decreasing frequency ) in : the lung ( 5060% ) , bone ( 3040% ) , liver ( 3040% ) , adrenal gland ( 5% ) , contralateral kidney ( 5% ) , retoperitoneum ( 5% ) and brain ( 5% ) . more unusual sites that have been reported include the pancreas , peritoneum , bowel , thyroid and muscle ( fig . cross - sectional imaging ( ct or mri ) is used for preoperative tumour assessment , with ct being the imaging modality of choice for assessing metastatic disease . bowel opacification is usually achieved with a positive oral contrast medium ( e.g. dilute gastrograffin ) , which is essential for the detection of peritoneal deposits . intravenous contrast is also routinely administered unless contraindicated , with imaging performed in the portal venous phase . conversely , local pelvic spread is better assessed with pelvic mri than ct ( see table 5 ) . table 5summary table of local , nodal and haematogenous spread in ovarian carcinomalocal spreaduterus and broad ligament ( via the fallopian tube);direct invasion of the rectum , colon , bladder and pelvic side walllymph node spreadvia lymphatics travelling along with the ovarian vessels to terminate in common iliac and para - aortic nodes;via the broad ligament to terminate in the internal iliac and obturator nodes;via the round ligament to terminate in the external iliac and inguinal nodestrans - coelomic spreadcommon sites : undersurface of the diaphragm , liver surface , pouch of douglas , omentum , serosal bowel surfaceshaematogenous spreadthis occurs late during the disease;liver > lungs , kidney , bonenoteascites arise from increased production from tumour surfaces and/or occlusion of retroperitoneal lymph nodes summary table of local , nodal and haematogenous spread in ovarian carcinoma primary ovarian cancer has variable appearances , presenting either as a solid mass or mixed solid / cystic lesion ( fig . ovarian cancer spreads locally to adnexal structures , the uterus and the contralateral ovary , although bilateral ovarian cancer may occur in 1150% of cases . invasion of the pelvic side wall is indicated when the tumour is seen encasing the iliac vessels or is within 3 mm of the pelvic sidewall . 8ovarian carcinoma : coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis ( asterisk ) , with large - volume para - aortic adenopathy ( double asterisk ) , ascites and small peritoneal deposits ( arrows ) . although para - aortic nodal involvement is seen in metastatic ovarian carcinoma , the large volume is rather unusual ; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient ( arrows ) ; c axial cect in the same patient as ( b ) showing a thick omental cake ( arrows ) ; d axial cect in the same patient as ( b ) showing multiple enlarged ( short axis diameter > 5 mm ) cardiophrenic nodes ( arrows ) ovarian carcinoma : coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis ( asterisk ) , with large - volume para - aortic adenopathy ( double asterisk ) , ascites and small peritoneal deposits ( arrows ) . although para - aortic nodal involvement is seen in metastatic ovarian carcinoma , the large volume is rather unusual ; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient ( arrows ) ; c axial cect in the same patient as ( b ) showing a thick omental cake ( arrows ) ; d axial cect in the same patient as ( b ) showing multiple enlarged ( short axis diameter > 5 mm ) cardiophrenic nodes ( arrows ) peritoneal dissemination is the commonest mode of spread , being found in approximately 70% of patients at presentation . common sites include the greater omentum , paracolic gutters , the pouch of douglas , the liver capsule ( fig . less commonly , implants may be seen within the mesentery , along the porta hepatis , lesser sac , splenic surface and gastrosplenic ligament [ 1618 ] . ascites are often present , due to either increased production from tumour surfaces or reduced peritoneal resorption from invasion of lymphatics by tumour cells . peritoneal deposits on ct have a variety of appearances including enhancing nodular soft tissue lesions , linear / plaque - like thickening of peritoneal reflections ( fig . 8c ) , tiny calcifications or mixed solid and cystic or purely cystic lesions [ 19 , 20 ] . the involvement of bowel serosa can lead to ( asymmetric ) bowel wall thickening , tethering of bowel loops and bowel obstruction . liver surface implants ( subcapsular deposits ) may be seen , with deposits causing characteristic scalloping of the liver capsule . metastatic mucinous tumours of the ovary can lead to pseudomyxoma peritoneii , although in many of these cases , the primary mucinous tumour actually arises from the gastrointestinal tract with secondary involvement of the ovary . lymphatic spread occurs via three routes and is usually seen in conjunction with peritoneal spread . routes include : ( 1 ) along the ovarian vessels to the common iliac and para - aortic nodes ( fig . 8a ) ; ( 2 ) via the broad ligament and parametria to the internal iliac and obturator nodes ; and ( 3 ) rarely via the round ligaments to the external iliac and inguinal nodes . cardiophrenic nodes ( defined as > 5 mm short axis diameter on ct ) are characteristically involved as they are the main lymphatic drainage route from the peritoneal cavity ( fig . haematogenous dissemination is rare in ovarian carcinoma with a malignant pleural effusion being the most common manifestation . at presentation , other sides of spread include the spleen , kidneys , adrenals , lungs , brain and bone . mri is traditionally used for local staging of prostate cancer because of its high contrast resolution ( as in rectal cancer ) . it is able to differentiate organ - confined disease from tumour that demonstrates extracapsular spread . direct involvement of the seminal vesicles or adjacent organs is also well seen on mri . ct is used for nodal and distant staging of prostate cancer ( see table 6 ) . table 6summary table of local , nodal and haematogenous spread in prostate cancerlocal spreaddirect extension through the prostate capsule into the seminal vesicles and bladder baselymph node spreadorder of nodal involvement : obturator , presacral , internal iliac , common iliachaematogenous spreadbone > > lung and liver;spinal bone metastases are the commonest site ( due to the direct communication between the presacral and periprostatic veins)notesdenonvillier s fascia forms a relative natural barrier to rectal spread;tumours at the apex of the prostate are more likely to demonstrate extracapsular extension because of relatively little capsule at this level summary table of local , nodal and haematogenous spread in prostate cancer classically , two lymph node metastatic patterns have been reported . 9a ) and para - aortic lymph nodes , and this pelvic nodal involvement is sequential with spread to the obturator nodes , presacral nodes , internal iliac and common iliac nodes . pattern 2 involves para - aortic nodes only , with this second pattern of nodal spread felt to be due to haematogenous dissemination . 9prostate carcinoma : a coronal reformat cect showing irregular enlarged prostate tumour ( white arrow ) extending into the bladder with enlarged left pelvic side wall node ( black arrow ) , liver metastases ( double arrows ) and lesion in the left pubic ramus with large soft tissue component ( * ) . note the bones are sclerotic in keeping with diffuse bony metastases ; b sagittal reformat cect in a different patient on bone windows , showing multiple sclerotic metastases in the thoracolumbar spine prostate carcinoma : a coronal reformat cect showing irregular enlarged prostate tumour ( white arrow ) extending into the bladder with enlarged left pelvic side wall node ( black arrow ) , liver metastases ( double arrows ) and lesion in the left pubic ramus with large soft tissue component ( * ) . note the bones are sclerotic in keeping with diffuse bony metastases ; b sagittal reformat cect in a different patient on bone windows , showing multiple sclerotic metastases in the thoracolumbar spine distant spread occurs in one third of patients with involved sites including bone ( 90% ) , lung ( 50% ) , liver ( 25% ) ( fig . osteoblastic sclerotic metastases rather than lytic bone lesions are usually seen on ct . in order of decreasing frequency , skeletal metastases are seen within the vertebrae ( fig . spinal involvement is initially more common within the lumbar region and less likely within the cervical spine ( 97% cf . pulmonary metastases occur in between 527% of patients at presentation and are more often seen in association with the second pattern of nodal involvement . pulmonary involvement can either appear as lymphangitis carcinomatosa ( due to direct invasion of the pulmonary lymphatics ) or as pulmonary nodules ( due to haematogenous spread ) [ 22 , 24 ] . this review has described the typical patterns of tumour spread for the more common cancers occurring within the abdomen and pelvis . knowledge of the characteristic sites of spread in such cancers is felt to be essential for the radiologist when reporting staging and restaging ct scans . it is hoped that the summary tables in particular will act as a useful aide - mmoir to the reporting radiologist . correct identification of metastatic sites of disease is important , as this will not only have an impact on patient management , but also a bearing on patient prognosis .

for the diagnosis of systemic autoimmune rheumatic diseases ( sard ) , patients are screened for anti - nuclear antibodies ( ana ) . ana , as assessed by indirect immunofluorescence ( iif ) , have a poor specificity . this hampers interpretation of positive results in clinical settings with low pretest probability of sard . we hypothesized that the utility of positive ana iif results increases from primary to tertiary care . we retrospectively determined ana , anti - ena , and anti - dsdna antibody prevalence in patient cohorts from primary ( n = 1453 ) , secondary ( n = 1621 ) , and tertiary ( n = 1168 ) care settings . results reveal that from primary care to tertiary care , ana prevalence increases ( 6.2 , 10.8 , and 16.0% , resp . ) . moreover , in primary care low titres ( 70% versus 51% and 52% in secondary and tertiary care , resp . ) are more frequent and anti - ena / dsdna reactivities are less prevalent ( 21% versus 39% in secondary care ) . typically , in tertiary care the prevalence of anti - ena / dsdna reactivities ( 21% ) is lower than expected . from this descriptive study we conclude that positive ana iif results are more prone to false interpretation in clinical settings with low pretest probabilities for sard , as in primary care . whether alternative approaches , that is , immunoadsorption of anti - dfs70 antibodies or implementation of anti - ena screen assays , perform better , needs to be determined . backgroundmultidetector computed tomography ( mdct ) has become the main investigation of choice for staging of many cancers.aimthe purpose of this pictorial review is to discuss the imaging appearances on ct of some of the more common cancers arising within the abdomen and pelvis and to describe their typical sites of local , nodal and haematogenous tumour spread.methodscancers arising from the stomach , pancreas , colon , kidney , ovary and prostate will be reviewed.resultsawareness of the characteristic sites of tumour spread is important to allow accurate identification of all sites of disease.conclusionthis will clearly have an impact on both patient management and prognosis .

the long - term use of immunosuppressive agents for prevention of allograft rejection increases the risk of malignancy approximately 100 times as high as that in the general population . the prevalence rate of post - transplant malignancies in total differs between geographical areas ; for example , in europe , that rate is 1.6% and in australia is 24% , with a mean of 6% . skin cancers , mostly squamous cell carcinoma ( scc ) , are the most common tumors among persons have solid organ transplantation . but , however , iranian studies found that the most common malignancy after kidney transplantation was kaposi sarcoma ( ks ) among the iranian patients . ks is a skin tumor of multicentre origin , characterized histologically by endothelium - lined vascular spaces and spindle - shaped cells . ks presents as single or multiple lesions on mucosal surfaces , including the skin , lungs , gastrointestinal tract and lymphoid tissues . the etiopathogenes of ks is complex and poorly understood , but is almost certainly dependent on human herpes virus type 8 ( hhv -8 ) infection in immunosuppressed , immunogenetically susceptible individuals . although the treatment of ks is controversial , it should ideally address these pathogenic issues . the current guideline is reduction of immunosuppression as first - line treatment , but these recommendations are based on anecdotal experience or uncontrolled studies . perhaps the most fundamental controversy that has implications for all aspects of the disease surrounds the nature of ks : i.e. whet her it is a true malignancy or reversible hyperplasia . the aim of present study was to investigate the frequency of ks in patients with kidney transplantation in 21 years period . an observational prospective follow - up study with a retrospective component , carried out in the imam reza hospital from kermanshah university of medical sciences ( kums ) during the period 19912012 . patients with pre - transplant neoplasm will be excluded from the analysis ( n = 46 ) . patients who had received transplants were identified through the hospital 's transplant registry . for each patient , information includes donor and recipient characteristics , patient and graft survival and cancer incidence after transplantation , data of serologic tests such as hiv were received . the period of follow - up for each patient starts on the day of transplantation and continues until death or last reported contact . following the methodology used in similar studies , patients will not be removed from the analysis at the time of graft failure for several reasons . we usually suspect ks when a kidney recipient presents with multiple hyperpigmented cutaneous nodules that may be associated with gastrointestinal discomfort and pulmonary symptoms resistant to conventional therapies . then a battery of endoscopic , bronchoscopic , radiologic and pathologic tests is used to diagnose ks . there were two main distinct periods of immunosuppressive regimen : the first period was from 1991- 2002 azathioprine , cyclosporine and prednisolone . the second period was from 2002 onwards during which the patients received triple immunosuppressive therapy consisting of cellcept , cyclosporine and prednisolone at the same dosages mentioned above . induction therapy using anti - thymocyte globulin ( atg ) or anti - lymphocyte globulin ( alg ) was preserved to the high risk patients in the early phase of transplantation or treatment of acute rejection ; okt-3 was not used in any of the studied populations . the doses of immunosuppressive agents were reduced , with changed drugs to sirolimus , or the agents were withdrawn upon diagnosis of ks . the method of reduction of immunosuppression and decision on which the agent to be reduced or withdrawn were dependent on the individual patient 's health condition , response to treatment and his / her physician 's judgment . from march 1991 to december 2012 , 1487 kidney transplantations were performed . among them , 67 malignant diseases were diagnosed in 64 patients with an overall incidence of 4.5% and ks was diagnosed as more frequent malignancy in 17 ( 25.37% ) patients ( table 1 ) . of these 17 ks patients , 10 were males and 7 were female with median age of 47.8 years old . the mean time between transplantation and non - ks malignant tumors was 34.4 21.8 months ( range 12140 months ) , while in ks patients it was 18.7 25.2 months , which was statistically significantly different ( p < 0.05 ) . after detection of ks in 12 patients , we perform serum antibody detection against hhv . among them , 8 ( 66.6% ) were seropositive . data of characteristics of transplant patients with kaposi 's sarcoma were summarized in the table 2 . type and frequency of most frequent post - transplant malignancies ( n = 67 ) characteristics of transplant patients with kaposi 's sarcoma there was no significant increase in the number of ks patients under cellcept compared to azathioprine or in the interval to development of ks . the sites of cutaneous involvement were lower extremities in 9 ( 52.9% ) , followed by upper - limb involvement in 5 ( 29.41% ) patients . other ks lesions occurred on trunk in 4 ( 23.5% ) and hard palate in 2 ( 11.7% ) patients . immunosuppression was reduced in 10 ( 58.8% ) patients ( that 8 ( 80% ) patients of them received sirolimus ) and thoroughly withdrawn in the remainder ( including three cases of visceral involvement ) . in 2 ( 11.7% ) patients immunosuppression was discontinued . in 11 ( 91 .6% ) of 12 patients , ks skin lesions improved with therapy ( excluded are five patients who died soon after ks diagnosis ) . patients received additional therapy arbitrarily if , in the opinion of the treating clinician , lesions were not improving . in the patients with hhv seropositive , ganciclovir were prescribed . the renal prognosis of patients who did not succumb to disseminated disease was related to the management of immunosuppression . all two patients in whom immunosuppression was discontinued had functioning grafts when ks was diagnosed , but all grafts were acutely rejected . one patient had azathioprine and one patient had cellcept immunosuppressive therapy and there is no significant difference between these two regimens ( p > 0.05 ) . dialysis was re - instituted in these patients a mean of 5 weeks after discontinuation of all immunosuppression . other patients had good response to reducing immunosuppression and remained their grafts in period of study . from march 1991 to december 2012 , 1487 kidney transplantations were performed . among them , 67 malignant diseases were diagnosed in 64 patients with an overall incidence of 4.5% and ks was diagnosed as more frequent malignancy in 17 ( 25.37% ) patients ( table 1 ) . of these 17 ks patients , 10 were males and 7 were female with median age of 47.8 years old . the mean time between transplantation and non - ks malignant tumors was 34.4 21.8 months ( range 12140 months ) , while in ks patients it was 18.7 25.2 months , which was statistically significantly different ( p < 0.05 ) . after detection of ks in 12 patients , we perform serum antibody detection against hhv . among them , 8 ( 66.6% ) were seropositive . data of characteristics of transplant patients with kaposi 's sarcoma were summarized in the table 2 . type and frequency of most frequent post - transplant malignancies ( n = 67 ) characteristics of transplant patients with kaposi 's sarcoma there was no significant increase in the number of ks patients under cellcept compared to azathioprine or in the interval to development of ks . the sites of cutaneous involvement were lower extremities in 9 ( 52.9% ) , followed by upper - limb involvement in 5 ( 29.41% ) patients . other ks lesions occurred on trunk in 4 ( 23.5% ) and hard palate in 2 ( 11.7% ) patients . immunosuppression was reduced in 10 ( 58.8% ) patients ( that 8 ( 80% ) patients of them received sirolimus ) and thoroughly withdrawn in the remainder ( including three cases of visceral involvement ) . in 2 ( 11.7% ) patients immunosuppression was discontinued . in 11 ( 91 .6% ) of 12 patients , ks skin lesions improved with therapy ( excluded are five patients who died soon after ks diagnosis ) . patients received additional therapy arbitrarily if , in the opinion of the treating clinician , lesions were not improving . in the patients with hhv seropositive , ganciclovir were prescribed . the renal prognosis of patients who did not succumb to disseminated disease was related to the management of immunosuppression . all two patients in whom immunosuppression was discontinued had functioning grafts when ks was diagnosed , but all grafts were acutely rejected . one patient had azathioprine and one patient had cellcept immunosuppressive therapy and there is no significant difference between these two regimens ( p > 0.05 ) . dialysis was re - instituted in these patients a mean of 5 weeks after discontinuation of all immunosuppression . other patients had good response to reducing immunosuppression and remained their grafts in period of study . post - transplantation ks is a well - known complication after renal transplantation with a possible negative impact on the patient 's and graft long term survival . the ks incidence peaks during the first year post - transplantation . in our study , 82.3% of all ks cases were diagnosed in the first 2 years after receiving a renal allograft , which is compatible with previous studies . the results of this study showed lower incidence of ks ( 1.1% ) in our transplant population than that reported from other regional countries . further , ks is frequent in african renal transplant recipients , where 13.3% of all transplanted patients developed ks . we investigated the ks was the most frequent post - transplantation malignancies . in confirm to our study , saudi arabian and turkish studies were reported the most frequent post - transplantation malignancies were ks with 87.5% and 80.0% , respectively . also , nafar et al . , found that the most common malignancy after kidney transplantation was ks among the iranian patients . the risk of death from dissemination of malignancy should be weighed against the risk of graft rejection . according our results , in 11 patients ( 91.6% ) ks skin lesions improved with reduction ( including 6 patients with sirolimus therapy ) or discontinued of immunosuppression with a graft loss rate of 11.7% . in compare to our study , reduction of immunosuppression resulted in complete remission of ks in 28% of patients from saudi arabia , and 61% of italian patients . duman et al . , report on complete remission after reduction of immunosuppressive drugs in all patients [ 12/12 ] , with a graft loss rate of 20% in turkey . reduction of immunosuppression allows for the immune system to reduce viral replication producing clinical remission of disease . new antiviral agents have recently been introduced as a promising therapeutic option in patients with ks . however , prospective studies that will determine the efficacy of this approach are warranted . sirolimus is a potent immunosuppressive drug that has been recently reported as an effective agent in the treatment of ks . cutaneous ks lesions disappeared in all patients three months from the initiation of sirolimus therapy . sirolimus may become the first choice immunosuppressant in renal transplant recipients with ks for pro - viding optimal immunosuppression and inhibiting the progression of malignancy . hhv-8 has been described in patients with hiv infection and ks . the results of serologic studies support the notion that infection with hhv-8 is nearly universal in patients with ks , since specific antibodies are detectable in 70% to 90% of all patients with ks and almost 100% of immunocompetent patients with the disease . also it 's clear that pre - transplantation hhv-8 seropositivity is found to be associated with an increased risk of post - transplant ks . immunosuppressive treatment may induce reactivation of latent virus infection , playing an important role in the development of combined iatrogenic and endemic ks . it is also possible that hhv-8 may be transmitted from the donor to induce sarcoma development in the organ recipient . we investigated that about 67% of our ks patients were seropositive for hhv after transplantation . laboratory studies of the susceptibility of hhv-8 to antiviral drugs suggest that the virus is resistant to acyclovir and penciclovir , but sensitive to ganciclovir , foscarnet and cidofovir . ks is a common long - term complication in renal transplant recipients , with an increased incidence compared with the general population . given that candidates for organ transplantation who are seropositive for hhv-8 -and thus at risk for ks- can now be identified , chemoprevention should be available in this high - risk population . such strategies in hhv-8-seropositive candidates for organ transplantation should be directed against the virus itself , and the immunosuppressive regimen should be carefully monitored to avoid the possibility of rejection . breast cancer is the most common cancer among american women , except for skin cancers . the chance of developing invasive breast cancer at some time in a woman 's life is a little less than 12% . it is the second leading cause of cancer death in women , exceeded only by lung cancer . the chance that breast cancer will be responsible for a woman 's death is about 3% . although clinical signs of disseminated disease occur in fewer than 10% of women at the time of diagnosis , the disease relapses in the form of metastasis within 5 years of surgery in about half of apparently localized tumors . it is difficult to predict the occurrence of distant metastases since breast cancer is a heterogeneous disease encompassing complex pathologic entities . a dynamic interaction between tumors and the immune system is essential for tumor survival , growth , and metastasis . tumors are infiltrated with large number of immune cells that constitute a major cell population in the tumor microenvironment . tumor cells depend on their microenvironment to provide signals for growth , anti - apoptosis , angiogenesis , and metastasis . however , tumor cells are also under the surveillance due to their recognition by immune cells as foreign . analysis of the interactions between tumor cells and the host 's immune system has led to the realization that tumor cells have devised multiple strategies to evade immune attack . development of an invasive cancer , however , is not only a result of the genetic changes in the tumor cell but also the result of genetic and epigenetic changes within the host . host cells , including inflammatory cells , endothelial cells , and fibroblasts , are recruited and activated in the microenvironment of transformed cells . the acute inflammatory response might succeed in eliminating the malignant cells , but if not , a chronic inflammatory process develops in conjunction with the dying tumor cells . the subsequent reciprocal interactions between these responding normal host cells and genetically altered cells result in the development of an invasive cancer . there is a constant interplay between the innate and adaptive immune systems , which leads to a protective immune response against pathogens and transformed cells and contributes effectively to discrimination between self and nonself . persistent protumor immune responses ( inflammation ) , now generally accepted as initiating primary tumor development , are also being recognized as mediators of cancer metastasis . thus , novel anticancer therapeutic strategies targeting molecular and/or cellular mechanisms regulating these collaborative interactions may provide efficacious relief for metastatic disease . this paradox was first resolved by matzinger in 1994 , who proposed that our immune system is designed to combat danger , rather than mediate recognition of nonself over self . pathogen - associated molecular patterns ( pamps ) and endogenous molecules created upon tissue injury , since called damage - associated molecular patterns ( damps ) , signal the threat of either infection or injury to the organism , independently of their nonself- or self - identity [ 710 ] . damage - associated molecular patterns ( damps ) include endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix ( ecm ) molecules that are upregulated upon injury or degraded following tissue damage . among the cellular receptors that sense these danger signals , toll - like receptors ( tlrs ) represent a key molecular link between tissue injury , infection , and inflammation . tlrs are critical in bridging innate and adaptive immune responses and play a significant role in cancer immunosurveillance . innate immune cells including natural killer ( nk ) , natural killer t ( nkt ) , and t cells play a critical role in protecting the host against cancer . macrophages and dendritic cells ( dcs ) , in particular , function as major sensors of invading pathogens and transformed cells via the tlrs . adaptive immunity is crucial to the elimination of pathogens and tumor cells in the late phase of host defense responses and generates more specific tumor immunity and immunological memory . tlrs are known to regulate cancer immunity and tolerance by controlling the suppressive function of regulatory t ( treg ) cell and through innate immune responses mediated by other immune cells [ 1113 ] . tlr signaling , critical for innate and adaptive immune responses , has been thought to be restricted to immune cells . however , many studies suggest that tumor cells bear tlrs and that tlr signaling promotes tumor growth and immune evasion [ 1517 ] . tlr activation by damps may initiate positive feedback loops where increasing tissue damage enhances proinflammatory responses leading to chronic inflammation . as tlrs are widely expressed on tumor cells and immune cells and play important roles in the initiation and progression of cancer , they may thus serve as an important target and have an effective perspective on breast cancer treatment . tlrs 1 , 2 , 4 , 5 , and 6 are expressed on the cell surface ; tlrs 3 , 7 , 8 , and 9 are found almost exclusively within endosomes . different tlrs exhibit specificity for pathogen - derived ligands ; tlrs 2 , 3 , 4 , 5 , 7 , and 9 recognize bacterial lipoproteins , double - stranded rna / poly ( i : c ) , lipopolysaccharides ( lps ) , flagellin , single - stranded rna , and cpg - containing dna , respectively [ 1823 ] . tlr10 is expressed in humans but not in mice , tlr8 is not functional in mice and tlrs 11 , 12 , and 13 are expressed in mice but not in humans . there are several studies which suggest that damp - mediated inflammation plays a vital role . necrotic cells were found to induce proinflammatory and tissue repair gene synthesis and cause dc maturation in a tlr2-dependent manner , as a result of the release of their intracellular contents . other intracellular molecules such as heat shock proteins including hsp70 , gp96 , hsp22 , and hsp72 and high - mobility group box-1 protein ( hmgb1 ) as well as ecm molecules such as biglycan , tenascin - c , versican , and fragments of ecm molecules including oligosaccharides of hyaluronic acid ( ha ) and heparan sulfate ( hs ) have been shown to activate tlrs . tlr1 , along with tlr2 , was found to be important for the activation of professional antigen - presenting cells by -defensin-3 , a host - derived antimicrobial peptide . self - nucleic acids have also been described as endogenous danger signals , namely , mrna recognized by tlr3 , single - stranded rna ( ssrna ) sensed by tlr7 and 8 , and igg - chromatin complexes recognized by tlr9 . tlr2 , 4 , 7 , and 8 were shown to be activated by antiphospholipid antibodies ( apl ) isolated from patients with apl syndrome . the signaling pathways utilized by various tlrs differ , which results in varied cellular responses . for example , tlr3 , the receptor for double - stranded rna couples to the adaptor protein trif . in contrast , other tlrs couple to the adapter myeloid differentiation primary response gene 88 ( myd88 ) [ 25 , 26 ] . the traf6 in turn activates tak1 that phosphorylates and activates the ikk complex resulting in the release and translocation of nf-b to the nucleus . tak1 also activates stress - activated protein kinase ( sapk ) pathways and activates c - jun - nh2-kinases ( jnk ) and p38 . the myd88-coupled tlrs induce the synthesis of cytokines such as tnf- , il-6 , and il-1 , key mediators of the inflammatory response [ 27 , 28 ] . tlr4 , the receptor for lps , is unique in that it activates both myd88-dependent and trif - dependent pathways . the link between inflammation and cancer is well documented [ 29 , 30 ] . several inflammatory diseases , including inflammatory bowel disease , increase the risk of cancer . conversely , in tumors that are epidemiologically unrelated to overt inflammatory conditions ( such as breast cancer ) , the activation of oncogenes can trigger the production of inflammatory molecules and the recruitment of inflammatory cells . in the tumor microenvironment , inflammatory cells and molecules influence almost every aspect of cancer progress , including the metastatic ability of tumor cells . there is biological heterogeneity among tumors with regard to cellular infiltrates , identifying subsets of mononuclear inflammatory cells both at the tumor centre and at the invasive front , which seem to be associated with the occurrence of distant metastasis . intratumour leucocytes from peripheral blood penetrate the tumor architecture after their phenotypic modification , from the invasive front to the tumor centre . this seems to be a dynamic process in which inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize the host immune response towards specific phenotypes impacting on tumor progression . previously , there were six recognized hallmarks of cancer , namely , unlimited replicative potential , self - sufficiency in growth signals , insensitivity to growth inhibitors , evasion of programmed cell death , ability to develop blood vessels , and tissue invasion and metastasis . a group of cytokine proteins , including il-1 , il-6 , tnf- , and rankl , activate inflammation and are known to augment tumor cells ' ability to metastasize by affecting several steps in the cells ' dissemination and implantation at secondary sites [ 29 , 32 , 33 ] . inflammatory cytokines lie downstream of the master gene transcription factor nf-b , for promoting inflammation which is itself activated by them . there is strong evidence that the tumor microenvironment is inflammatory and that activation of the innate immune system plays a role in the progression of cancer [ 34 , 35 ] . a major source of inflammatory cytokines in the tumor microenvironment , are specialized white blood cells called macrophages . tumor - associated macrophages assist the malignant behaviour of tumor cells , not only by producing cytokines , but also by secreting growth factors and matrix - degrading enzymes [ 3638 ] . it has long been suggested that there may be common pathways of inflammation shared by responses to infection and to malignancy . recent evidence indicates that tlrs on macrophages may be critical elements in these common pathways . myd88 has been reported to activate not only ap-1 and nf-b subunit p65 and p50 , but also c - rel , c / ebp , and c / ebp. in case of lps signaling through tlr4 , where nf-b and ap-1 activities are relatively preserved in myd88-deficient macrophages , the specific defect in c - rel and the profound defect in c / ebp/ activation likely accounts for the reduction of il-12 p40 , il-6 , and tnf. the absence of both c / ebp/ specifically in tlr signaling impairs key proinflammatory cytokines without affecting other nf-b - dependent genes such as ib . toll - like receptors are expressed on cells of the immune system but there is growing evidence that tlrs are also expressed on tumor cells , where they may influence tumor growth and host immune responses . activation of tlrs expressed on tumor cells may have profound consequences for tumor growth by factors released after tlr activation . tumor immune evasion may be facilitated by inhibitory cytokines , inflammatory factors , proteinases , and other small molecules such as nitric oxide . recent evidence suggests that tlrs also contribute to tumor - cell resistance to apoptosis and increased invasiveness . the human breast cancer cell line mda - mb-231 was found to express tlr1-tlr10 at both the mrna and protein levels . knockdown of tlr4 gene in mda - mb-231 resulted in a dramatic reduction of breast cancer cell viability and inhibition of il-6 and il-8 cytokines compared with vector control . another study highlights the role of tlr9 in highly invasive mda - mb-231 breast cancer cell line which when activated promotes mda - mb-231 cell invasion by increasing the activity of matrix metalloproteinase 13 ( mmp13 ) , but not mmp8 . samples of mammary carcinomas with recurrence have also exhibited a significant increase in the mrna levels of tlr3 , tlr4 , and tlr9 . a significant percentage of tumors also showed tlr4 expression by mononuclear inflammatory cells ( 21.6% ) and tlr9 expression by fibroblast - like cells ( 57.5% ) . tumors with high tlr3 expression by tumor cell or with high tlr4 expression by mononuclear inflammatory cells ( mics ) , but not tlr9 high fibroblast like cells were significantly associated with higher probability of metastasis . this study highlights the importance of the tumor stromal cells in tumor behavior , and how tlr - induced inflammation on inflammatory cells drives metastatic cascade . synthetic tlr9-ligands ( cpg - sequence containing oligonucleotides ) stimulated tlr9 expressed on cancer cells as well as various normal cells , including mesenchymal stem cells and stimulated their invasion in vitro . this invasion was mediated via downregulation of tissue inhibitor of matrix metalloproteinase-3 ( timp-3 ) and through matrix metalloproteinase-13 ( mmp-13 ) activation . expression of tlr9 isoforms a and b have been detected in clinical breast cancer specimens . expression of tlr9 and its invasive effects on breast cancer cells has been found to be regulated by estrogen receptor- ( er ) and sex steroid hormones . tlr9 expression was also found to be affected by commonly used hormonal cancer therapy bicalutamide . activation of tlr signaling on tumor cells by their ligands can also trigger apoptosis and may have therapeutic effects . for example , in a randomized clinical trial for the efficacy of poly ( a : u ) dsrna , therapeutic effect was mediated through tlr3 expressed on tumor cells , and could therefore represent an effective targeted treatment in patients with tlr3-positive cancers . the predictive value of tlr3 expression by tumor cells for the efficacy of poly ( a : u ) dsrna was determined in 194 breast cancer patients enrolled in a randomized clinical trial . however , conventional chemotherapy or in vivo injection of poly ( a : u ) , alone or in combination , failed to reduce tumor growth unless an immune - chemotherapeutic regimen of vaccination against tumor antigens was included . recently , tlr5 has been found to be highly expressed in breast carcinomas and activation of tlr5-signaling pathway was found to be overly responsive in breast cancer cells by inhibiting cell proliferation and an anchorage - independent growth . in addition , the secretion of soluble factors induced by flagellin , was found to the growth - inhibition of breast cancer cells in an autocrine fashion . this inhibitory activity was further confirmed in vivo using mouse xenografts models of human breast cancer cells . sites of chronic inflammation are often associated with the establishment and growth of various malignancies including breast cancer . enhanced neutrophilic and granulocytic infiltration in lungs and bone of the proarthritic and arthritic mice and subsequent increase in circulating levels of proinflammatory cytokines , such as macrophage colony stimulating factor ( m - csf ) , interleukin-17 ( il-17 ) , interleukin-6 ( il-6 ) , vascular endothelial growth factor ( vegf ) , and tumor necrosis factor - alpha ( tnf - alpha ) were found to contribute to the increased metastasis . breast cancer - associated secondary metastasis was found to be significantly increased in pro - arthritic and arthritic conditions . breast cancer metastasis was found to be significantly reduced by blocking the il-17 and cox-2 pathway . inflammatory tlr signaling has also been shown to promote the attraction and generation of th17 cells induced by tumor cells and tumor - derived fibroblasts . enhanced migration of th17 cells to tumor sites was reported to be due to the expression of chemokines and tumor - derived fibroblasts . there are currently approximately twenty drugs in preclinical development , with a further dozen or so in clinical trials . there are clearly many options for the targeting of tlrs , because the key function of tlrs is to induce cytokines , which are well validated in these diseases and are successfully being targeted in the clinic . tlrs occur early in pathways and so inhibiting them might be more potent than inhibiting their downstream cytokine targets . are a feasible option , but only for those on the cell surface , such as tlr2 , tlr4 , and tlr5 . small - molecule antagonists ( e.g. , eritoran against tlr4 or odn - based inhibitors of tlr7 ) might be a better prospect , but it is hard to predict their off - target effects and efficacy . because there are kinases on the signaling pathways , these might also be sensitive to inhibition . one major concern here , however , is that such inhibitors might block multiple tlrs and therefore give rise to unwanted immunosuppression . studies on knockout mouse indicate that there is less redundancy in tlrs in relation to inflammation . imiquimod is already approved for its antiviral effects , whereas mpl is approved as a vaccine adjuvant . in terms of antagonism , effects of tlr inhibitor , eritoran have been found to be significant but somewhat marginal . to further develop more effective tlr therapeutic targeting strategy , there are a few more tasks : further identifying and determining the pathogenesis of challenging medical conditions like cancer ; analysis of genetic sequence , molecular structure , epigenetic observations , and functional activities on both animal model and human clinical studies ; design of clinical study based on study indication , dosing regimens , drug delivery route or format consideration , and pharmacokinetics ; timely and objective assessment of adverse events with details . targeting tlrs will therefore in all likelihood prevent the induction of many immune and inflammatory proteins . the wide tissue distribution of tlrs , however , may make it difficult to determine whether an agonist or an antagonist will be most effective therapeutically . metastasis is regulated not only by intrinsic genetic changes in malignant cells , but also by the microenvironment . several studies have demonstrated that sites of chronic inflammation are often associated with the establishment and growth of various malignancies . toll - like receptors ( tlrs ) have emerged as sensors that can detect a variety of invading pathogens and malignant cells . since their discovery a decade ago , tlrs have been shown to be critical for efficient innate and adaptive immunity and the framework of tlr - mediated signaling pathway has been explained . however , tlr activation may be a two - edged sword , with both antitumor and pro - tumor consequences . the general expression of functionally active tlrs by tumor cells and inflammatory cells in the stroma by putative endogenous ligands suggests that tlr signaling may be continually activated and may contribute to tumor progression and metastasis . understanding tlr function in tumor biology may lead to discovery of new therapeutic targets in cancer therapy .

introductionthe long - term use of immunosuppressive agents for prevention of allograft rejection increases the risk of malignancy approximately 100 times as high as that in the general population and kaposi 's sarcoma ( ks ) is a relatively common malignancy after kidney transplantation . the aim of present study was to investigate the frequency of ks in patients with kidney transplantation in 20 years period.material and methodsin this study charts and pathology reports of 1487 recipients for kidney allografts treated at imam reza hospital between 1991 and 2012 were reviewed . the spss software package version 16 ( spss inc . , chicago , illinois , usa ) was used for the statistical analysis.resultsthere were 17 of 1487 incident cases of ks kidney transplant population at our hospital in period of study . there is no significant difference between age and gender of patients . the mean time between transplantation and non - ks malignant tumors was 34.4 21.8 months ( range 12140 months ) , while in ks patients it was 18.7 25.2 months , which was statistically significantly different ( p < 0.05 ) . after detection of ks in 12 patients , we perform serum antibody detection against hhv . among them , 8 ( 66.6% ) were seropositive.conclusionks is a common long - term complication in renal transplant recipients , with an increased incidence compared with the general population . given that candidates for organ transplantation who are seropositive for hhv-8 -and thus at risk for ks- can now be identified , chemoprevention should be available in this high - risk population . breast cancer remains a major cause of death in women in the developed world . as toll - like receptors ( tlrs ) are widely expressed on tumor cells and play important roles in the initiation and progression of cancer , they may thus serve as important targets and have an effective perspective on breast cancer treatment . expression of tlrs on breast cancer cells and mononuclear inflammatory cells can promote inflammation and cell survival in the tumor microenvironment . inflammation and cancer are related . it is well known that persistent inflammatory conditions can induce cancer formation , due to production of cytokines and chemokines , which play a crucial role in promoting angiogenesis , metastasis , and subversion of adaptive immunity . tlr signaling in tumor cells can mediate tumor cell immune escape and tumor progression , and it is regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression . this paper delineates the expression of various tlrs in promotion of inflammation and development of mammary tumors . understanding the mechanisms through which tlrs on breast cancer cells and inflammatory cells regulate growth , survival , and metastatic progression can make them potential targets for breast cancer therapy .

forensic odontology has established itself as an important and often indispensible science in medicolegal matters and identification of the dead . the forensic importance of dental tissue has been well recognized because tooth is the hardest of all human tissues . they are well preserved for a long period even after death , hence dental remains are the most stable biological evidence encountered in crime cases and yield useful information . blood grouping has been one of the major factors for identification of biological materials in forensic investigations and is a widely used technique in forensic laboratories . the presence of abo blood group and rhesus factor is applied to inherited antigens detected on red cell surface by specific antibodies . once the blood group and rhesus factor are established , it remains unchanged throughout life . kind in 1960 discovered the presence of abo blood group in saliva by absorption - elution ( ae ) method . blood grouping from dried stain by elution procedure was described more than 50 years ago but not employed widely in forensic serology , until 1960 , when kind refined this technique . ae technique / procedure originally devised by siracusa is now employed in all forensic laboratories because it is proven to be most sensitive , reliable and reproducible . pulp tissue is one of the most protected tissues being surrounded from all sides by dental hard tissues . pulp contains numerous blood vessels and blood group antigens are certainly present in tooth pulp . the distribution of abo substances from the pulp cavity wall to the dentin edge and the enamel gradually reduces , because of fewer possibilities of diffusion of antigens from both blood and saliva . the existence of blood group antigens in tooth dentin and enamel and their nature has been substantiated by infusion sedimentation phenomena combined with inherently present antigens . this theory describes the infusion of water - soluble antigens from saliva into the tooth tissue . the presence of abo blood group and rhesus factor antigen in soft and hard dental tissues makes a possible contribution in human identification even in decomposed bodies . therefore , blood group and rhesus factor determination for biological evidence on tooth material is of great importance in forensic odontology . the aim of this study was to determine the abo blood grouping and rhesus factor from dentin and pulp of extracted teeth using ae technique at 0 , 3 , 6 , 9 and 12 months after extraction . a brief case history with relevant medical history was recorded from patients selected for study and consent was taken . the blood groups were determined for all the study participants using capillary blood by slide agglutination method . carious teeth and grossly decayed teeth were excluded ; and teeth extracted for periodontal , and orthodontic purposes were included in the study . the extraction procedure was carried out under local anesthesia following the aseptic protocol in the department of oral and maxillofacial surgery . the extracted teeth were dried and stored in labeled bottles for a span of 3 , 6 , 9 and 12 months . the pulp was scooped with a spoon excavator and dentin was powdered using a straight fissure bur [ figure 1 ] . the blood grouping from teeth was performed by ae technique using powdered dentin and dental pulp . the pulverized dentin powder and pulp were divided into three equal parts and were taken into six sterile test tubes containing 2 ml of saline and labeled , respectively . to each of these test tubes , three drops of antiserum a , b , d was added respectively , and the test samples were sufficiently soaked with antiserum for 2 h and left standing at room temperature . each sample was washed five times with cold saline solution by centrifuging it at 3000 rpm for 5 min and the supernatant was removed with pipette . then two drops of fresh saline were added to the sample , and the test tubes were heated in a water bath at a temperature of 5055c for 10 min to elude the antibodies . ( a ) dissected teeth samples , ( b ) pulverized dentine and ( c ) dried pulp tissue a drop of 0.5% red cell suspension of known blood group a , b and o was freshly prepared and immediately put into respective test tubes . the samples were incubated at 37c for 30 min to enhance agglutination , and then , it was centrifuged at 15002000 rpm for 1 min . by gentle shaking of the test tube , the presence or absence of red cell agglutination was ascertained macroscopically and microscopically at a magnification of 4 [ figures 2 and 3 ] . macroscopic observation of the agglutination in dentin ( l ) and pulp ( r ) samples microscopic observation of the agglutination in dentin ( l , unstained , x100 ) and pulp ( r , unstained , x400 ) samples ( 4 ) data were analyzed in proportion . for comparison , chi - square test or fisher 's exact test was used for small sample . two - tailed p < 0.05 was considered as statistically significant , and 0.01 was considered as highly statistically significant . data were analyzed in proportion . for comparison , chi - square test or fisher 's exact test was used for small sample . two - tailed p < 0.05 was considered as statistically significant , and 0.01 was considered as highly statistically significant . abo blood grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased . in dentine , the sensitivity ranged from 100% to 73% and pulp sensitivity ranged from 100% to 80% . there was no significant difference in the sensitivities of dentin and pulp [ table 1 ] . abo blood grouping for pulp and dentine rh factor grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased up to 9 months . in dentine , the sensitivity ranged from 100% to 40% and pulp sensitivity ranged from 100% to 23% . overall pulp showed better sensitivity than dentin except at 12 months time period where dentin showed better sensitivity than pulp . moreover , in comparison with the sensitivities of dentin and pulp , the p values obtained were not significant [ table 2 ] . rh factor blood grouping for pulp and dentine when compared the sensitivity of abo and rh factor blood grouping of dentin and pulp in accordance to time period dentin and pulp showed almost similar results from 0 to 9 months . at 12 months both dentin and pulp showed vast difference in the sensitivities of abo and rh blood grouping with significant p value [ table 3 ] . comparison of abo and rh factor blood grouping for dentin and pulp the overall mean sensitivity of dentin for abo blood grouping is 88% and for rh factor , blood grouping is 79% , with a significant p = 0.03 . the overall mean sensitivity of pulp for abo blood grouping is 90% and for rh factor , blood grouping is 82% with a significant p = 0.04 . dentin and pulp showed the p = 0.58 for abo blood grouping and 0.46 for rh factor blood grouping [ table 4 and figure 4 ] . overall comparison of blood group systems and teeth components graph of comparisons of the overall sensitivity of abo and rh factor blood grouping of dentin and pulp lattes have rightly said the fact that belonging to a definite blood group is a fixed character of every human being and can be altered neither by lapse of time nor by intercurrent disease . human identification is the mainstay of civilization , and the identification of unknown individuals has always been of paramount importance to society . the use of blood group substances in medico - legal examination is grounded on the fact that once a blood group is established in an individual , it remains unchanged for a lifetime . for several decades , the technique ae is the most sensitive and the most widely employed . according to kind , nickolls and periera and outtridge , ae has proved to be markedly more sensitive than the absorption - inhibition test . ae has shown more success rate than mixed - agglutination for certain antigens . rh blood group is considered to be the most complex genetically of all the blood type systems since it involves 45 different antigens on the surface of red cell . teeth are used for blood grouping and are considered as a hallmark for identification of biological materials in forensic investigations . considering this fact in our present study , an attempt was made to detect abo and rh factor antigen from dentin and pulp over a time period of 0 , 3 , 6 , 9 and 12 months . for abo blood grouping , both dentin and pulp showed 100% sensitivity with the samples tested immediately after extraction and the sensitivity gradually reduced as time period prolonged . pulp showed better sensitivity than dentin but the difference was statistically insignificant , suggesting both dentin and pulp have almost equal antigenic potential although pulp is better , the sensitivity weakened as the time period increased . till date , the various studies conducted by smeets et al . , shetty and premlata , ballal and david and ramnarayan et al . at different time period showed pulp to be better tool than dentin and there was a decrease in the sensitivity of the dentin and pulp as the time periods increased . the overall decrease in the sensitivity could be due to dehydration , the loss of pulp antigens , insufficient quantity of pulp , calcification of the canals , cell lysis ; contamination of the tooth or time lapse for the procedure . for rh factor blood grouping , both dentin and pulp showed 100% sensitivity when the samples were tested immediately after extraction and the sensitivity gradually reduced as time period prolonged . pulp showed better results than dentine , but the difference was statistically insignificant , suggesting both dentin and pulp have almost equal antigenic potential which weakened as the time period increased , similar to abo antigens . this may be attributed to autolysis , dehydration and the loss of pulp antigens similar to abo antigens . is the only study available in english language literature that was conducted for determination of rh factor antigens along with abo blood group antigens in freshly obtained pulp samples . in our study , both pulp and dentin samples were used and tested at 0 month to an extensive time period of 12 months . abo and rh factor blood grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased . at 12 months , both dentin and pulp showed a drastic decrease in the sensitivity for rh factor blood grouping than the abo blood grouping with a statistically significant difference . the sensitivity of pulp was better than dentin in both the blood grouping systems with an insignificant p value . abo blood grouping was better than rh factor grouping in both the teeth components and the p values obtained were significant . this indicates that the antigenicity of pulp is better than dentin in both abo and rh blood groups , and abo antigens were better expressed than rh factor blood group antigens . the outcome of our study showed that abo and rh blood group antigens could be detected up to 12 months , and there are no studies in the english literature to compare this study . this shows that pulp contains more amounts of antigen than the dentin and its antigenicity decreases with time . blood grouping on teeth is not a straight forward technique ; the concentrations of blood group antigens are low in the teeth when compared to other tissues and body fluids . in this study , it is assumed that the origin of blood group antigen in dental hard tissue is based on the infusion sedimentation phenomenon combined with inherently present antigens . considering all the factors that support the presence of blood group antigens in dentin and pulp and also the pitfalls of false positive result or mistyping of blood group , over a period in this study , we came to a close consequence that the results obtained with pulp were better than that of dentine . another aspect to be highlighted here is abo and rh factor antigens were detected from both dentin and pulp even up to 12 months after extraction of the teeth . blood grouping has been one of the bases for identification of biological materials in forensic investigations and abo blood grouping is a widely used technique in forensic laboratories . the presence of abo blood group antigens along with rh factor antigens in pulp and hard dental tissues makes it a potential substance in the identification of highly decomposed bodies or body part where teeth and bones are the only significant tissue remains . blood group substances in the hard dental tissues thus remain unaffected even in adverse environmental conditions . teeth were used as a mode of identification of blood group in this study because teeth are one of the most indestructible parts of the body and exhibit the least turnover of natural structure . the presence of abo blood group and rh factor antigen in soft and hard tissue makes it possible for identification of the deceased . ae test to identify blood groups in teeth may be of immense value not only in the identification of accused but also in the investigation of mass disaster and fire victims . on the basis of results obtained in our study , both dentin and pulp are reliable sources of blood group determination for upto 12 months for abo and rh factor blood grouping , especially where teeth ensues to be the only remnants existing for individual identity . although expression of abo blood groups and rh factor was seen in both dentin and pulp , intensity of abo blood groups and rh factor was higher in pulp than dentin and abo blood group antigens were better expressed than rh factor antigens in both dentin and pulp . blood group determination from teeth warrants advance exploration as the establishment of identification of a person from the skeletal remains is of paramount importance to a forensic odontologist . till date , in english language literature no studies have been conducted to detect abo and rh factor blood group antigens from tooth material for up to 12 months . this study detected blood groups antigens of abo and rh systems from tooth material over this extensive time period of 12 months . this study is thus a quantum of what has been known and learned and how much more needs to be understood in this challenging branch of forensic odontology . cystic echinococcosis ( ce ) is a severe zoonosis caused by the cyclophyllidean cestode echinococcus granulosus . the disease has a worldwide distribution , with endemic regions in many countries of the mediterranean basin , north and east africa , western and central asia , china , south america , and australia [ 1 , 2 ] . although the distribution of echinococcus granulosus is considered worldwide , it is higher in developing countries in tropics and subtropics , especially in rural communities where there is close contact between dogs and various domestic animals . in some western countries , ce is considered as a reemerging zoonosis due to its resurging prevalence [ 4 , 5 ] . the worldwide distribution of the disease is partly due to the easy adaptability of the parasite to several domestic and wild intermediate hosts . clinically , there are three broad morphological forms of echinococcosis that are recognized : cystic echinococcosis caused by e. granulosus , alveolar echinococcosis caused by e. multilocularis , and polycystic echinococcosis caused by e. vogeli and e. oligathrus [ 79 ] . the sheep strain ( defined as g l on mitochondrial genotypic grounds ) is generally considered as the most widespread strain of e. granulosus in the world and the one mainly involved in ce in humans . at least five out of ten strains of e. granulosus strains ( g 1 to g 10 ) have been found to be infective to humans in sub - saharan africa . , disease consequences may include poor quality of life ( disability adjusted life years ( dalys ) ) , costs of medical treatment , lost opportunity for income generation , and mortality in some cases while in animals there is reduced productivity and monetary losses due to abattoir condemnations of organs [ 14 , 15 ] . the dalys for human cystic echinococcosis was recently estimated to be more than that for onchocerciasis and almost the same as that for africa trypanosomiasis . the annual ce - associated economic losses on a global basis have been recently estimated to be at least over us$2 billion . in zambia , like in most sub - saharan africa , echinococcosis has been reported in many parts of the country , although not much information is currently available making it one of the neglected tropical diseases . in western province of zambia , hydatid cysts are reported to have been diagnosed from cattle carcasses during meat inspection although most of these reports are inconclusive . however , there has been no comprehensive study carried out thus far to describe echinococcosis infections in both the intermediate and final hosts and also to determine the economic and public health significance . based on circumstantial evidence , it is assumed that the disease has serious public health and socioeconomic implications given the interactions that exist between cattle , dogs , and humans and also the uncontrolled disposal of abattoir waste and remains from animal slaughters . however , this assertion needed to be supported by well - structured studies . the aim of this study therefore was to determine the prevalence of hydatidosis in cattle presented for slaughter at abattoirs in western province of zambia and assess economic losses due to organ condemnation using a cross - sectional epidemiological survey with the view to identifying intervention measures aimed at reducing transmission of the disease between humans and different animals hosts . the study was conducted in western province of zambia from october 2007 to november 2008 . western province lies between longitudes 22 degrees and 25 degrees east and latitude 13 degrees 30 mins and 17 degrees 45 mins south . the province covers an area of 126,386 km , which represents about 17% of the total land surface of zambia which covers 752,000 km ( figure 1 ) . about 10% ( 12,950 km ) of the total land area consists of a vast sandy upland . the province has a dry and cold winters ( april to july ) , hot and dry season ( august to october ) , and hot and wet summers ( november to march ) . the annual flooding of the zambezi plains controls the pattern of life for the people and livestock in western province with people practice transhumant subsistence livelihood . thus , during flooding , the largest population of cattle and people are concentrated along the edges of the plains . western province has a cattle population of approximately 452,400 , making it one of the largest cattle producing areas in zambia , while the dog population is estimated at 65,315 with mongu having highest number of dogs at 16,210 followed by kalabo ( 13,496 ) , shangombo ( 11,732 ) , sesheke ( 8,638 ) , kaoma ( 6,254 ) , senanga ( 4,750 ) , and lukulu ( 4,236 ) . dogs , generally , belong to specific households where feeding is supplemented but often have the freedom to roam and scavenge in the neighbourhood . all cattle are slaughtered within the province , mostly in mongu and senanga abattoirs , due to a movement ban imposed in 1998 as a result of the outbreak of contagious bovine pleural pneumonia ( cbpp ) . therefore , data obtained from cattle that are slaughtered in mongu is a good representation of the true provincial picture . the study was conducted as a two - tier study involving a prospective abattoir survey and a retrospective review of meat inspection reports at zambeef and starbeef abattoirs in mongu . a retrospective study was carried out based on a review of postmortem reports findings during meat inspection at the abattoirs in the last eleven years ( 19942007 ) . data was obtained from district veterinary offices and abattoir reports on meat inspection and movement of livestock carried out in the previous 11 years in western province . information collected included number of cattle slaughtered , breed and type of organs condemned , number and weight of condemned organs . the aim of this was to provide baseline information and a retrospective understanding of the prevalence , dynamics and spatial distribution of the disease in western province and also to estimate the annual economic loss due to organ condemnation . this study was conducted between october 2007 and october 2008 at zambeef and starbeef abattoirs in mongu district . cattle that were slaughtered at the two abattoirs were sourced from all the seven districts of western province except sesheke district . all 4061 cattle that were slaughtered during the study period were included in the survey . the slaughtered cattle were subjected to thorough postmortem inspection and lesioned organs were identified and samples were collected . prior to commencement of the prospective study , meat inspectors at the two slaughterhouses underwent an in - house refresher training in recognition of hydatid cysts in various organs of the carcasses according to the procedures recommended by fao / unep / who ( 1994 ) . each animal that was slaughtered was uniquely identified using stock movement permits that included the veterinary camp of origin in the district and further information was obtained by interviewing the owner . the age of the animals was obtained by interviewing the owners in cases where the ultimate owner brought the cattle ; otherwise the age was estimated using dentition as described by jenkins . visceral organs including lungs , liver , heart , spleen , and kidneys were examined , through visual observation , palpation and systematic incision in each carcass according to procedures recommended by fao / unep / who ( 1994 ) . hydatid cysts where identified through visual inspection and palpation of organs during meat inspection and enumerated . a sample of hydatid cysts during inspection was removed whole and collected in polythene bags . a separate polythene bag was used for hydatid cysts obtained from one animal and was uniquely labelled and stored in ice before transportation , within one hour , to mongu regional laboratory for viability determination . cattle were classified as positive for hydatidosis if it was found with one or more hydatid cysts in any of the internal organs . at mongu regional laboratory , the collected cysts were individually grossly examined for degeneration and calcification as described by oostburg et al . . the cyst wall was carefully incised with a scalpel blade and the contents poured into a petri dish . the contents were examined under a microscope ( 40x magnification ) for the presence of protoscoleces . the germinal layer was also put in glycerine and placed between two microscopic glass slides and examined for the presence of protoscoleces . cysts that did not contain protoscoleces contained pus or were calcified were considered as sterile or not viable . further , the viability of the protoscolices was checked under the microscope using the dye exclusion principle after staining with 0.1% eosin stain for 15 minutes . the protoscolices that took up the stain were classified as dead while those that did not were considered to be alive and viable [ 18 , 19 ] . the loss attributed to condemnations of offal due to echinococcus was determined using a modification of the formula as described by yamene ( 1990 ) cited by getaw et al . . this was on the basis of the average price of wholesome and intact visceral organs obtained from zambeef and starbeef abattoirs mongu . data was stored in microsoft excel spread and transferred to stata statistical packages version 10 ( stata corp . infections in cattle for both prospective and retrospective data was determined as proportion of the test - positive subjects against the total number tested . apparent prevalence estimates were converted into true prevalence values by taking into account the sensitivity and specificity of the test methods as described in dohoo ( 2003 ) . the annual economic loss as a result of condemned organ was estimated by taking into account the average number of cattle slaughtered per annum at the zambeef and starbeef abattoir and the percentage of condemned organs using the following formula described by yemane ( 1990 ) as cited by getaw et al . . ( 1)annual loss=(npsiluclu)+(npsilicli)+(npsiheche)+(npsikicki ) , where nps : total number of positive animal slaughter , ilu : prevalence of lung hydatidosis , ili : prevalence of liver hydatidosis , ihe : prevalence of heart hydatidosis , iki : prevalence of kidney hydatidosis , clu : cost of lung , cli : cost of liver , che : cost of heart , and cki : cost of kidney . a retrospective study was carried out based on a review of postmortem report findings during meat inspection at the abattoirs over a period of eleven years from 1994 to 2007 ( with exemption of 1997 , 1998 , and 2002 where data was missing ) . during this period , 158 , 456 bovines were slaughtered and inspected , and 4689 cases of bovine hydatidosis were recorded ( table 2 ) . the overall combined prevalence of bovine echinococcosis during the period under review was estimated at 3.0% ( table 1 ) which was close to prevalence observed in our prospective study . annual prevalence ranged from the lowest at 1.56% ( n = 12641 ) in 2006 to the highest at 4.7% ( n = 2633 ) in 2001 . a review of the postmortem records over an eleven year period revealed that the distribution of hydatid cysts in bovine was highest in lung at 93.47% ( 95% ci : 92.7594.14 ) followed by liver at 6.55% ( 95% ci : 5.887.29 ) and spleen with the lowest at 0.02% ( 95% ci : 0.000.12 ) prevalence . a total of 4061 cattle from mongu ( n = 2441 ) , senanga ( n = 577 ) , kalabo ( n = 653 ) , lukulu ( n = 335 ) , shangombo ( n = 47 ) , and kaoma ( n = 8) were slaughtered at the zambeef and starbeef abattoirs between october 2007 and november 2008 . out of this , 84 ( 2.1% ) carcasses ( table 3 ) were diagnosed positive for hydatidosis during postmortem inspections . there was variation in prevalence of hydatidosis according to the district of origin , where cattle coming from mongu had the highest prevalence of cyst positive cases ( 2.5% ) compared to senanga ( 2.1% ) , kalabo ( 1.4% ) , and lukulu ( 0.6% ) ( table 3 ) . sex was found to be positively associated with hydatidosis ( p = 0.035 ) with female cattle being more likely to test positive than males ( odds ratio = 1.62 ) . on the other hand , hydatidosis was independent of age ( p = 0.31 ) where the mean age among the positives was 7.8 years ( range : 7.47.6 ) and that among the negatives was 7.5 years ( range : 7.38.3 ) . in terms of distribution of hydatid cysts by organ , 51.2% were found in lungs , 47.6% were in livers , while 1.2% were in the kidneys . mukukutu camp in senanga district accounted for the highest prevalence at 4.0% ( 95% ci 3.811.8% ) , while lukulu central camp in lukulu district had the lowest prevalence at 0.3% ( 95% ci 0.20.9% ) . on comparison of camps in different districts , it was observed that in mongu , the highest prevalence of bovine hydatidosis was in limulunga veterinary camp at 2.9% ( 95% ci 1.44.4% ) with the lowest prevalence in luandui camp at 1.5% ( 95% ci 0.53.7% ) . in senanga district , the highest prevalence was in mukukutu camp at 4.0% ( 95% ci 3.811.8% ) and the lowest was mouyo camp at 1.6% ( 95% ci 0.043.2% ) . in kalabo district , the highest prevalence was observed in sikongo camp at 3.3% ( 95% ci 1.28.0% ) . in lukulu district , the highest prevalence was in mbanga camp at 1.8% ( 95% ci 1.75.3% ) and lowest in lukulu central camp at 0.3% ( 95% ci 0.20.9% ) . the overall median number of cysts in an organ was 6 ( range 221 ) , in the lungs the median was 6 ( range 221 ) , and liver the median was 4 ( range ; 35 ) . the number of hydatid cysts that were examined in the lung was 108 while in the liver it was 16 . the lung had a highest density of cysts per organ compared to the liver ( table 4 ) . there was no significant difference in viability rate of hydatid cysts recovered from the lung ( 43.5% ) and in liver ( 43.8% ) . the prices used in the estimation of annual economic loss from condemned organs , were the 2011 average prices for wholesome and intact visceral organs obtained from zambeef butchery in mongu . while the average weights of the various organs were calculated from the data obtained from the abattoir prospective study . the average weight of a lung was estimated at 2.92 kg , liver and spleen were 3.34 kg and 2.00 kg , respectively . the average cost of lung was zmk ( zambian kwacha ) 12,000 per kg , liver zmk 18,000 per kg , and spleen zmk 12,000 . the cost of one lung = average weight cost / kg ( 2.92 @ 12000 ) = zmk 35,040 ; cost of liver = average weight cost / kg ( 3.34 @ 18000 ) = zmk 60,120 ; cost of spleen= average weight cost / kg ( 2 @ 12000 ) = zmk24 , 000 , average annual slaughter= 14,405 . in this study , we investigated the prevalence of hydatidosis based on pm findings at two abattoirs in western province of zambia . it is therefore noted that the prevalence estimates provided here may have some bias as abattoir sample populations is not always representative of the reference populations where animals are drawn . this is often so because animals brought for slaughters are those that are old or out of production . considering the reduced sensitivity of pm inspection - based diagnosis , there is always a possibility that some positive cases were missed resulting in underreporting the actual disease burden . despite these short - comings , abattoir survey data is routinely used to estimate disease burden because of easy feasibility of conducting abattoir surveys compared to field surveys based on random study designs . besides , abattoir data provides opportunity for developing intervention strategies by timely diagnosis and condemning carcasses infected with zoonoses likely to enter the food chain . the observed prevalence of hydatid cysts in cattle sampled at the two abattoirs in mongu was found to be low ( 2.1% ) and was comparable to that observed during the retrospective survey ( 3.0% ) . furthermore , the findings in this study were in agreement with that observed in a study done in sudan which reported a prevalence of 3% in cattle . in arusha tanzania , a study by nonga and karimuribo reported a prevalence of 4.2% in cattle . similarly , for instance , rkia azlaf and allal dakkak reported prevalence of 23.0% bovine hydatidosis in morocco and so did kebede in ethiopia who reported a prevalence of 22.1% . in our study , the distribution of hydatidosis varied according to district with mongu reporting the highest prevalence compared to other districts . the reason for the high prevalence in mongu could be attributed to a high numbers of cattle and dog populations coupled with a high number of home slaughters during ceremonies , which in some cases are not inspected by the veterinary department staff . there is an increased dog and cattle interaction due to high populations and free range rearing of cattle which are often herded by boys with dogs ; this increases contact of cattle with dog faeces . further , dog access to slaughterhouse waste in mongu abattoirs is likely to increase exposure of both cattle and dogs in the district . sex was found to be positively associated with hydatidosis ( p = 0.035 ) with female cattle being more likely to test positive than males . , in iran , who observed that the prevalence was higher in females than males . there was a significant difference in the prevalence of hydatid cysts between carcasses slaughtered in 2007 and 2008 ( p = 0.024 ) , where prevalence of bovine hydatidosis in 2007 was 1.3% ( 95% ci : 0.691.93 ) and in 2008 was 2.4% ( 95% ci : 1.82.9 ) . this could be as a result of more animals coming from areas of higher prevalence of bovine hydatidosis such as mongu and senanga in 2008 than in 2007 . however , this could not be fully ascertained due to absence of trace back information during the period under review . the lung was found to be the most affected organ ( 51.2% ) compared to the liver ( 47.6% ) and the kidney ( 1.2% ) . this is in agreement with what was reported by getaw et al . who observed that the lung had a higher prevalence at 55.2% and the liver at 37.1% while the kidney was the least affacted organ . the results are also in agreement with findings by cadmus and adesokan ( 2009 ) in nigeria , and kebede et al . who did their study in ethiopia . however , our results are at odds with the findings from a study conducted in libya where researchers reported higher prevalence in the liver than in the lung and al - khalid ( 1998 ) cited by dakkak who showed that in libya , 75% of the positive bovine hydatidosis cases were in the liver and 37.5% in the lung and 12.5% in the spleen . the reason why the lung and liver are mostly affected could be due to the fact that the lungs and livers are the first capillary beds encountered by migrating echinococcus oncospheres via the portal vein route before any other peripheral organs . the lungs however have a larger capillary bed than any other organs and this could account for the observed higher prevalence than seen in the other organs . in humans , however , the liver is most commonly affected . the explanation to this differences in the predirection sites between cattle and human is beyond the scope of this study . cysts viability study revealed that the overall percentage of viable cyst in this study was 43.5% which is comparable to findings by other researchers like ibrahim who found cyst viability of 47.8% in sheep and 24% in goats . , who did not observe any viable cyst from their survey and berhe who found a lower viability rate of 10.7% in cattle in tigray region of ethiopia . the possible reason why no viable cysts were observed by rinaldi et al . could be due to the differences in immunological responses by different individual hosts or deworming of the animals by use of antihelmintics . out of a total of 19 hydatid cyst infested organs that were investigated ( 15 lungs and 4 livers ) for cyst fertility , viability , and density , it was found that the lung had a higher average density of cysts infestation ( 7 cysts per lung ) ( table 4 ) while the liver had a low hydatid cyst density ( 4 cysts per liver ) . this was however different from findings by ibrahim in saudi arabia who observed that the liver had a higher cyst density . the difference in the cyst density could mainly be attributed to the higher vascularisation of lung tissue compared to liver . the other reason in the difference in cyst density could be as a result of the soft texture of the lung tissue in comparison to liver , which has a harder texture thus restricting hydatid cyst development . the number of hydatid cysts that were examined in the lung was 108 while in the liver was 16 . most of the dead cysts in the liver were found to be calcified compared to the lung . , kebede et al . , and berhe who reported a higher fertility rate of pulmonary and lower fertility rate in hepatic cysts . this could probably be due to the various metabolic reactions that take place in the liver as compared to lungs . however ibrahim found a higher fertility rate in liver at 38.8% than in the lung at 25.1% and so did dalimi et al . who reported a higher fertility rate in the liver than in the lung . the high percentage of viable cysts indicates that there is a high risk of dog exposure in situations where offal are carelessly disposed of and dogs have access to the infected offal , like the situation is in western province of zambia . this points to a possible intervention area in which dogs should be prevented from ingesting infected with cysts such as the lungs and liver . in this study , the annual economic loss as a result of condemnation of organs due to bovine hydatidosis was low at k 15 , 894,039.00.(3,311 us$ ) per annum . the loss was found to be low due to the low prevalence of hydatidosis in cattle in western province of zambia . the total annual loss could be greater than the estimated amount bearing in mind that this only took into account direct losses and not indirect losses as a result of weight loss due to ce and other losses such as reduced milk production and reduction in reproduction in cattle . the main thrust of the study was to describe the hydatidosis situation in western province so as to increase public health awareness , describe the socioeconomic impact , and to recommend possible mitigation measures . it should be noted however , that echinococcosis is a disease of multiple hosts and the objective of this study could only be addressed by the application of conventional observational studies . this study has demonstrated that hydatidosis is an important disease and is endemic in western province . the disease also causes considerable economic losses as a result of offal condemnations . despite the low infection rate demonstrated by the current study , there are certain socioeconomic conditions that are favourable for the existence of ce , and therefore ce still remains one of the most important helminth zoonotic disease hence the need for increased attention in control and prevention of the disease . livestock and dog echinococcosis prevalence studies and surveillance can help map out ce risk landscape profiles that will determine communities at greatest risk to human ce . a molecular analysis of human and animal hydatidosis would be desirable in order to effectively map out epidemiology of the disease and determine the spread of the disease . a specific study of the disease in dogs could also help in knowing the prevalence in the definitive host . wildlife species have not been shown to harbour e. granulosus in zambia . in view of the extensive livestock / wildlife interface areas in the province , furthermore , a study in small ruminants such as sheep and goats may improve epidemiological understanding of the disease in zambia . with the current study having been done on cattle , it is also suggested that in areas where there is a presence of large numbers of wildlife definitive hosts such as observed in some parts of kalabo , lukulu , and kaoma districts , increased effort should be made to sample some of the possible wildlife definitive host of echinococcus spp . effort should be made to ascertain the host specificity of local strains of the parasite in respect to cattle and other domestic animals . to effectively come up with a control program , possible wildlife reservoirs , and the survival of eggs under the local climatic and soil conditions have to be investigated .

objective : the aim of the study was to determine blood groups and rhesus factor from dentin and pulp using absorption - elution ( ae ) technique in different time periods at 0 , 3 , 6 , 9 and 12 months , respectively.materials and methods : a total of 150 cases , 30 patients each at 0 , 3 , 6 , 9 and 12 months were included in the study . the samples consisted of males and females with age ranging 1360 years . patient 's blood group was checked and was considered as control . the dentin and pulp of extracted teeth were tested for the presence of abo / rh antigen , at respective time periods by ae technique.statistical analysis : data were analyzed in proportion . for comparison , chi - square test or fisher 's exact test was used for the small sample.results:blood group antigens of abo and rh factor were detected in dentin and pulp up to 12 months . for both abo and rh factor , dentin and pulp showed 100% sensitivity for the samples tested at 0 month and showed a gradual decrease in the sensitivity as time period increased . the sensitivity of pulp was better than dentin for both the blood grouping systems and abo blood group antigens were better detected than rh antigens.conclusion:in dentin and pulp , the antigens of abo and rh factor were detected up to 12 months but showed a progressive decrease in the antigenicity as the time period increased . when compared the results obtained of dentin and pulp in abo and rh factor grouping showed similar results with no statistical significance . the sensitivity of abo blood grouping was better than rh factor blood grouping and showed a statistically significant result . a cross - sectional study was conducted from october 2007 to november 2008 to estimate the prevalence of hydatidosis in slaughtered cattle from two abattoirs in mongu , western province , zambia , using prospective and retrospective data . out of the 4061 cattle examined during postmortem inspection , 84 ( 2.1% ) were positive for hydatidosis . no cases were detected from kaoma and shangombo districts ; however , prevalence ranged from 0.6% to 2.5% in districts where it was present . sex was found to be positively associated with hydatidosis ( p = 0.035 ) with female cattle being more likely to have hydatidosis ( or = 1.62 ) . in the retrospective study ( 1994 to 2007 ) , annual prevalence of hydatidosis ranged from 1.56% ( n = 12,641 ) in 2006 to 4.7% ( n = 2633 ) in 2001 with an overall prevalence of 3% ( 4689/158,456 ) . this value is comparable to that observed in cattle slaughtered between october 2007 and november 2008 ( 2.1% ) . hydatidosis was observed in the lungs ( 51.2% ) , liver ( 47.6% ) , and kidneys ( 1.2% ) . the percentage of viable cysts was 43.7% . this study confirms the presence of hydatidosis in cattle in western province of zambia and estimates economic losses due to organ condemnations . data presented herein provides a useful baseline for developing policy and intervention measures .

septic internal jugular vein - sigmoid sinus thrombosis is a rare condition that complicates local and regional infectious inflammatory processes occurring in the head and neck . these include deep neck infections , lemierre syndrome , and central venous catheterization ( cvc ) or cannulation17 ) . this is a life - threatening condition and prompt management is essential to decrease the potential for thrombosis - related morbidity and mortality . we report a case of internal jugular vein - sigmoid sinus thrombosis due to a misplaced central venous catheter . a 52-year - old woman presented with a severe bursting headache , vomiting , and a drowsy mentality . brain computed tomography ( ct ) scan revealed subarachnoid hemorrhage in the basal cistern with a small amount of hematoma at the left sylvian fissure ( fig . we identified an aneurysmal rupture of the middle cerebral artery on the left side by cerebral catheter angiography ( fig . postoperative chest x - ray was normal except for malposition of the central venous catheter in the right internal jugular vein ( fig . ten days later , the patient developed a fever , elevated white blood cell count to 227000 , and elevated c - reactive protein to 33.6 . the central venous catheter was removed , and antibiotics were administrated through a different intravenous route . despite these managements , the patient was stupor the next day . brain ct scan revealed cerebral infarction with hemorrhagic transformation in the right temporal lobe , and ct angiography did not identify vasospasm ( fig . retrospective analysis of the contrast - enhanced ct scan and ct angiography showed an " empty delta sign " and absence of venous flow within the right internal jugular vein - sigmoid sinus that was sufficient for diagnosis of sinus thrombosis ( fig . results of central venous catheter tip and blood culture was reported staphylococcus epidermidis and methicillin - resistant staphylococcus aureus , respectively . we could not start systemic heparinization due to hemorrhagic transformation of the cerebral infarction in the right temporal lobe . we administrated mannitol and steroids to manage increased intracranial pressure ; however , the patient died from severe pneumonia due to septic emboli after one week ( fig . 2c ) . indications for a cvc are the intravenous administration of drugs , parenteral nutrition , hemodialysis , and hemodynamic monitoring13 ) . at many institutions , cvc is routinely inserted before undergoing major surgery or treating patients with critical illnesses or cancer . the most frequently available anatomical sites for cvc are the subclavian and internal jugular veins . these procedures carry a substantial risk of mechanical lesions , such as arterial puncture , pneumothorax , cardiac tamponade , nerve lesions , or thrombotic or septic complications13 ) . malpositioning of the catheter tip may happen more often in the subclavian vein than the internal jugular vein . the reported incidence of primary misplacement of the catheter tip after infraclavicular subclavian vein catheterization varies from 5% to 24% even when inserted by experienced clinicians7 ) . inadvertent catheterization of the ipsilateral internal jugular vein is one of the most common misplacements , with a reported incidence of around 7%15 ) . the positioning of catheter tips within the cardiac silhouette is associated with increased risk of cardiac tamponade6 ) . also , positioning of the catheter tip in the subclavian vein is associated with a high risk of thrombus formation and vessel occlusion2 ) . the risk of thrombosis may increase when hyperosmolar parenteral nutrition fluid is administered through a misplaced central venous catheter into a internal jugular vein3,15 ) . moreover , malpositioned catheter tips can damage the endothelium and precipitate the formation of thrombi17 ) . when a cvc causes thrombosis , the risk of catheter - related sepsis may increase . in patients with a cvc , the risk of catheter - related infection was reported to range between 1% and 10%1 ) . contamination of a thrombus from the skin puncture site may result in septic endophlebitis , and occasional blood - borne infections may also contaminate the thrombus . embolic septic thrombi may involve the lungs and , less frequently , the joints , viscera , and brain17 ) . subclavian or internal jugular vein thrombosis associated with indwelling catheters will propagate into other vessels , but extension into the intracranial sinuses and veins is rare . three reports describe an association between cerebral venous sinus thrombosis and central venous hyperalimentation due to placement of the catheter tip in the internal jugular vein3,15,16 ) . the authors warn of the potential for thrombosis due to retrograde infusion into the valveless internal jugular - dural sinus system but also suggest that the small caliber of the vein may predispose to thrombosis . in our case , we found the malpositioned catheter tip in the internal jugular vein on follow - up chest x - ray , but it was ignored . all intravenous fluids ( e.g. , total parenteral nutrition , mannitol , antibiotics ) were administered through the misplaced catheter . the patient 's condition worsened , leading to thrombosis of the internal jugular vein secondary to sigmoid sinus thrombosis . since the advent of antibiotics fever , chills , otalgia , tenderness to percussion over the mastoid emissary vein , headache and vomiting are common but not pathognomonic features8,18 ) . occasionally , neurologic symptoms are related to increased intracranial pressure or infarct and present as deteriorating mental status , lethargy , seizures , hemiplegia , and coma , and may lead to death . rarely , remote septic conditions such as pneumonia are the presenting symptoms9 ) . because of the nonspecific signs and symptoms of disease and the masking effects of antibiotics , diagnosis is difficult . the diagnosis of sigmoid sinus thrombosis can be confirmed by ct , magnetic resonance imaging , or angiography . contrast - enhanced ct scans demonstrate multiple intraluminal filling defects and nonvisualization of the sinus18 ) . in addition , low density lumen , sharply defined dense vessel wall , or distension of the thrombosed vein , such as the " empty delta sign " , are positive signs for sinus thrombosis19 ) . vascular imaging with cerebral angiography is highly specific for the recognition of sinus thrombosis because it can detect the lack of blood flow in thrombosed cerebral veins and dural sinuses . chest x - rays may also demonstrate septic embolic pleura - pulmonary complications , which are often bilateral , by revealing nodular infiltrates with pleural effusion17 ) . retrospectively , we confirmed that contrast - enhanced brain ct scans revealed a low density lumen surrounding a sharply enhanced dense vessel wall at the sigmoid sinus on the right side . also , the right internal jugular vein and sigmoid sinus were not visualized on ct angiography . treatment consists of aggressive antimicrobial therapy , heparinization , anticoagulation , and decreasing intracranial pressure . antibiotic selections is directed toward the causative pathogen cultured at the initial site of infection . recent studies have shown heparinization to be safe and beneficial , despite the possibility of an increased risk of hemorrhage4,10,16 ) . currently , heparin is recommended as the initial drug of choice for cerebral venous sinus thrombosis followed by long - term anticoagulation with warfarin5,10,12 ) . although these procedures allow for rapid clot removal and reduction of venous hypertension , hemorrhagic complications can occur , leading to be high morbidity and mortality14 ) . occlusion of the cerebral veins due to thrombosis may induce localized brain edema and venous infarction resulting in elevated intracranial pressure . finally , death frequently results from increased intracranial pressure caused by obstruction of venous and cerebrospinal outflow11 ) . in our case culture results of the subclavian catheter tip and blood were confirmed as staphylococcus epidermidis and methicillin - resistant staphylococcus aureus , respectively , and we switched from broad - spectrum antibiotics to vancomycin . unfortunately , we could not start heparinization due to concurrent cerebral hemorrhage of being transformed from cerebral infarction . severe pneumonia due to septic emboli eventually developed in both lung fields , and the patient died . diagnosis of internal jugular vein - sigmoid sinus thrombosis is challenging due to vague clinical features . therefore , if patients with malpositioned cvcs present with symptoms of fever , chills , headache , vomiting , increased intracranial pressure , mental deterioration , and focal neurologic deficits , intracranial sinus thrombosis should be considered . even vague symptoms may need to be investigated radiologically to discover a thrombosis early , when it can be treated . they are also used to treat multiple myeloma , bone metastases , and calcium disorders 2 . these patients reportedly often develop bisphosphonaterelated osteonecrosis of the jaw ( bronj ) that is caused by subsequent dental surgery 3 , 4 . in general , it is thought that nitrogencontaining bisphosphonates are much high risk of osteonecrosis than nitrogen notcontaining bisphosphonates . thus , medications for cancer , such as zoledronate ( zometa ) , are thought to have much high risk of osteonecrosis than medications for osteoporosis , such as alendronate ( bonalon ) 5 . besides bisphosphonates , other antiresorptive agents such as denosumab and antiangiogenic medications also cause osteonecrosis 6 . thus , the american association of oral and maxillofacial surgeons position paper published in 2014 and the committee recommended changing the nomenclature of bronj to the term medicationrelated osteonecrosis of the jaw ( mronj ) to accommodate the growing number of jaw osteonecrosis cases associated with other antiresorptive and antiangiogenic therapies 7 . according to the position paper , there are three categories of risk factors for mronj : ( 1 ) medicationrelated risk factors ( e.g. , bisphosphonate use ) , ( 2 ) local factors ( e.g. , dental surgery ) , and ( 3 ) demographic and systemic factors and other medication factors . in this case report , the patient developed mronj and died of sepsis , although he received shortterm oral bisphosphonate therapy and had not received any dental surgical treatment because he was completely edentulous . however , he had many systemic factors such as diabetes , cirrhosis , and steroid use for interstitial pneumonia . the presence of multiple systemic factors can be a very high risk for mronj , even though there are few medicationrelated factors and local factors 8 , 9 , 10 . the patient was a 59yearold man with a history of smoking and drinking , insulin use for diabetes , cirrhosis associated with chronic hepatitis c , steroid use for interstitial pneumonia , sepsis , and spinal disk herniation . this study was conducted according to the guidelines of hiroshima city hiroshima citizens hospital , and a written informed consent was obtained from this patient . white blood cell count ( wbc ) was 7800/l and creactive protein ( crp ) level was 15.523 he was treated with intravenous steroids ( i.e. , methylprednisolone sodium succinate 1000 mg / day for 3 days , prednisolone sodium succinate 20 mg twice daily for 4 days , and methylprednisolone sodium succinate 1000 mg / day for 3 days ) . this regimen was followed by oral steroids ( i.e. , prednisolone 60 mg / day for 2 months ) . to prevent steroidinduced osteoporosis , he was treated with oral bisphosphonate ( i.e. , alendronate 35 mg / week for 7 weeks ) . however , the administration of these drugs was stopped because of a right buccal space infection . the patient had severe swelling from the right buccal area to the infraorbital region , hypesthesia , and tenderness , but no spontaneous pain ( fig . there were ulcerations and exposed bone on the alveolar part of the right incisor and molar ( fig . computer tomography ( ct ) scanning with a radiocontrast agent showed swelling in the right buccal area , but showed no mandibular bone resorption ( fig . 2b ) . magnetic resonance imaging ( mri ) demonstrated an abnormal signal ( e.g. , low signal on t1weighted [ t1w1 ] mri and high signal on t2weighted [ t2w1 ] mri ) in the bone marrow of the right mandibular angle ( fig . based on these results , the patient was diagnosed as having right mandibular cellulitis , sepsis , and disseminated intravascular coagulation ( dic ) . we administered intravenous antibiotic treatment and irrigated part of the exposed bone with povidone iodine every weekday . at 3 days after the first visit , sequestrectomy and drainage therefore , we changed the antibiotics from meropenem hydrate ( meropen ; sumitomo dainippon pharma co. , ltd . , osaka , japan ) alone to meropenem hydrate and ampicillin / sulbactam ( unasyn s ; pfizer inc . one month after the first visit , swelling from right buccal area had nearly disappeared ( fig . 4b ) . exposed bone remained on the alveolar portion of the right mandibular molar and pus discharge was present ( fig . antibioticresistant bacteria ( grampositive bacilli ) were detected at that time ( table 1 ) . therefore , we changed the antibiotics to sitafloxacin ( gracevit ; daiichi sankyo , tokyo , japan ) ( fig . 3 ) . seven weeks after the first visit , his condition had taken a turn for the worse . , he died of multiorgan failure . finally , we diagnosed this patient as mronj . his multiple systematic factors and mronj caused lethal sepsis because it fulfilled the diagnostic criteria : previous treatment with bisphosphonate , exposed bone in the maxillofacial region that has persisted for 8 weeks and no history of radiation therapy to the jaws . ( b ) bisphosphonaterelated exposed necrotic bone in the right posterior mandibular ( during drainage ) . ( a ) no bone resorption is present on the right part of the mandibula in orthopantomogram analysis . ( b , c ) right buccal swelling is apparent , but there is no bone resorption of the mandibula , based on computer tomography ( ct ) analysis . ( d , e ) abnormal signal ( i.e. , a low signal on t1weighted imaging [ tiwi ] and a high signal on t2weighted imaging [ t2w1 ] ) of the bone marrow on the right angle part of the mandibula . bacterial identification and microbial sensitivity test the transition in the laboratory test values for the white blood cell count ( wbc ) and the creactive protein ( crp ) level , and the use of several antibiotics for cellulitis and medicationrelated osteonecrosis of the jaws ( mronj ) . ( a ) disappearance of swelling from the right buccal area to the infraorbital region . ( c ) discharge of pus by exposed bone on the alveolar part of the right mandibular molar . in recently years , there have been many reports on osteonecrosis of the jaw ( onj ) caused by bisphosphonates and by other antiresorptive and antiangiogenic therapies 6 , 11 , 12 . bisphosphonaterelated osteonecrosis of the jaw to medicationrelated osteonecrosis of the jaw 7 . denosumab , a rank ligand inhibitor , is an antiresorptive agent and used to treat osteoporosis , multiple myeloma , and giant cell tumor 13 . the manufacturer reports that the frequency of onj with denosumab is nearly the same as the frequency with zoledronic acid treatment 14 . thus , it may be that the development of onj is associated with the inhibition of bone resorption , rather than the use of a certain type of drug . however , it is considered that careful attention is required for the use of bisphosphonates in the present case due to the frequent use for the treatment of osteoporosis , osteopenia , and other diseases . many reports indicate that the incidence of onj is significantly higher with the use of intravenous ( iv ) bisphosphonates such as zoledronic acid ( zometa ; novartis , basel , switzerland ) than by the use of oral bisphosphonate 3 , 15 . the incidence of onj because of the use oral bisphosphonates was higher in japan than in europe and america 16 . the period of drug use is also important , and longterm use is considered a high risk for the development of onj . in our patient , the period of oral bisphosphonates use was only 7 weeks , but it caused severe symptoms . it may be that these symptoms were because of his multiple systemic factors . in this case thus , it is thought that there were another option not to use bisphosphonate for such a highrisk patient or at least before administration we had to perform a risk assessment for mronj enough . in addition , the presence of comorbid condition such as obesity , alcohol use , and smoking was a risk factor for mronj 9 . poor oral hygiene and oral infection such as periodontal disease are also risk factors 18 , 19 . the dentures were possibly contaminated with oral bacteria . in this case , we do not know whether the dentures were illfitting and there were denturerelated traumatic ulcers because he already had ulcerations and exposed bone when we first saw him . however , we think denturerelated traumatic ulcers might have promoted a mronj . osteonecrosis of the jaw has also been reported in patients with no history of surgery or in edentulous regions of the jaw 20 . physicians have to be careful with the use of bisphosphonates or other bone resorption inhibitors in patients , regardless of the presence or absence of teeth or surgical history . the most important thing to prevent mronj it required cooperation among physicians , nurses , dentists , dental hygienists , pharmacists , and other medical staff 7 . physicians who prescribe bisphosphonates or other bone resorption inhibitors have to provide a detailed explanation to patients of the risk of mronj and consult dentists if a patient has even a small risk factor . to prevent mronj , dentists and dental hygienists have to explain sufficiently to patients the importance of oral hygiene , and perform oral care and dental treatments before patients use these drugs . there is a tendency to believe that mronj is caused by longterm bisphosphonate use ( especially intravenous bisphosphonate ) and dental surgery . we found that multiple systemic factors can be a worse risk factor for mronj because it may cause lethal disease such as sepsis . physicians have to be careful when administering bisphosphonate , other antiresorptive agents , and antiangiogenic medications for patients with multiple systemic factors , even if the patients seem to have no problems in the oral cavity . ky : involved in study conception , design , and drafting manuscript ; as : involved in study conception , design , and is the chief doctor for the patient ; fo : is one of the main doctors for the patient and performed data acquisition ; mn : is supervisor and main doctor for the patient ; ks and ey : performed analysis and interpretation of data ; yh : is one of the main doctors for the patient and performed data acquisition ; si : made critical revision for this report ; st : made critical revision and is the corresponding author .

septic internal jugular vein - sigmoid sinus thrombosis ( ijv - sst ) associated with a malpositioned central venous catheter is a rare condition . it is potentially life - threatening and necessitates early diagnosis and rapid administration of appropriate medications . unfortunately , it is difficult to diagnose due to vague clinical presentations . several studies such as ct , mri , and cerebral angiography should be performed and carefully examined to help make the diagnosis . we report a case of septic ijv - sst due to a malpositioned central venous catheter . key clinical messagemedicationrelated osteonecrosis of the jaw ( mronj ) is developed even in the patients who are edentulous and treated with shortterm bisphosphonate therapy and oral administration . it sometimes causes lethal sepsis in patients who have multiple health problems such as diabetes , cirrhosis , steroid use for interstitial pneumonia , sepsis , and spinal disk herniation .

supported by nci 2p50 ca09825806 , nci u01 ca168394 , stand up to cancer / aacr dream team translational cancer research grant su2c - aacr - dt0209 , tcga gdac grant ( nih / nci u24 ca143883 ) to gbm ; mdacc uterine spore career development award ( nci p50ca098258 ) to lwt . to review the current knowledge about nonpharmacologic approaches in the prevention and early treatment of type 2 diabetes . this study reviewed the research reports dealing with nonpharmacologic interventions aimed at preventing type 2 diabetes with early lifestyle interventions . the results from the randomized controlled trials all show that people with impaired glucose tolerance who received enhanced lifestyle advice had significantly lower ( on average 50% reduced ) incidence of type 2 diabetes compared with those allocated to receive usual care . individuals who were able to correct their lifestyle habits as recommended for usual healthy life patterns were mostly protected against type 2 diabetes . thus , compelling evidence exists that most of the cases of type 2 diabetes can be prevented or at least the onset of the disease can be significantly delayed . randomized controlled trials have unequivocally demonstrated that lifestyle management is highly efficient in the prevention and also in the early management of type 2 diabetes . this evidence of lifestyle modification in diabetes prevention is stronger than for most other multifactorial diseases . in the early randomized intervention study in malmhus , sweden ( 19 ) , lower rates of type 2 diabetes was found in igt men randomized to dietary intervention compared with those who received no therapy . more recently , several trials have tested the efficacy of lifestyle intervention in prevention of type 2 diabetes . the feasibility of diet and exercise intervention in men with igt was assessed in another study in malm , sweden ( 20 ) . because the reference group comprised of men who did not want to join the intervention , the groups were not randomly assigned . the lifestyle intervention aimed at reducing the intake of refined sugar , simple carbohydrates , fat , saturated fat , energy , and alcohol and an increase in the intake of complex carbohydrates and vegetables . physical activity training consisted of two weekly 60-min sessions with various dynamic activities . by the end of the 5-year study period , 11 and 29% of the men in the intervention group and reference group had developed type 2 diabetes , respectively . overall , the progression to diabetes in these swedish men was relatively low , even in the reference group compared with the data from the observational studies ( 1 ) . the intervention resulted in significant changes in lifestyle and physiological parameters . in another study , 577 subjects with igt were assigned either to a control , exercise alone , diet alone , or exercise plus diet group in da - qing , china ( 14 ) , using a cluster - randomized trial design . participants were assigned to clinics for dietary intervention and were encouraged to reduce weight if bmi was 25 kg / m ( 61% of all participants ) aiming at 23 kg / m ; otherwise high - carbohydrate ( 5565% of energy ) and moderate - fat ( 2530% of energy ) diet was recommended . the participants were encouraged to increase their level of leisure - time physical activity by at least 12 units per day in clinics assigned to exercise intervention . one unit would correspond for instance to 30 min slow walking , 10 min slow running , or 5 min swimming . the cumulative 6-year incidence of type 2 diabetes was lower in each of the three intervention groups ( 4146% ) compared with 68% in the control group . the results of the finnish diabetes prevention study ( dps ) provided the first convincing evidence from a proper randomized controlled trial that type 2 diabetes can be prevented by lifestyle modification ( 21 ) . a total of 522 individuals with igt were randomized to either an intensive lifestyle or a control intervention : during an average of 3.2 years of follow - up , type 2 diabetes incidence was reduced by 58% in the lifestyle group . the lifestyle intervention goals were 1 ) reduction in weight of 5% , 2 ) total fat intake < 30% of energy , 3 ) saturated fat intake < 10% of energy , 4 ) fiber intake 15 g/1,000 kcal , and 5 ) moderate exercise for 30 min / day . during the first year of the study , body weight decreased on average 4.5 kg in the intervention group and 1.0 kg in the control group subjects ( p < 0.0001 ) . indicators of central adiposity and fasting glucose and insulin , 2-h postchallenge glucose and insulin , and a1c were all reduced significantly in the intervention group compared with the control group at 1-year examination ( fig . changes in clinical and metabolic characteristics among the intervention and control group participants of the dps . 2h - p - gluc , 2-h plasma glucose ; diast , diastolic blood pressure ; f - p - gluc , fasting plasma glucose ; s , serum ; syst , systolic blood pressure . diabetes prevention program ( dpp ) ( 22 ) recruited 3,234 individuals with igt ( and fasting plasma glucose 95 mg / dl ) who were randomized to receive intensive dietary and exercise counseling , metformin , or placebo . the main aims of the intervention were 7% weight reduction and 150 min / week moderate physical activity . the relative risk reduction after 2.8 years was 58% in the lifestyle intervention group compared with the placebo group . the effect of lifestyle was higher than the effect of metformin , which showed 35% relative risk reduction . during the first year of the intervention , weight reduction was 5.6 kg ( 6% ) , with slight , gradual regain to the end of the study at year 4 ( 23 ) . the indian diabetes prevention program ( 14 ) recruited 531 people with igt who were randomized into four groups ( control , lifestyle modification , metformin , and combined lifestyle modification and metformin ) . lifestyle modification included advice on physical activity ( 30 min of brisk walking per day ) and reduction in total calories , refined carbohydrates and fats , avoidance of sugar , and increase in fiber - rich foods . after median follow - up of 30 months , the relative risk reduction in type 2 diabetes incidence was with lifestyle modification , 26.4% with metformin , and 28.2% with lifestyle modification and metformin , as compared with the control group . thus , there was no added benefit from combining the pharmacologic and lifestyle interventions . the japanese trial ( 24 ) included 458 igt 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 / m ( otherwise , to maintain present weight ) , 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 . the cumulative 4-year incidence of type 2 diabetes in the intervention group was 67% lower than in the control group . body weight decreased by 2.2 and 0.4 kg in the intervention and control groups , respectively . the trials listed above have demonstrated the benefits of healthy lifestyle on delaying the deterioration of glucose tolerance to manifest type 2 diabetes , at least as long as the intervention continued . data on possible long - term effects of such active lifestyle counseling are scarce . the 12-year follow - up of the malm study ( 25 ) revealed that mortality among men in the former igt intervention group was lower than in the control group ( 6.5 vs. 14.0/1,000 person - years , p = 0.009 ) . in a median 7-year follow - up of the dps , the marked reduction in type 2 diabetes incidence was sustained ( 13 ) . more importantly , after a median postintervention follow - up of 3 years , type 2 diabetes incidence was 4.6 and 7.2 per 100 person - years in the intervention and control groups , respectively ( log - rank test , p = 0.0401 ) , i.e. , a 36% additional risk reduction . the absolute risk difference between groups increased during the postintervention period : intensive lifestyle intervention for a limited time can yield long - term benefits on type 2 diabetes risk in individuals with igt . the 20-year follow - up of the original da qing cohort showed that a lower type 2 diabetes incidence persisted in the lifestyle intervention groups ( combined ) compared with control participants . the risk reduction remained essentially the same also during the postintervention period ( 26 ) . ( 26 ) observed no statistically significant differences in cvd events , cvd , or total mortality between the control group and the combined intervention groups , but cvd mortality tended to be lower ( 17% ) among individuals who had received lifestyle intervention . in most of the published prevention trials , the main aim was to see if comprehensive lifestyle intervention reduces type 2 diabetes risk . in the chinese prevention study ( 14 ) , an attempt to determine whether diet or exercise intervention is more effective by randomizing the participating clinics to diet only , physical activity only , or diet plus physical activity intervention revealed no difference in outcome between the two interventions . in the dps , the risk of being diagnosed with diabetes was strongly associated with the number of lifestyle goals achieved ( 21 ) . success in achieving the intervention goals in the dps was estimated from the food records and exercise questionnaires . the success score ( from 0 to 5 ) there was a strong inverse correlation between the success score and the incidence of diabetes during the total follow - up . this was especially apparent when the success in achieving the goals was assessed at year 3 , which probably reflects the importance of sustained lifestyle changes ( 13 ) . the hazard ratios were 1.00 , 0.87 , 0.67 , 0.70 , and 0.23 , for success scores from 0 to 45 , respectively ( p for trend < 0.001 ) . the effects of various components of intervention are interesting , and therefore some post hoc analyses related to this issue were completed . the independent effects of achieving the success score components at 3-year examination were assessed by including each of the five lifestyle goal variables individually in a cox model ( table 1 ) . univariate hazard ratios for diabetes incidence ( 95% ci ) were 0.45 ( 0.310.64 ) for weight reduction from baseline , 0.65 ( 0.450.95 ) for intake of fat , 0.59 ( 0.311.13 ) for intake of saturated fat , 0.69 ( 0.490.96 ) for intake of fiber , and 0.62 ( 0.460.84 ) for physical activity , comparing those who did or did not achieve the respective goal . when all the five success score components were simultaneously included in the cox model , the multivariate - adjusted hazard ratios for diabetes ( 95% ci ) were 0.43 ( 0.300.61 ) for weight reduction , 0.80 ( 0.481.34 ) for intake of fat , 0.55 ( 0.261.16 ) for intake of saturated fat , 0.97 ( 0.631.51 ) for intake of fiber , and 0.80 ( 0.571.12 ) for physical activity . furthermore , weight change was significantly associated with the achievement of each of the other four lifestyle goals , and consequently , success score was strongly and inversely correlated with weight reduction ( 27 ) . multivariate logistic regression model to predict diabetes during a 10-year follow - up correspondingly , the reduction in body weight was reported to be the main determinant of risk reduction in the u.s . after adjustment for other components of the intervention , there was a 16% reduction in diabetes risk per 1 kg weight lost during the first year of the intervention . furthermore , lower percent of calories from fat and increased physical activity predicted weight loss , and increased physical activity was important to help sustain weight loss . achieving the physical activity goal of 150 min / week reduced diabetes risk , especially among those participants who did not achieve the weight reduction goal of 7% , with risk reduction of 44% compared with those who achieved neither the weight reduction nor the physical activity goal . these findings suggest that dietary composition and physical activity are important in diabetes prevention , but their effect on diabetes risk is primarily mediated through resulting weight reduction . nevertheless , because of multicolinearity it should also be noted that in the indian diabetes prevention program ( 15 ) and chinese prevention study ( 14 ) , the participants were relatively lean , and there was no large change in body weight , but despite that , a remarkable reduction in diabetes risk was apparent . thus , in these studies , components of the intervention other than weight control were responsible for the beneficial effects on diabetes risk . with compelling evidence that type 2 diabetes can be prevented or delayed , strategies to implement the primary prevention of type 2 diabetes both in high - risk subjects as well as at the population level are urgently needed . while type 2 diabetes prevention trials rigorously defined populations by explicitly characterizing their glycemic status , these studies did not include all groups at risk for developing type 2 diabetes . methods that can also define other groups at high risk for developing type 2 diabetes have been recently developed and are increasingly used in several countries ( 28 ) . the recent analysis of the dps has also shown that such people will significantly benefit from lifestyle interventions ( 27 ) . a prospective study based on the data from the u.k . estimated the association between the achievement of the five lifestyle goals used in the dps and the type 2 diabetes risk developing diabetes during a 4.6-year follow - up ( 29 ) . the incidence of type 2 diabetes was inversely related to the number of goals achieved ( p < 0.001 ) . none of the participants who met all five of the goals ( 0.8% of the total population ) developed diabetes , whereas the risk of diabetes was highest in those who did not meet any of these goals . if the entire population were able to meet one more goal , the total incidence of diabetes is predicted to decrease by 20% . this finding suggests that health promotion interventions that result in an increase in healthy lifestyle in the general population might significantly reduce the growing burden of type 2 diabetes . groups that will be the targets for prevention efforts can be identified through several reasonably effective strategies . however , there is no universal well - tested method that will identify all at high risk for developing type 2 diabetes , and there may be some variation in the optimal strategies for different populations and regions around the world . it is also important to realize that the identification of people having a high risk of type 2 diabetes or asymptomatic type 2 diabetes is not identical with the diagnosis of type 2 diabetes . in practice , we can identify people at high risk with simple and cost - efficient tools . the main question , however , is how to implement an efficient preventive strategy in individuals identified to be at high risk , i.e. , how to translate the results of the recent successful type 2 diabetes prevention trials to a real - life setting ( 30 ) . much attention has been put on the biochemical methods for the assessment of glycemia in the early diagnosis of type 2 diabetes , but much less on the coverage of the detection of asymptomatic type 2 diabetes . the evidence is compelling that without applying an oral glucose tolerance test or an assessment of postprandial glucose , a large proportion of early cases of type 2 diabetes will remain unrecognized ( 31 ) . the international diabetes federation consensus group recently prepared a document on the prevention of type 2 diabetes ( 32 ) . this shows that the international diabetes community is now ready to accept the principle that the primary prevention of type 2 diabetes must be considered as an essential part of public health policy for diabetes . the american diabetes association consensus development conference in 2006 outlined principles regarding impaired fasting glucose ( ifg ) and igt and interventions to be applied among such individuals ( 26 ) . the american diabetes association consensus group also recommended lifestyle intervention initially for people with ifg or igt ( weight control and physical activity ) but does not mention diet at all . if both ifg and igt are present as well as additional risk factors ( and most of such people have additional risk factors ) , then a combination of lifestyle intervention and metformin is recommended . however , the evidence is not there to show that the combination of lifestyle and metformin is effective ; on the contrary , the results from the indian diabetes prevention program suggest that there is no additional benefit from metformin over and above lifestyle intervention ( 15 ) . it is not clear how much antidiabetic drugs can help in preventing progression from ifg or igt to overt diabetes and what is their overall costs and risk / benefit ratio in the long term . it is evident that long - term effects of lifestyle interventions are highly beneficial and that long - term costs are very low ( 13,26 ) . ( 33 ) in their analysis stress that the real answer to reductions in incidence and prevalence of diabetes is in social policy , not in medical care .

pik3r1 ( encoding the p85 subunit of phosphatidylinositol 3-kinase ) is the 11th most frequently mutated gene across tumors . we recently reported neomorphic p85 mutants that induce signaling cascades not predicted by the canonical functions of p85 , suggesting the need to functionally annotate specific mutations in cancer genes for effective genome - informed personalized therapy . objectiveto review the current knowledge about nonpharmacologic approaches in the prevention and early treatment of type 2 diabetes.research design and methodsthis study reviewed the research reports dealing with nonpharmacologic interventions aimed at preventing type 2 diabetes with early lifestyle interventions.resultsthe results from the randomized controlled trials all show that people with impaired glucose tolerance who received enhanced lifestyle advice had significantly lower ( on average 50% reduced ) incidence of type 2 diabetes compared with those allocated to receive usual care . individuals who were able to correct their lifestyle habits as recommended for usual healthy life patterns were mostly protected against type 2 diabetes . thus , compelling evidence exists that most of the cases of type 2 diabetes can be prevented or at least the onset of the disease can be significantly delayed.conclusionsrandomized controlled trials have unequivocally demonstrated that lifestyle management is highly efficient in the prevention and also in the early management of type 2 diabetes . this evidence of lifestyle modification in diabetes prevention is stronger than for most other multifactorial diseases .

the function of sensory receptors ( nrec ) in the movement control , muscle coordination and perception of the space position of temporo - mandibular joint ( tmj ) is fundamental although the presence of nrec in the tmj is still debated : some authors have reported on the lack of nervous fibers in the articular disk 1,2 , while florid innervation of tmj has been reported in several studies on animal models and in human 3 - 6 which suggested that 6 the concentration of sensory receptors within tmj is higher in the areas supporting higher strong tensions during articular movements ( chewing , biting , speaking ) . in discordance , other authors disclosed the presence of mechanical nrec in the articular disk of human tmj 7,8 also distinguishing receptors in capsulated and uncapsulated on the bases of morphological features . the aim of this study is to ascertain the presence and the distribution of nrec in human tmj by using of immunohistochemical investigations in healthy and pathological tmj such as arthritis and arthrosis . the study was approved by the bioethics committee of the department of odontology and surgery of university of bari . 10 samples of capsular and pericapsular soft tissues with the disk were obtained from healthy patients ( six men and four women with a mean age of 39 years ) who suffered surgery of tmj because of accidental trauma of the temporo - mandibular region ; the remaining 7 cases ( four men and three women with a mean age of 57 years ) were patients surgically treated for severe degenerative lesions of tmj ( chronic arthritis and arthrosis ) . all specimens were immediately fixed in neutral buffered formalin and embedded in paraffin ; 5 micron thick sections were cut and stained with haematoxylin - eosin , pas , gomori 's reticulin and azan - mallory trichrome ; consecutive sections were used for the immunohistochemical detection of the antigens listed in table 1 . all the antibodies used are commercially avalaible from dako italia spa , milan , italy ( glial fibrillary acidic protein = gfap ; myelin basic protein = mbp ; neurofilaments = nf ; neuron specific enolase = nse ; synaptophysin ; s-100 protein = s-100 ) and from becton dickinson , burlingame , usa ( leu-7 ) . in all cases the immunohistochemical alkaline phosphatase - anti - alkaline phosphatase ( apaap ) method was performed 9 while some sections for anti - neurofilaments antibodies were treated for 10 minutes with 1% saponin in phosphate buffered saline ( pbs ) , ph 7.2 , before the application of primary antibodies . with histochemical techniques and h&e stain , nrec were not easily detectable ( figures 1 - 8 ) ; however , these were used to identify the corresponding tissue in immunostained slides . in all specimens , such types of nrec have been detected by immmunohistochemistry ( table 2 ) : a ) globular receptors with a thin capsule closely resembling ruffini 's ones ; they exhibited strong immunoreactivity in their core for s-100 , nse and leu-7 and were abundant in the superficial peri - articular muscles and in the peri - articular fibrous capsule ; b ) elongated , onion - like receptors with a thick capsule mimicking pacini 's receptors , positive for mbp , s-100 , nse and leu-7 , particularly abundant in deep muscle fibres and in peri - articular dense fibrous tissues ; c ) fusiform capsulated receptors , morphologically similar to golgi 's receptors , located within peri - articular fibrous tissues and ligament and fibromuscolar resections , strongly positive for s-100 , mbp and leu-7 and weaker gfap reactivity . in all these types of nrec ( a+b+c ) and especially in the core , punctate reactivity for synaptophysin and for neurofilaments was also evident ; d ) free and thin nervous endings in high density within subsynovial connective tissues , in intra- and periarticular fibrous tissues and along the perimisial , endomisial sarcolemma of striated muscle fibres were detected , showing immunoreactive for neurofilaments , nse , synaptophysin and to s-100 antibodies . in the articular fibrous cartilage ( articular disk ) any previously described nrec was identified ; only s-100 protein seemed to react with chondrocytes both in normal and diseased tissues . furthermore , chondrocytes of healthy individuals appeared round - shaped , with distinct cell borders and central nuclei with an evident s-100 reactivity both in nucleus and in cytoplasm . in diseased tmj , instead , chondrocytes showed a different morphoology , especially after s-100 immunostaining : they had an elongated cytoplasm with one or more thick dendritiform processes of variable length but with a strong reactivity only for s-100 protein . the number of dendritiform chondrocytes was higher in specimens of diseased patients than in healthy patients and seems undergo a reactive reparative proliferation of discal and peridiscal tissues . few studies are reported in literature on the precise identification and distribution of nrec in articular and peri - articular tissues of tmj . in the past , some authors 3 - 6 identified ruffini's - like , pacini 's like and golgi 's like receptors both in articular and periarticular tissues , using conventional or histochemical methods usually performed to identify nerve fibres and receptors 7,8 . by using immunofluorescent techniques , other authors 3 demonstrated the presence of nervous fibers in the periarticular fibrous tissues , which seemed to run along the blood vessels reaching the fibrous cartilage of tmj and ending to the inside . our study confirmed the results of other preceding reports on the presence of several different types of nrec in periarticular soft tissues of tmj , allowing additionally a precise immunohistochemical identification of ruffini's - like , pacini's- like and golgi's - like receptors in skeletal muscles and tendons , in periartcular dense fibrous connective tissues and in subsynovial tissues . in fact , nrec appeared nse , s-100 and mbp immunoreactive showing gfap and and leu-7 immunoreactivity to lower degrees . free nervous endings , immunohistochemically positive for neurofilaments , nse , and s100 protein , have been detected only in periarticular soft tissues ( higher density in muscles and in the vascular venous plexus in the posterior part of discal ligaments and in the trilaminar zone ) and not in cartilagineous disk . the latter , besides , appeared constituted by s100 immunoreactive chondrocytes both in healthy individuals and in patients with chronic degenerative tmj lesions . in pathological patients with severe disk damage number and morphology of chondrocytes were severely different in comparison to normal tissues 11 ; chondrocytes , in fact , were more numerous with a rough and thick elongated cytoplasmic processes conferring a dendritic - like appearance . in some instances , dendritic processes were extremely long and consequently their cytoplasm of origin could not be detected in a single section but only on consecutive sections of the same specimen . in no instances this cellular component and their prolongments in cartilagineous disks showed neurofilaments , nse or synaptophysin immunoreactivity . morphological changes of chondrocytes in patients with chronic arthritic disease of tmj we observed , conferring them the appearance of neural cells and their axons , have been previously described in literature 10 . this misleading feature is further emphasized by the occurence of s-100 immunoreactivity in dendritic chondrocytes , as well as in normal chondrocytes . nevertheless , s-100 positive cells in the articular cartilage of tmj bear rare , thick and coarse cytoplasmic processes in which no one of the antigens commonly found in peripheral nerve fibres ( e.g. leu-7 , mbp , neurofilaments , nse and synaptophysin ) , except for s-100 , could be detected . in contrast , peripheral nerve fibres usually exhibit long , thin and varicose cytoplamic prolongments in which a variable combination of the above mentioned antigens is usually detectable in conjunction with s-100 reactivity . the results of our study indicate that free nervous endings described by other authors 3 - 8 are definitely proven to be nrec , and it appears likely to stress that , especially in pathological conditions , chondrocytes and their prolongments might morphologically resemble nrec in the articular disk , although their immunophenotype is rather different with the lack of expression of typical neural antigens . although immunochemistry can be easily used to study distribution and location of nrec in articular tissues , we also suggest that ultrastuctural and immuno - ultrastructural studies should be performed in order to definitely assess if chondrocytes is the exclusive cell type of articular cartilage or if also nrec could be present in tmj . areview on the history of mental disorders shows their wide prevalence among human beings always . a glance on statistics reveals that mental disorders and abnormalities are affecting the humans at an increasing pace . the world health organization ( who ) in its report revealed that the incidence of mental disorders is 10% among the young adults . noorbala et al . , in a study on mental health among individuals > 15 years of age ( n = 19,370 ) who were randomly selected , reported that 34.2% of the subjects had different types of mental disorders . on the other hand , university students as a major social group and human resource are the future investment in each society and their mental health is of great importance . the above - mentioned statistics show a high prevalence of the disorders among university students . on the one hand , the university entrance process influences students mental health through educational stressors , immigration to major cities , being far from the family , facing financial problems , etc . on the other hand , if these students face an additional stress due to the stressful nature of their major , their mental health should be considered more . among the stressful educational courses are nursing and midwifery . based on a study on the association between mental health and educational stressors in nursing students of kerman nursing school in 2011 , an increase in the number of students referrals to counseling centers shows their social and cultural growth on one hand , as asking help is believed to be a sign for mental growth , but on the other hand , it reveals the disturbing factors decreasing students academic performance . in a study conducted in private universities of malaysia , therefore , experts of education , psychologists , sociologists , and educational policy makers should seek an appropriate pattern to facilitate students overall growth and their mental health improvement . regard , realizing the need of humans , scientists tried to explore and reconstruct more humanistic methods consistent with human beings psychological characteristics to reach their mental health . therefore , in the recent past , some psychologists have turned to new educational and treatment methods , namely , neurolinguistic programming ( nlp ) . in addition , a wide spectrum of human behaviors are mediated and regulated by human language . the importance of nlp lies in the fact that this programming is a collection of skills based on psychological characteristics of the human beings through which the individuals obtain the ability to use their personal capabilities as much as possible . in an article titled neurolinguistic methodology in controlling examination anxiety reported that anxiety is one of the destructive factors of students mental health in every level and can reduce educational function . they introduced nlp as one of the efficient methods to control pre - exam anxiety . as mentioned earlier , students of medical group , including nursing and midwifery , tolerate high stress due to the stressful nature of their course , not only during their education but also at their working environment , and need decision making in critical situations . with regard to their various employment opportunities and working conditions in hospitals , which can affect their mental health , the researcher conducted a study with an aim to define and compare the efficacy of nlp training on mental health in nursing and midwifery students in islamic azad university , tehran medical branch in 2011 - 2012 . the sample size was calculated as 30 subjects in each group by alpha = 0.05 , z = 1.96 , and delta = 0.80 . due to subjects dropping out of the study in the midwifery group , finally 52 subjects entered the study in both groups . both groups were homogenous concerning age range , sex , education level , and the faculty and the university . group comparison was conducted with regard to adequacy of sample size(based on sample size formula ) . the first section was on personal characteristics including age , sex , course of study , year of entering university , marital status , place of birth , residence , head of household , parents education and family monthly income , history of psychotropic drug , stimulants , and narcotics consumption , and history of a disastrous experience in the past 6 months . the second section consisted of 28-item goldberg general health questionnaire , which had four subscales of physical signs , anxiety , social dysfunction , and depression . the total score of general health was obtained for the subjects by adding up the scores of these four sub - scales . questions 1 - 7 were connected with physical signs , 814 with anxiety , 15 - 21 with social function , and 2128 were connected with depression . each question had four options , with scores ranging from 0 to 3 ( never = 0 , normal = 1 , almost more than normal = 2 , and actually more than normal = 3 ) . the total scores ranged 0 - 84 , of which scores > 23 showed low general health . reported the general validity of this test as 88% and that of its subscales to be between 50% and 81% . they also reported the reliability of its subscales as : physical signs = 0.78 , anxiety = 0.86 , social dysfunction = 0.77 , and depression = 0.88 . then , the educational program in the form of a 1-month program containing five 120-min educational sessions was held . the sessions were in lecture form and as questions and answers accompanied with slide show and short films . an education booklet and a cd were prepared from valid references prior to each educational session and distributed among the attendants . during the sessions , some skills of nlp were taught : ( 1 ) defining a goal , ( 2 ) time management , and ( 3 ) self - assertion . then , 1 month after the last educational session , subjects mental health was measured with the same questionnaire . data were analyzed by descriptive ( absolute and relative distribution table , mean and sd ) and inferential ( paired t - test ) statistical tests . ethical considerations of the present study included obtaining consent from the subjects and giving them explanations about the goal of the study . they were also assured of confidentiality of their data and the mere use of the data for analysis . some limitations , which were out of the researchers control , were personal differences ( different levels of motivation , comprehension , interest of the subject , and iq ) and social and cultural differences , which could have affected the subjects level of learning . subjects honesty in responses and the relevancy of their answers were out of control of the researchers . lack of a control group due to less number of volunteers and educational classes interference with students own classes were the other limitations of this study . about 57.7% of the subjects were nursing students and 42.35% were midwifery students , 80.8% were single and 19.2% were married , 78.8% were born in tehran and 21.2% in other cities , 19.2% consumed psychotropic medications and 13.5% had a history of referring to a psychiatrist , and 30.8% smoked in their leisure time . mean scores of nursing students mental health dimensions were 56.23 ( 14.7 ) before nlp education and 17.36 ( 9.44 ) after nlp education . score of mental health showed a significant reduction of 39 scores after education compared to before education . in other words , nlp education had a significant effect on the reduction of mental health scores ( p < 0.001 , t = 18.42 ) [ table 1 ] . with regard to subscales , the mean score of physical signs dimension was 14.30 ( 4.78 ) before education and 4.76 ( 3.63 ) after education ( p < 0.001 ) . the mean score of anxiety was 15.10 ( 4.22 ) before education and 4.76 ( 3.63 ) after education ( p < 0.001 ) . the mean score of depression was 10.73 ( 5.38 ) before education and 1.01 ( 2.27 ) after education ( p < 0.001 ) . the mean score of social function was 16.10 ( 2.52 ) before and 8.53 ( 3.63 ) after intervention ( p < 0.001 ) . the mean score of depression was 10.37 ( 5.38 ) before and 1.01 ( 2.27 ) after intervention ( p < 0.001 ) . a reduction of mean score observed in all dimensions after education shows the positive effect of nlp education . the mean score of mental health among midwifery students was 53.72 ( 15.97 ) before education and 23.63 ( 12.06 ) after education , which is significantly less than pre - test values , showing that nlp was effective on the subjects mental health ( p < 0.001 ) [ table 2 ] . with regard to mental health dimensions , the mean score of physical signs was 11.36 ( 5.10 ) before education and 5.54 ( 4.30 ) after education ( p < 0.001 ) . the mean score of anxiety was 14.54 ( 6.32 ) before education and 5.54 ( 4.35 ) after education ( p < 0.001 ) . the mean score of social function was 16.36 ( 3.23 ) before education and 10.01 ( 3.54 ) after education ( p < 0.001 ) . the mean score of depression was 11.45 ( 5.56 ) before education and 2.54 ( 3.71 ) after education ( p < 0.001 ) . determination and comparison of nursing students health status before and after education determination and comparison of midwifery students health status before and after education in the nursing group , the mean score of mental health was 56.23 ( 14.7 ) before education and 17.36 ( 9.44 ) after education , while in the midwifery group , it was 53.72 ( 15.97 ) before and 23.63 ( 12.06 ) after education . there was no significant difference in the mean scores of general health before education concerning the course of study ( p = 0.561 ) , but the difference was significant after education in nursing and midwifery subjects such that nursing students mental health was more influenced by nlp education compared to that of midwifery students ( p = 0.041 ) . in the nursing group , the mean score of physical signs was 14.30 ( 4.78 ) before education and 4.76 ( 3.63 ) after education and in the midwifery group , it was 11.36 ( 5.10 ) before education and 5.54 ( 4.30 ) after education , with no significant difference in the mean scores of physical signs concerning the course of study before and after nlp education ( p = 0.584 ) . in the nursing group , the mean score of anxiety was 15.10 ( 4.22 ) before education and 3.06 ( 3.01 ) after education and in the midwifery group , it was 14.54 ( 6.32 ) before and 5.54 ( 4.35 ) after education , showing a significant difference concerning the course of study ( p = 0.019 ) such that anxiety in the nursing group was significantly higher than in the midwifery group before education . after nlp education , the anxiety in nursing group was less than in midwifery group , revealing the effect of nlp education on the nursing group . in the nursing group , the mean score of social function was 16.10 ( 2.52 ) before education and 8.53 ( 3.63 ) after education and in the midwifery group , it was 16.36 ( 3.23 ) before education and 10.01 ( 3.54 ) after education , revealing no significant difference in the mean scores of social function with regard to the course of study before and after education ( p = 0.153 ) . in the nursing group , the mean score of depression was 10.73 ( 5.38 ) before education and 1.01 ( 2.27 ) after education and in the midwifery group , it was 11.45 ( 5.56 ) before education and 2.54 ( 3.17 ) after education , showing no significant difference ( p = 0.153 ) . the mean scores of mental health pre - test were significantly different in the groups with respect to the variables of place of birth , interest in the selected course , interest in the present course , mental disease , consumption of psychotropic medications , referring to a psychiatrist , existence of a disastrous event 6 months prior to study , and smoking and alcohol consumption ( p = 0.05 ) . mental health was lower among the students of tehran compared to that in students of other cities . it was also lower among the students who were not interested in their course of study compared to those who were . the students with a history of mental disease and referral to a psychiatrist had lower mental health compared to those with no such history . mental health among the students experiencing a disastrous event in the past 6 months , or consuming psychotropic medication , cigarettes , or alcohol was lower than that of others . mean scores of students mental health showed a significant difference with regard to smoking a water pipe ( p = 0.005 ) . mental health was lower among the students who always smoked cigarette and a water pipe compared to those who did not . comparison of pre - test post - test mean scores of students mental health showed no significant difference in the variables associated with family ( p > 0.05 ) . in the nursing group , the mean score of mental health was 56.23 ( 14.7 ) before education and 17.36 ( 9.44 ) after education , while in the midwifery group , it was 53.72 ( 15.97 ) before and 23.63 ( 12.06 ) after education . there was no significant difference in the mean scores of general health before education concerning the course of study ( p = 0.561 ) , but the difference was significant after education in nursing and midwifery subjects such that nursing students mental health was more influenced by nlp education compared to that of midwifery students ( p = 0.041 ) . in the nursing group , the mean score of physical signs was 14.30 ( 4.78 ) before education and 4.76 ( 3.63 ) after education and in the midwifery group , it was 11.36 ( 5.10 ) before education and 5.54 ( 4.30 ) after education , with no significant difference in the mean scores of physical signs concerning the course of study before and after nlp education ( p = 0.584 ) . in the nursing group , the mean score of anxiety was 15.10 ( 4.22 ) before education and 3.06 ( 3.01 ) after education and in the midwifery group , it was 14.54 ( 6.32 ) before and 5.54 ( 4.35 ) after education , showing a significant difference concerning the course of study ( p = 0.019 ) such that anxiety in the nursing group was significantly higher than in the midwifery group before education . after nlp education , the anxiety in nursing group was less than in midwifery group , revealing the effect of nlp education on the nursing group . in the nursing group , the mean score of social function was 16.10 ( 2.52 ) before education and 8.53 ( 3.63 ) after education and in the midwifery group , it was 16.36 ( 3.23 ) before education and 10.01 ( 3.54 ) after education , revealing no significant difference in the mean scores of social function with regard to the course of study before and after education ( p = 0.153 ) . in the nursing group , the mean score of depression was 10.73 ( 5.38 ) before education and 1.01 ( 2.27 ) after education and in the midwifery group , it was 11.45 ( 5.56 ) before education and 2.54 ( 3.17 ) after education , showing no significant difference ( p = 0.153 ) . the mean scores of mental health pre - test were significantly different in the groups with respect to the variables of place of birth , interest in the selected course , interest in the present course , mental disease , consumption of psychotropic medications , referring to a psychiatrist , existence of a disastrous event 6 months prior to study , and smoking and alcohol consumption ( p = 0.05 ) . mental health was lower among the students of tehran compared to that in students of other cities . it was also lower among the students who were not interested in their course of study compared to those who were . the students with a history of mental disease and referral to a psychiatrist had lower mental health compared to those with no such history . mental health among the students experiencing a disastrous event in the past 6 months , or consuming psychotropic medication , cigarettes , or alcohol was lower than that of others . mean scores of students mental health showed a significant difference with regard to smoking a water pipe ( p = 0.005 ) . mental health was lower among the students who always smoked cigarette and a water pipe compared to those who did not . comparison of pre - test post - test mean scores of students mental health showed no significant difference in the variables associated with family ( p > 0.05 ) . the results obtained showed that nlp education was effective on the mental health of nursing and midwifery students ( p < 0.001 ) . these results are consistent with the results of sahebalzamani et al . , who conducted a study on the effect of life skills training on general health in students and showed that life skills training is effective on nursing and midwifery students mental health ( p < 0.01 , t = 11.2 ) . our results are consistent with the result of zamini et al . who studied the effect of nlp education on self - efficiency and problem solving among students . our results showed that nlp education is notably effective on students mental health , indicating that students general health can be promoted through nlp education . the results showed that the mean scores of mental health were 56.23 ( 14.7 ) and 53.72 ( 15.97 ) in nursing and midwifery groups , respectively , before education , showing no significant difference with regard to the course of study ( p = 0.561 ) . but they were 17.36 ( 9.44 ) and 23.63 ( 12.06 ) in nursing and midwifery groups , respectively , after education , showing a significant difference with regard to the course of study ( p = 0.041 ) . it can be concluded that mental health was higher in the nursing group after they were given nlp education , compared to the midwifery group . in other words , nlp education had a notable effect on nursing students mental health promotion . results of this study concerning mental health dimensions showed that anxiety was more influenced by education in nursing students , which needs further studies investigating more dimensions in this regard . the obtained results are consistent with of the result of sahebalzamani et al . reporting the effect of expression skill education on self - esteem and decisiveness of high - school girls ( p = 0.000 ) . with regard to the increasing changes and sophistication of society and development of social communications , preparation of individuals , especially the young generation , for facing difficult situations is an essential issue . since iran has a young population , and unfortunately , corruption and social assaults have increased in recent years , the policy makers of education and training system and the related authorities should essentially have plans to lower the corruption and assault and to increase job opportunities in the society and create jobs for students . our results showed that nlp education can promote individuals mental health level . by providing nlp education skills including setting a goal , time management , and the skill of self - expression in the form of nlp nlp education , as public education , can increase the level of knowledge and have a positive effect on the various dimensions of human life . as students are the future investment of the society , their mental health is of great importance . a family is the first place for children to experience a common life and so ciability . therefore , based on the findings , it is suggested to consider nlp education along with other educational courses , not only to promote individuals general health but also to lower depression , anxiety , social function reduction , and physical problems , which will prevent mental and physical disorders .

aim : a study was performed on the articular disk and periarticular tissues of the temporo - mandibular joint ( tmj ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors ( nrec ) in these sites . methods : the study was carried out on tissue samples obtained from 10 subjects without tmj disease and from 7 patients with severe tmj arthritis and arthrosis . we use antibodies directed against following antigens : gliofibrillary acidic protein ( gfap ) , leu-7 , myelin basic protein ( mbp ) , neurofilaments 68 kd ( nf ) , neuron specific enolase ( nse ) , s-100 protein ( s-100 ) and synaptophysin ( syn ) . results : this study revealed that ruffini's - like , pacini's - like and golgi's - like receptors can be demonstrated in tmj periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk . we observed elongated cytoplamic processes of chondrocytes that demonstrated strong s-100 immunoreactivity but they were unreactive with all other antibodies . these cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease tmj . conclusion : the results of this study confirm that different nrec are detectable in tmj periarticular tissues but they are absent within the articular disk . in the latter site , only condrocytic processes are evident , especially in diseased tmj , and they might have been confused with nervous endings in previous morphological studies . nevertheless the absence of immunoreactivity for nf , nse and syn proves that they are not of neural origin . background : neurolinguistic programming ( nlp ) refers to the science and art of reaching success and perfection . it is a collection of the skills based on human beings psychological characteristics through which the individuals obtain the ability to use their personal capabilities as much as possible . this study aimed to investigate the efficacy of nlp training on mental health in nursing and midwifery students in islamic azad university tehran medical sciences branch.materials and methods : in this quasi - experimental study , the study population comprised all nursing and midwifery students in islamic azad university , tehran medical branch , of whom 52 were selected and assigned to two groups through random sampling . data collection tool was goldberg general health questionnaire ( 28-item version ) . after primary evaluation , nlp training was given in five 120-min sessions and the groups were re - evaluated . the obtained data were analyzed.results:in the nursing group , paired t - test showed a significant difference in the scores of mental health ( with 39 points decrease ) , physical signs ( with 7.96 scores decrease ) , anxiety ( with 10.75 scores decrease ) , social function ( with 7.05 scores decrease ) and depression ( with 9.38 scores decrease ) . in the midwifery group , it showed a significant difference in mental health ( with 22.63 scores decrease ) , physical signs ( with 6.54 scores decrease ) , anxiety ( with nine scores decrease ) , and depression ( with 8.38 scores decrease).conclusions : this study showed that nlp strategies are effective in the improvement of general health and its various dimensions . therefore , it is essential to conduct structured and executive programs concerning nlp among the students .

polycystic ovary syndrome ( pcos ) which was first reported in 1935 is known as one of the most common endocrine hormones disorders in the women of the reproductive age afflicting as many as % 10 of them . the name of this syndrome has been derived from the appearance of the ovaries of the afflicted women ( an ovary of enlarged size and full of cysts ) . these cysts are in the form of follicles filled with liquid sacs full of developed ovum . due to the lack of a set of standard and unified diagnostic criteria , the rate of prevalence of this syndrome reported in the different studies is varied . some studies which used sonography as their diagnostic criteria found that the prevalence of pcos ranged between 21% and 22% respectively . on the contrary , in some other studies using oligomenerrhea and hyperandrogenemia manifestations as the pcos 's diagnostic criteria , this syndrome accounts for 30 - 40% of the common causes of infertility resulting from ovary dysfunctions . in addition , pcos is considered as the most leading cause of ovary disorders . as recent studies indicate , a person may have pcos without showing one or some of the above - mentioned symptoms , some other symptoms and signs which can be enumerated for pcos are as follows : increased androgenic hormones , metabolic syndrome ( insulin resistance ) , fat - related disorders , type 2 diabetes , cardiovascular disease , endometrial cancer and blood pressure . the patients with pcos also suffer from anxiety and depression some symptoms which are believed to be mostly a result of disorders occurred in the reproduction cycle which is naturally completed by releasing an ovum every month . the exact cause of pcos is not yet completely known , however , some factors such as insulin resistance / hyperinsulinaemeia , obesity , heredity and genetics factors , environmental factors such as exposure to high levels of masculinizing hormones , embryonic life cycle - related factors , hyperandrogenemia , gonadotropins secreting and functioning , hyperprolactinemia , hypothyroidism or thyroid - related dysfunctions , diet all may play a part in developing pcos . according to previous studies , the eating habits are not different between patients with pcos and healthy persons but some foods seem to affect hormone parameters . in the year 2008 , one case - control study was carried out in al - zahra hospital of the city of tabriz , iran in which 60 women were studied , the results showed that the intake amounts of ca , mg , d vitamin , dairy , fruits and nuts and seeds were remarkably low among the women with pcos . the frequency of their milk and dairy intakes products was also lower compared with the healthy women ( p < 0.05 ) . another study by billaudel et al . revealed that d vitamin and calcium play a critical role in insulin secretion and insulin - resistance in human and animal models . another research on 103 women with pcos and 103 healthy women provided some evidence on the decreased amount of adiponectine , calcium , d vitamin in the patients suffering from pcos with having a higher thyroglobulin . there was also a significant correlation between adioponectrine and calcium concentrations ( oh - d 25 ) . in one prospective study on 3157 young women aged 18 - 30 , pereira et al . have reported in 2002 that the amount of dairy intake and insulin - resistance were indirectly related . a study by pereira et al . found an inverse relationship between consuming more than 4 units of dairy products per day and metabolic syndrome in the females . as indicated in previous research , diet and dairy intake which itself is a source of calcium not only can trigger pcos condition , but also may directly affect obesity and insulin - resistance , two factors which themselves act as the causes of pcos . numerous studies have demonstrated the association between the diet and its components and risk factors developing various diseases . however , previous studies did not address the relationship between nutrition choices and type of diet chosen by the patients . the adverse health effects of pcos such as infertility , diabetes , cardiovascular diseases and the growth in its prevalence , can impose huge financial and health costs on the society and can affect affecting many families . therefore , however , the number of studies which have addressed the relationship between dairy intake and pcos is limited . hence , the present study intends to explore the relationship between the amount of dairy intake and pcos . as a descriptive cross - sectional study , the present research tried to find the relationship between the amount of dairy consumption and pcos condition in 400 women who referred to shahid beheshti hospital clinic in 2013 . as far as exclusion and inclusion criteria of the study is concerned , it is noteworthy that the criteria for the inclusion in the study were willingness to participation , lack of precocious puberty , not having uterus cancer and not being pregnant . on the contrary , the subjects were excluded from the study if they were unwilling to participation , did not respond to more than 30 food items included in the food frequency questionnaire ( ffq ) , diagnosed with typical ( chronic ) diseases having to follow a specific diet . at the start of the study , the data related to the food intakes were checked by the researcher using ffq , ( in the ffqs the person were asked how often they consumed certain foods from a given l of foods ) the validity and reliability of which were previously confirmed . the data on the history of developing ovary dysfunctions ( any endocrine disorders related to reproductive system ) , genetics background ( having first related family members with ovarian disease ) , the age of the first menstruation , the history of thyroid diseases , the patients medication history ( any kind of medicine used or any surgery ) , the period of developing pcos and physical activity were collected by a nutritious expert using a self - developed questionnaire . to do so , the weight ( kg ) of the participants was measured with clothes but without shoes . the height of the participants was measured in a normal condition without wearing shoes using a strip tape . the body mass index ( bmi ) was calculated by dividing the weight ( kg ) value by the square root of height ( m ) value . the pcos condition was identified through sonography diagnostic assessment with checking patients clinical manifestations performed by a trained authority . data analysis was performed by descriptive analysis and logistic regression tests using spss software version 15 . for age , age at menarch and bmi t - test was applied . mann - whitney t - test was applied for kcal while for other variables chi - square test was applied [ table 1 ] . as shown in table 2 for all variables mann - whitney test were used . the study 's confidence coefficient was considered at 95% and the level of strength of the study at 80% . furthermore , the ratio of pcos - afflicted patients and the sampling error were assumed to be 50% and 0.07% , respectively . frequency ( % ) and central tendency ( spread ) of some health condition characteristics of participants frequency ( % ) and central tendency ( spread ) of food intake characteristics of sample population study as a descriptive cross - sectional study , the present research tried to find the relationship between the amount of dairy consumption and pcos condition in 400 women who referred to shahid beheshti hospital clinic in 2013 . as far as exclusion and inclusion criteria of the study is concerned , it is noteworthy that the criteria for the inclusion in the study were willingness to participation , lack of precocious puberty , not having uterus cancer and not being pregnant . on the contrary , the subjects were excluded from the study if they were unwilling to participation , did not respond to more than 30 food items included in the food frequency questionnaire ( ffq ) , diagnosed with typical ( chronic ) diseases having to follow a specific diet . at the start of the study , a written information - consent form was signed by the participants . the data related to the food intakes were checked by the researcher using ffq , ( in the ffqs the person were asked how often they consumed certain foods from a given l of foods ) the validity and reliability of which were previously confirmed . the data on the history of developing ovary dysfunctions ( any endocrine disorders related to reproductive system ) , genetics background ( having first related family members with ovarian disease ) , the age of the first menstruation , the history of thyroid diseases , the patients medication history ( any kind of medicine used or any surgery ) , the period of developing pcos and physical activity were collected by a nutritious expert using a self - developed questionnaire . the weight and height of the participants were also measured . to do so , the weight ( kg ) of the participants was measured with clothes but without shoes . the height of the participants was measured in a normal condition without wearing shoes using a strip tape . the body mass index ( bmi ) was calculated by dividing the weight ( kg ) value by the square root of height ( m ) value . the pcos condition was identified through sonography diagnostic assessment with checking patients clinical manifestations performed by a trained authority . data analysis was performed by descriptive analysis and logistic regression tests using spss software version 15 . for age , age at menarch and bmi t - test was applied . mann - whitney t - test was applied for kcal while for other variables chi - square test was applied [ table 1 ] . as shown in table 2 for all variables mann - whitney test were used . the study 's confidence coefficient was considered at 95% and the level of strength of the study at 80% . furthermore , the ratio of pcos - afflicted patients and the sampling error were assumed to be 50% and 0.07% , respectively . frequency ( % ) and central tendency ( spread ) of some health condition characteristics of participants frequency ( % ) and central tendency ( spread ) of food intake characteristics of sample population study this study was carried out on 400 women of an mean standard deviation ( sd ) age of 29.5 4.8 years old and an mean sd bmi of 24.1 4.8 admitted to the shahid beheshti hospital . the prevalence of pcos in the persons in the study was found to be 9.9% . based on the primary analyses on the data ( descriptive analysis ) , there was relationship between the history of ovarian diseases , genetics and the use of medications and pcos . no relationship was observed between pcos and other variables [ table 1 ] . as for the history of ovarian diseases , there was statistically significant difference between the patients with pcos and healthy people ( p < 0.001 ) . as for the family history , a similar result was obtained so that 22.6% of the patients with pcos were revealed having a family history of pcos . on the contrary , there was no significant relationship between physical activity and pcos ( p = 0.86 ) . a significant relationship was also observed between the use of medications and pcos ( p 0.001 ) . the pcos - afflicted patients and healthy persons did not show any difference in terms of weight and bmi . the average age of the patients with pcos was 26.7 compared to 29.8 as the average age of the healthy persons . the two groups also differed in terms of the amount of calorie intake with 4247 kcal for the subjects with pcos and 6410 kcal for the healthy subjects . the average age of the first menstrual period was found to be 13.35 years among the healthy individuals and 13.02 years among the individuals afflicted with pcos [ table 1 ] . the average sum of dairy intake was 581.5 and 611 g for the healthy persons and patients with pcos , respectively [ table 2 ] . the results from using logistic regression revealed that there was no association between genetics and pcos ( p = 0.13 ) [ table 3 ] . on the contrary ( p < 0.001 odds ratio [ or ] = 0.2 confidence interval [ ci ] ( 95% ) = 0.082 - 0.490 ) , age ( p < 0.001 , or = 0.85 ci ( 95% ) = 0.787 - 0.934 ) and the use of medication as three confounding variables ( p = 0.001 or = 0.17 ci ( 95% ) = 0.064 - 0.477 ) and pcos [ table 3 ] . based on the results , bmi and pcos were not significantly related but their relationship may be remarkable ( p = 0.068 ) . every one - unit increase in the bmi was accompanied by an increase in the risk of affliction with pcos . although like bmi , this relationship was not significant but remarkable ( p = 0.088 ) . after modifying the effects of confounding variables , a direct significant relationship was found between the intake of milk and pcos ( p = 0.028 ) so that every 1-unit increase in the milk intake led to a % 1-increase in the risk factors [ table 3 ] . in the present study we evaluated the effects of dairy product on pcos . according to the results of this study , no significant relationship was observed between each component of dairy foods group and other variables with pcos . however , after modifying the effects of confounding variables , it was found that the milk intake and pcos were directly related . very few studies have been conducted on exploring the direct effect which dairy foods may have on pcos . the results of empirical research on the effect of dairy foods on the weight which itself is believed to be one of the effective factors of infliction with pcos are inconsistent without producing a rigid and strong outcome . the results of available clinical evaluations especially those who address the effects of a diet rich in dairy foods on the weight do not yield a fixed outcome . the results of some previous studies are in line with our results . according to adebamowo et al . , there is a relationship between slim / low - fat or free - fat milk and increased frequency of acne , a component of the pcos . two intervention studies ( thompson et al . and harvey - berino , et al . ) provided no evidence on an additional weight loss when choosing a diet rich in dairy foods compared with a low - dairy diet . the results of these studies were similar to those of one earlier research by zemel et al . ( 2004 ) with a study period of 12 weeks . however , the periods of these two recent studies in which no association was observed between the variables in question was 48 weeks and 52 weeks , respectively . the findings of a 3-year prospective study on a large number of children and young adults revealed that the subjects with a higher intake of milk had a higher bmi . although this reported increase was very trivial per year , adjusting for a number of body weight factors , it was statistically significant . chavarro et al . in their prospective cohort study found that high intake of low - fat dairy products may lead to an increase in the women 's risk of ovulation - related infertility , while incorporating more high - fat dairy foods may decrease its risk . ( 1994 ) reported a positive association between intakes of milk and age - related decreased fertility rates in 31 nations . to put it differently , consuming milk gunther et al . in their study divided 155 women of normal weight into three sub - categories as per their amount of dairy foods intake within 1 year , while their diets during intervention were the same in terms of caloric value . although the results of the study did not support the hypothesis related to the dairy foods intake and weight loss , following 51 of the healthy women participating in the study for a 6-month period after the study revealed that compared to those with a low intake of dairy foods , those who had a diet of high dairy intake had a lower fat accumulation . results of some previous studies are not in line with our findings . in two studies by zemel a considerable decrease was reported for bmi and waist circumference with a notable decrease in the plasma insulin . in one case - control study , dixon , et al . argued that there is an intricate association between obesity and dairy products intake concluding that for the children of higher age , there was an inverse association between the intake of dairy foods and obesity . by analyzing a number of databases , a group of cross - sectional studies on the adults and children almost demonstrated the inverse relationship between the intake of dairy foods and initial obesity . the samples used in these studies were mostly selected from among post - menopausal middle - aged women . carruth and skinner in their research on a group of preschool children concluded that the number of servings estimated per day is obviously inversely related to the fat accumulation and positively related to the individuals slimness . in particular , each serving resulted in an additional weight loss up to 0.9 - 1.1 kg . as for consuming dairy foods and insulin resistance relationship , a cross - sectional study conducted on 496 samples of participants aged 20 - 68 aged in 2009 can be enumerated . this study tried to assess the amounts of consumed dairy foods using a food history questionnaire the validity of which had been checked . based on the results , compared with other groups , the samples situated on the top quarter of the intake of dairy products had the lowest mean scores for insulin resistance markers . although the observed differences were not statistically significant , eating whole - fat dairy products was inversely associated with homa - ir . in contrast , there was no relationship between low - fat dairy products and insulin resistance markers . a case - control study by keshavarzi demonstrated that the risk of infertility among the women who ate 3 or more glasses of milk per day was lower than those with no intake of milk as much as 70% . most of these studies were done on obese subjects , children or on adolescents . on the whole , the findings of this study indicated that there was no statistically significant relationship between consuming milk , yogurt , sherbet / frozen yogurt , cheese and cream and pcos . conversely , it became evident that pcos and the history of ovarian diseases ( p < 0.001 ) , age ( p < 0.001 ) and the use of medication ( p = 0.001 ) as three confounding variables were inversely related . furthermore , although no statistically significant relationship was obtained between bmi and pcos , it was at the threshold of being significant ( p = 0.068 ) . every one - unit increase in bmi is associated with a 10% increase in the risk of pcos . of course , this relationship was no significant . similar to bmi , this relationship was insignificant ( being at the threshold of significance , p = 0.0880 . after modifying the effects of confounding variables , a statistically significant relationship was observed between milk intake and risk of pcos ( p = 0.028 ) hence that every 1-unit increase in the amount of milk consumed caused a 1%-increase in the risk of pcos . there is no study addressing the relationship between the history of ovarian diseases and pcos . as for the relationship between the use of medication and pcos , the research suggest that since the medications used by the majority of the participants were metformin , anti - fertility as well as thyroid medications , they helped prevent developing pcos due to their effect on androgen secretion and luteinizing hormone / follicle - stimulating hormone ratio . based on past research , the higher the age , makes the lower the possibility of pcos . affecting ovulation however , it is noteworthy that yielding inconsistent results , few studies have addressed this issue . if the women with pcos or those who are suspicious of having it make some changes in their diets , for instance choosing low - fat dairy foods instead of high - fat ones , a positive relationship between these changes and anovulatory infertility would be expected . foods factors relevant to the augmentation of the phenotype attributes of pcos may increase infertility risk due to disturbed ovulation . the current study revealed that the average intake of milk and pcos had significant relationship . to explain more , on the average the total intake of low- and free - fat milk was estimated to be 52 61 ( gr ) and 55 132 ( gr ) for the healthy and patient individuals , respectively . the average amount of low- and free - fat milk consumption was found to be 12.4 39.35 ( gr ) for the healthy persons and 76.55 ( gr ) for the pcos patients . furthermore , the individuals with pcos condition , revealed to have a higher consumption of low- and free - fat milk . as per the results of the past studies , there is a positive relationship between low - fat milk and pcos . low - fat dairy products may increase the level of insulin like growth factor i ( igf - i ) . as some researchers have controversies the igf - i existing in the human ovarian cells may stimulate the cell activity - related changes observed in the pcos . it is not yet known whether the change in the level of igf - i caused by diet may contribute to the pcos 's clinical manifestations or not . as the research suggest , the fatty acids of dairy products may have potential positive effects on the ovary function . in more details , the substance soluble in the fat available in the dairy products plays the essential role in creating such effects . compared with low - fat dairy products , whole milk and fat - rich dairy products since estrogen can decrease the level of igf - i , their existence in high - fat dairy foods may explain the observed association . the frequent increased insulin sensitivity in the high - fat dairy consumers may improve their ovulation performance . eating low - fat dairy foods has also been accompanied by an excess of androgens secretion known as one of components of pcos . one of the weak points of this study is its cross - sectional design which fails to identify the cause - effect relationships . in addition , hormone tests such as thyroid , prolactine and homa - ir tests were not taken from the subjects . in contrast , some strength points that can be mentioned for this study include : the large number of the samples , lack of similar study in iran directly addressing the relationship between dairy products intake and pcos , evaluating the foods intakes using a validated ffq , using sonography test known as the most reliable method for pcos diagnose . based on the findings of the study , milk intake and prevalence of pcos may be related in some way . due to the adverse health effects of this condition , its high prevalence in the society as well as the shortage of research on the effectiveness of diets on preventing and treating it , the need to more research is felt . in the future research , it is better to address laboratory practices such as thyroid , prolactine and sexual hormones , as well . milrinone , a phosphodiesterase iii inhibitor currently used to treat pediatric patients with cardiac diseases , increases the level of cyclic adenosine monophosphate in the myocardium and vascular smooth muscle . high levels of cyclic adenosine monophosphate enhance the contractility of the myocardium by increasing calcium influx and relaxing vascular smooth muscles,1 ) therefore increasing cardiac output and decreasing afterload . milrinone also has a lusitropic property , which improves myocardial relaxation.2 ) unlike other inotropes such as dopamine , dobutamine , and epinephrine , milrinone is not associated with an increase in myocardial oxygen consumption.3 ) milrinone is widely used due to its inotropic , vasodilatory , and lusitropic properties . it is frequently prescribed after cardiac surgery due to its efficacy in preventing low cardiac output syndrome.4 ) milrinone is also commonly administered to pediatric patients with myocarditis as a bridge therapy for patients with heart failure who are waiting for cardiac transplantation.5)6 ) milrinone has been shown to improve heart contractility in patients with septic shock.7 ) milrinone is currently one of the most commonly used off - label cardiovascular medications in children.8 ) although a few reports showed the efficacy and safety of short - term milrinone treatment ( for about 35 hours ) in children with heart disease,9)10 ) the safety and efficacy of long - term use of milrinone in children is limited due to a lack of sufficient evidence - based studies in pediatric populations . currently , milrinone is widely used for 3 days , which is usually based on extrapolation from studies in adults or clinical experience . therefore , the objective of this study was to evaluate the safety and efficacy of the current pattern of milrinone administration for 3 days in pediatric patients . a retrospective analysis was conducted using data collected from patients who received milrinone treatment for 3 days at the seoul national university children 's hospital from january 2005 to december 2012 . patients were divided into two groups based on the indication for milrinone treatment : group a consisted of patients who received milrinone to prevent low cardiac output syndrome after cardiac surgeries whereas group b consisted of patients who received milrinone treatment due to acute heart failures from causes other than cardiac surgeries . patients with the following conditions or those who underwent the following treatments were excluded from the study : 1 ) severe left ventricular outflow obstructive diseases such as aortic stenosis ; 2 ) hypertrophic or restrictive cardiomyopathies ; 3 ) milrinone administration immediately after cardiopulmonary resuscitation ; 4 ) multi - organ failures ; 5 ) simultaneous administration of 3 inotropes other than milrinone to increase blood pressure ; 6 ) renal replacement therapies including peritoneal dialysis ; 7 ) myocardial infarctions ; 8) univentricular heart surgeries . however , patients receiving 3 inotropes were included if inotropes were added after an interval of > 2 hours consecutively . patients ' characteristics including age , sex , height , weight , and body surface area were recorded . the following parameters were analyzed to identify the clinical application of milrinone : 1 ) initial infusion rate ; 2 ) maintenance continuous infusion rate ; 3 ) total duration of milrinone therapy ; 4 ) concomitantly infused inotropes . the operation and anesthesiology records of patients who had undergone cardiac surgeries were also studied to identify data related to anatomic diagnosis , type of surgery , time of cardiopulmonary bypass , and time of aortic cross - clamping . systolic blood pressure , heart rate , and echocardiographic data ( the left ventricular internal dimension at end - diastole , ejection fraction , and fractional shortening ) obtained before or immediately after starting milrinone treatment were compared to the values prior to treatment cessation . in cases where appropriate echocardiographic data could not be obtained on the day milrinone treatment was discontinued , echocardiographic data obtained within a week of the treatment endpoint were used . the safety of milrinone was determined based on the occurrence of adverse events such as hypotension , arrhythmia , chest pain , headache , hypokalemia , and thrombocytopenia . hypotension was defined as a sudden decrease in blood pressure within two hours after starting milrinone that necessitated the addition of inotropes or cessation of milrinone . arrhythmia was classified as supraventricular tachycardia , ectopic atrial tachycardia , atrial fibrillation / flutter , accelerated junctional rhythm , junctional ectopic tachycardia , ventricular tachycardia , or ventricular fibrillation . evidence of chest pains or headache during milrinone use were collected from the patient 's chart . potassium levels and platelet counts 24 to 48 hours after starting milrinone infusion were estimated . changes in hemodynamic parameters and echocardiographic data were analyzed using a pairwise comparison test and the wilcoxon signed - rank test . the frequency of hypokalemia and thrombocytopenia after milrinone infusion was analyzed using the mcnemar test . univariate and multivariate logistic regression analysis was used to evaluate the relationships between diverse variables , including the practical use of milrinone and the development of arrhythmia . for statistical analyses , descriptive data are presented as means and standard deviation , whereas categorical variables are presented as proportions . data manipulation and statistical analyses were performed with statistical package for the social sciences ( spss ) 19.0 for windows ( spss inc . , somers , ny , usa ) and excel 2010 ( microsoft ) . the study protocol was approved by the institutional review board of seoul national university hospital and patient consent was waived due to the study 's retrospective design . a retrospective analysis was conducted using data collected from patients who received milrinone treatment for 3 days at the seoul national university children 's hospital from january 2005 to december 2012 . patients were divided into two groups based on the indication for milrinone treatment : group a consisted of patients who received milrinone to prevent low cardiac output syndrome after cardiac surgeries whereas group b consisted of patients who received milrinone treatment due to acute heart failures from causes other than cardiac surgeries . patients with the following conditions or those who underwent the following treatments were excluded from the study : 1 ) severe left ventricular outflow obstructive diseases such as aortic stenosis ; 2 ) hypertrophic or restrictive cardiomyopathies ; 3 ) milrinone administration immediately after cardiopulmonary resuscitation ; 4 ) multi - organ failures ; 5 ) simultaneous administration of 3 inotropes other than milrinone to increase blood pressure ; 6 ) renal replacement therapies including peritoneal dialysis ; 7 ) myocardial infarctions ; 8) univentricular heart surgeries . however , patients receiving 3 inotropes were included if inotropes were added after an interval of > 2 hours consecutively . patients ' characteristics including age , sex , height , weight , and body surface area were recorded . the following parameters were analyzed to identify the clinical application of milrinone : 1 ) initial infusion rate ; 2 ) maintenance continuous infusion rate ; 3 ) total duration of milrinone therapy ; 4 ) concomitantly infused inotropes . the operation and anesthesiology records of patients who had undergone cardiac surgeries were also studied to identify data related to anatomic diagnosis , type of surgery , time of cardiopulmonary bypass , and time of aortic cross - clamping . systolic blood pressure , heart rate , and echocardiographic data ( the left ventricular internal dimension at end - diastole , ejection fraction , and fractional shortening ) obtained before or immediately after starting milrinone treatment were compared to the values prior to treatment cessation . in cases where appropriate echocardiographic data could not be obtained on the day milrinone treatment was discontinued , echocardiographic data obtained within a week of the treatment endpoint were used . the safety of milrinone was determined based on the occurrence of adverse events such as hypotension , arrhythmia , chest pain , headache , hypokalemia , and thrombocytopenia . hypotension was defined as a sudden decrease in blood pressure within two hours after starting milrinone that necessitated the addition of inotropes or cessation of milrinone . arrhythmia was classified as supraventricular tachycardia , ectopic atrial tachycardia , atrial fibrillation / flutter , accelerated junctional rhythm , junctional ectopic tachycardia , ventricular tachycardia , or ventricular fibrillation . evidence of chest pains or headache during milrinone use were collected from the patient 's chart . potassium levels and platelet counts 24 to 48 hours after starting milrinone infusion were estimated . changes in hemodynamic parameters and echocardiographic data were analyzed using a pairwise comparison test and the wilcoxon signed - rank test . the frequency of hypokalemia and thrombocytopenia after milrinone infusion was analyzed using the mcnemar test . univariate and multivariate logistic regression analysis was used to evaluate the relationships between diverse variables , including the practical use of milrinone and the development of arrhythmia . for statistical analyses , descriptive data are presented as means and standard deviation , whereas categorical variables are presented as proportions . data manipulation and statistical analyses were performed with statistical package for the social sciences ( spss ) 19.0 for windows ( spss inc . , the study protocol was approved by the institutional review board of seoul national university hospital and patient consent was waived due to the study 's retrospective design . in total , 2299 eligible admissions were identified during the study period . of these admissions , a total of 730 admissions ( 684 patients ) met the inclusion criteria and were selected for the present study . twenty - seven patients received milrinone twice and one patient received milrinone thrice after serial cardiac surgeries . most patients ( 715 , 97.9% ) received milrinone to prevent low cardiac output after cardiac surgeries . there were 715 admissions in group a and 15 admissions in group b. the male to female ratio was 5.6:1 . the mean age of patients at the time of milrinone infusion was 0.82 years ( table 1 ) . in total , 429 admissions ( 60% ) in group a underwent cardiac surgeries at less than 3 months of age . the most common cardiac defect was ventricular septal defect in group a ( 42.4% ) , followed by tetralogy of fallot ( 11% ) and coarctation of the aorta with ventricular septal defect ( 6.4% ) ( table 2 ) . accordingly , the most common type of surgery was closure of the ventricular septal defect , followed by repair of tetralogy of fallot and coarctoplasty . in group b , the duration of milrinone treatment ranged from 3 to 64.4 days . in 149 ( 20.4% ) two or more inotropes other than milrinone were used in 425 ( 59.4% ) admissions . the most commonly infused inotrope in group a was dopamine ( 83.4% ) , whereas all patients in group b received dobutamine ( table 3 ) . systolic blood pressure , fractional shortening , and ejection fraction after milrinone treatment were significantly ( p<0.05 ) increased in group a when compared to values obtained immediately after cardiac surgeries . however , there was no significant difference in systolic blood pressure , fractional shortening , and ejection fraction before and after milrinone treatment in group b. in both groups , heart rate was significantly ( p<0.05 ) reduced after milrinone use ( table 4 ) . there were no reports of adverse events in the majority of admissions ( 78.7% ) ( table 5 ) . although diverse arrhythmias occurred in 75 ( 10.3% ) admissions , only 3 ( 0.4% ) required reduction or discontinuation of milrinone infusion to alleviate arrhythmia . two of them developed junctional ectopic tachycardia and one of them developed ectopic atrial tachycardia . one - third of patients in group b as well as approximately 10% of patients in group a developed arrhythmia . in total , 41 ( 58.6% ) of the 70 arrhythmias in group a were classified as junctional ectopic tachycardia , the development of arrhythmia was not influenced by the initial infusion rate , maintenance continuous infusion rate , or duration of milrinone treatment . based on univariate logistic regression analysis of group a data , the following parameters were associated with the development of arrhythmia : male gender , age 3 months , and cardiopulmonary bypass time 180 minutes . multivariate analysis of group a data in - dicated that male gender and cardiopulmonary bypass time 180 minutes influenced the development of arrhythmia . however , the incidence rate did not increase significantly ( p=0.390 ) , which was 108/730 ( 14.4% ) at the initial point and 118/730 ( 16.2% ) at 24 to 48 hours after starting milrinone treatment . thrombocytopenia did increase slightly but significantly ( p=0.039 ) , which occurred in 4/729 ( 0.5% ) at the initial point and 12/729 ( 1.6% ) at 24 to 48 hours after starting milrinone treatment . most patients in group a with thrombocytopenia required only a few platelet transfusions after cardiac surgeries . platelet count was increased and maintained 50000/dl spontaneously in 2 admissions , after 1 platelet transfusion in 6 admissions , and after 2 platelet transfusion in 2 admissions . the cardiopulmonary bypass time ( 249.272.8 minutes vs. 153.170.4 minutes ; p=0.004 ) were significantly longer in group a patients with thrombocytopenia than in patients without thrombocytopenia . the initial infusion rate , maintenance continuous infusion rate , and duration of milrinone treatment were not associated with thrombocytopenia . two patients in group b had a low platelet count due to additional medical illnesses such as hemophagocytic lymphohistiocytosis and fungal infection . in total , 2299 eligible admissions were identified during the study period . of these admissions , a total of 730 admissions ( 684 patients ) met the inclusion criteria and were selected for the present study . twenty - seven patients received milrinone twice and one patient received milrinone thrice after serial cardiac surgeries . most patients ( 715 , 97.9% ) received milrinone to prevent low cardiac output after cardiac surgeries . there were 715 admissions in group a and 15 admissions in group b. the male to female ratio was 5.6:1 . the mean age of patients at the time of milrinone infusion was 0.82 years ( table 1 ) . in total , 429 admissions ( 60% ) in group a underwent cardiac surgeries at less than 3 months of age . the most common cardiac defect was ventricular septal defect in group a ( 42.4% ) , followed by tetralogy of fallot ( 11% ) and coarctation of the aorta with ventricular septal defect ( 6.4% ) ( table 2 ) . accordingly , the most common type of surgery was closure of the ventricular septal defect , followed by repair of tetralogy of fallot and coarctoplasty . in group b , the duration of milrinone treatment ranged from 3 to 64.4 days . in 149 ( 20.4% ) two or more inotropes other than milrinone were used in 425 ( 59.4% ) admissions . the most commonly infused inotrope in group a was dopamine ( 83.4% ) , whereas all patients in group b received dobutamine ( table 3 ) . systolic blood pressure , fractional shortening , and ejection fraction after milrinone treatment were significantly ( p<0.05 ) increased in group a when compared to values obtained immediately after cardiac surgeries . however , there was no significant difference in systolic blood pressure , fractional shortening , and ejection fraction before and after milrinone treatment in group b. in both groups , heart rate was significantly ( p<0.05 ) reduced after milrinone use ( table 4 ) . there were no reports of adverse events in the majority of admissions ( 78.7% ) ( table 5 ) . although diverse arrhythmias occurred in 75 ( 10.3% ) admissions , only 3 ( 0.4% ) required reduction or discontinuation of milrinone infusion to alleviate arrhythmia . two of them developed junctional ectopic tachycardia and one of them developed ectopic atrial tachycardia . one - third of patients in group b as well as approximately 10% of patients in group a developed arrhythmia . in total , 41 ( 58.6% ) of the 70 arrhythmias in group a were classified as junctional ectopic tachycardia , whereas the most common arrhythmia identified in group b was ventricular tachycardia . the development of arrhythmia was not influenced by the initial infusion rate , maintenance continuous infusion rate , or duration of milrinone treatment . based on univariate logistic regression analysis of group a data , the following parameters were associated with the development of arrhythmia : male gender , age 3 months , and cardiopulmonary bypass time 180 minutes . multivariate analysis of group a data in - dicated that male gender and cardiopulmonary bypass time 180 minutes influenced the development of arrhythmia . however , the incidence rate did not increase significantly ( p=0.390 ) , which was 108/730 ( 14.4% ) at the initial point and 118/730 ( 16.2% ) at 24 to 48 hours after starting milrinone treatment . thrombocytopenia did increase slightly but significantly ( p=0.039 ) , which occurred in 4/729 ( 0.5% ) at the initial point and 12/729 ( 1.6% ) at 24 to 48 hours after starting milrinone treatment . most patients in group a with thrombocytopenia required only a few platelet transfusions after cardiac surgeries . platelet count was increased and maintained 50000/dl spontaneously in 2 admissions , after 1 platelet transfusion in 6 admissions , and after 2 platelet transfusion in 2 admissions . the cardiopulmonary bypass time ( 249.272.8 minutes vs. 153.170.4 minutes ; p=0.004 ) were significantly longer in group a patients with thrombocytopenia than in patients without thrombocytopenia . the initial infusion rate , maintenance continuous infusion rate , and duration of milrinone treatment were not associated with thrombocytopenia . two patients in group b had a low platelet count due to additional medical illnesses such as hemophagocytic lymphohistiocytosis and fungal infection . in the present study , we reviewed the current patterns of milrinone use that continued for 3 days . of a total of 2299 eligible admissions in which milrinone was used , 730 ( 31.8% ) admissions involved the use of milrinone for 3 days . although the korean national insurance covers only 35 hours of milrinone use , we found that milrinone was frequently prescribed for a longer duration in actual settings , suggesting a significant off - label use . the frequent use of milrinone in pediatric patients undergoing heart surgery was supported by the findings of the prophylactic intravenous use of milrinone after cardiac operation in pediatrics ( primacorp ) trial , which demonstrated that low cardiac output syndromes could be prevented by high - dose milrinone infusion after pediatric cardiac surgeries.10 ) although the initial and maintenance milrinone infusion rates determined in the present study were lower than those previously reported in the primacorp study , our values were similar to those reported in other european practices.4)10 ) in terms of efficacy issue , fractional shortening and ejection fraction were both improved after milrinone infusion in group a patients , which is in consistent with previous findings.9)11 ) chang et al.9 ) reported that milrinone treatment in neonates with low cardiac output after congenital heart surgery improved the cardiac index without changing myocardial oxygen consumption . duggal et al.11 ) documented that milrinone therapy improved the left and right myocardial performance index , a reliable index that reflects myocardial contractility . our findings revealed no significant changes in fractional shortening and ejection fraction after milrinone infusion in group b , which is in contrary to previously reported data in adults.12 ) the severity of the disease in group b is likely to have some influence on the results . seven ( 46.7% ) of the 15 admissions in group b involved patients who died due to underlying cardiac disease itself , reflecting the severity of disease in group b. in terms of safety , several adult studies have shown detrimental effects of milrinone on hemodynamic stability in chronic heart failures . the prospective randomized milrinone survival evaluation13 ) trial revealed that oral administration of milrinone to patients with heart failure increased mortality and induced serious adverse cardiovascular events including hypotension . the outcomes of a prospective trial of intravenous milrinone for exacerbations of chronic heart failure trial reported that sustained hypotension requiring additional treatment was more common in patients who received milrinone infusion for exacerbation of chronic heart failure.14 ) however , in the present study , only 1 ( 6.7% ) of 15 admissions in group b needed a reduction in milrinone dose due to a decrease in blood pressure , with the incidence of hypotension being lower than that reported previously in adult data . furthermore , only 3 ( 0.4% ) of the 715 admissions in group a experienced hypotension that required a reduction in milrinone infusion rate . this result was in consistent with previous pediatric data that reported an infrequent occurrence of hypotension in the milrinone treatment group after cardiac surgery.10 ) milrinone use has been known to be associated with postoperative arrhythmia in adults that increased mortality and morbidity after cardiac surgery.15 ) arrhythmia is a concern following milrinone use in the pediatric population as well . smith et al.16 ) recently reported that approximately half of the pediatric patients receiving milrinone after cardiac surgery experienced arrhythmia . in the present study , various arrhythmias occurred in 70 ( 9.8% ) of 715 admissions in group a. however , only 3 ( 0.4% ) admissions required modification of milrinone infusion to manage arrhythmias . our finding is in consistent with that from other studies,17)18 ) indicating that the incidence of arrhythmia due to milrinone use in pediatric patients is relatively low after cardiac surgery . postoperative arrhythmia is associated with age , cardiopulmonary time , aortic cross clamping time , and type of repair.19)20 ) in the present study , the initial infusion rate , maintenance infusion rate , and duration of milrinone treatment were not associated with the development of arrhythmia . however , in consistent with the previous findings , a longer cardiopulmonary time affected the development of arrhythmia in group a ( table 6 ) . junctional ectopic tachycardia is the most common type of arrhythmia after cardiac surgery.20 ) in the present study , the incidence rate of junctional ectopic tachycardia in group a was 5.7% . this result is in accordance with previous studies , which reported that the incidence rate of junctional ectopic tachycardia after cardiac surgery range from 1.4% to 14.7%.21)22 ) the relationship between junctional ectopic tachycardia and milrinone use has not been elucidated thus far.23 ) a study in adults showed that atrial arrhythmia developed more frequently in patients with chronic heart failure who received milrinone treatment.14 ) however , the independent cause - effect relationship of milrinone with arrhythmia in pediatric patients with heart failure has not yet been established . five ( 33.3% ) of the 15 admissions in group b involved patients who mainly developed ventricular arrhythmia . in the present study , there was no significant difference in initial infusion rate , maintenance infusion rate , or duration of milrinone treatment between patients with or without arrhythmia in group b. in fact , heart failure due to myocarditis or dilated cardiomyopathy itself increased the vulnerability of the patients to tachyarrhythmia and conduction disturbance.24 ) ichikawa et al.25 ) reported that 3 of 7 patients with fulminant myocarditis developed ventricular tachycardia or ventricular fibrillation . friedman et al.26 ) also documented the high incidence rate of arrhythmia in pediatric idiopathic dilated cardiomyopathy . the incidence rate of thrombocytopenia in the present study was lower than that in previous studies . ten ( 1.4% ) of the 715 admissions in group a involved patients who developed thrombocytopenia after milrinone treatment , which required a few platelet transfusions . ramamoorthy et al.17 ) previously reported that milrinone treatment resulted in a high incidence ( 58% ) of thrombocytopenia ( platelet count < 100000/dl ) that did not often necessitate platelet transfusions . bishara et al.18 ) further documented that 12.7% of admissions developed thrombocytopenia with milrinone treatment . however , it is difficult to identify milrinone - induced thrombocytopenia , because various factors could confound the observations , including the cardiac surgery itself . cardiopulmonary bypass is known to be associated with thrombocytopenia that results from hemodilution and mechanical disruption.27 ) in the present study , patients who developed thrombocytopenia had a longer cardiopulmonary bypass time and aorta - clamping time . thus , patients with a more complex heart disease and longer bypass times during cardiac surgery would be more likely to develop thrombocytopenia . in contrast to amrinone , the first phosphodiesterase inhibitor used that was known to have adverse effect on platelets , milrinone did not alter platelet count or function in patients after cardiac surgery.28 ) the fact that two ( 13.3% ) patients in group b developed thrombocytopenia could be due to complications such as hemophagocytic lymphohistiocytosis and fungal sepsis , rather than due to milrinone use . the utility of chronic continuous milrinone infusion for heart failure as bridge therapy to heart transplantation was proposed in a recent adult study.6 ) mcmahon et al.29 ) demonstrated that long - term support with milrinone was safe and effective in infants with cardiomyopathy awaiting heart transplantation . outpatient continuous parenteral inotropic therapy , including milrinone , has also been used in inotropic - dependent children with advanced heart failure.30 ) although these studies used small patient populations , the incidence of complications was sufficiently low to indicate the safe and effective potential of milrinone use in infants and children with heart diseases . this study had a retrospective design which did not have a control group containing patients who did not receive milrinone . moreover , all arrhythmias could not be included due to the study 's retrospective nature . furthermore , because the exclusion criteria of this study were relatively strict to appropriately clarify the efficacy and safety of milrinone use and reduce confounding factors , the patients who would be more susceptible to milrinone use could not be included . in surgically treated patients with congenital heart disease , we should have also considered the natural improvement of cardiac function after the corrective operation , which could be a confounding factor for evaluating the efficacy of milrinone use in this study . finally , only a few echocardiographic data including ejection fraction and fractional shortening were used to evaluate milrinone efficacy due to the retrospective study design . if other echocardiographic data , such as tissue doppler imaging findings , were available , a more accurate evaluation of the hemodynamic effects of milrinone would have been possible . this is the largest retrospective study , to date , to evaluate the safety and efficacy of long - term milrinone use ( 3 days ) in pediatric patients with cardiac diseases . such long - term milrinone use is common in actual clinical practice , and milrinone is generally used for > 35 hours in infants and children with heart disease . our data revealed that the use of milrinone for 3 days was effective in preventing low cardiac output after cardiac surgery when combined with other inotropes . this study had a retrospective design which did not have a control group containing patients who did not receive milrinone . moreover , all arrhythmias could not be included due to the study 's retrospective nature . furthermore , because the exclusion criteria of this study were relatively strict to appropriately clarify the efficacy and safety of milrinone use and reduce confounding factors , the patients who would be more susceptible to milrinone use could not be included . in surgically treated patients with congenital heart disease , we should have also considered the natural improvement of cardiac function after the corrective operation , which could be a confounding factor for evaluating the efficacy of milrinone use in this study . finally , only a few echocardiographic data including ejection fraction and fractional shortening were used to evaluate milrinone efficacy due to the retrospective study design . if other echocardiographic data , such as tissue doppler imaging findings , were available , a more accurate evaluation of the hemodynamic effects of milrinone would have been possible . this is the largest retrospective study , to date , to evaluate the safety and efficacy of long - term milrinone use ( 3 days ) in pediatric patients with cardiac diseases . such long - term milrinone use is common in actual clinical practice , and milrinone is generally used for > 35 hours in infants and children with heart disease . our data revealed that the use of milrinone for 3 days was effective in preventing low cardiac output after cardiac surgery when combined with other inotropes .

background : polycystic ovary syndrome ( pcos ) is the most common endocrine disorder in reproductive women . nearly 10% of young women in this period involved . although factors such as insulin resistance , hyper insulinemia , obesity and dietary are suggested to be associated with pcos , cause of pcos is not completely understood . dairy products ( a key component of the usual diet ) of participants can also affect the factors of this disease and may have beneficial effects on treatment of pcos . however , research in this area is scarce . the purpose of this study was to evaluate the relationship between dairy products consumption and pcos.methods:this descriptive cross - sectional study of 400 women was conducted in shahid beheshti hospital of isfahan university of medical science , iran . dietary intake was evaluated by validated food frequency questionnaire . other variables such as ovarian disease , inherited predisposition , age at menarche , physical activity and history of other diseases were evaluated using questionnaire . data analysis was performed by a logistic regression test using spss software version 15 predictive analytics software and solutions.results:there were a significant association between pcos and ovarian disease ( p < 0.001 ) , age ( p < 0.001 ) and using medication ( p = 0.001 ) . body mass index ( bmi ) was inversely associated with pcos , but it was not significant ( p = 0.068 ) . there was a significant direct relationship between milk consumption and risk of pcos after adjusting for confounding factors ( p = 0.028).conclusions : the findings of this study indicated that ovarian disease and medication use is directly linked to pcos . dairy consumption was not significantly correlated with pcos . however , after adjustment for confounders , there was an direct relationship between milk consumption and risk of pcos . background and objectivesmilrinone is often used in children to treat acute heart failure and prevent low cardiac output syndrome after cardiac surgery . due to the lack of studies on the long - term milrinone use in children , the objective of this study was to assess the safety and efficacy of the current patterns of milrinone use for 3 days in infants and children with heart diseases.subjects and methodswe retrospectively reviewed the medical records of patients aged < 13 years who received milrinone for 3 days from january 2005 to december 2012 . patients ' characteristics including age , sex , height , weight , and body surface area were recorded . the following parameters were analyzed to identify the clinical application of milrinone : initial infusion rate , maintenance continuous infusion rate , total duration of milrinone therapy , and concomitantly infused inotropes . the safety of milrinone was determined based on the occurrence of adverse events such as hypotension , arrhythmia , chest pain , headache , hypokalemia , and thrombocytopenia.resultswe assessed 730 admissions ( 684 patients ) during this period . ventricular septal defects were the most common diagnosis ( 42.4% ) in these patients . milrinone was primarily used after cardiac surgery in 715 admissions ( 97.9% ) . the duration of milrinone treatment varied from 3 to 64.4 days ( 7 days in 149 admissions ) . ejection fraction and fractional shortening of the left ventricle improved in patients receiving milrinone after cardiac surgery . dose reduction of milrinone due to hypotension occurred in only 4 admissions ( 0.5% ) . although diverse arrhythmias occurred in 75 admissions ( 10.3% ) , modification of milrinone infusion to manage arrhythmia occurred in only 3 admissions ( 0.4% ) . multivariate analysis indicated that the development of arrhythmia was not influenced by the pattern of milrinone use.conclusionmilrinone was generally administered for 3 days in children with heart diseases . the use of milrinone for 3 days was effective in preventing low cardiac output after cardiac surgery when combined with other inotropes , suggesting that milrinone could be safely employed in pediatric patients with heart diseases .

brown - squard syndrome ( bss ) , which occurs due to dysfunction of the spinothalamic tract , typically reflects the hemisection of the spinal cord at the cervical or thoracic level . the syndrome mainly occurs as a result of penetrating trauma , syringomyelia , hematomyelia , tumor , severe discs , or blunt trauma . among the multiple etiologies , the most common cause is penetrating trauma , such as a gunshot7,8 ) . therefore , most management guidelines focus on penetrating cervical injuries and/or vertebral artery ( va ) injury12 ) . non - missile penetrating spinal cord and va injuries are rare because of the bony structures that protect the spinal cord and va14 ) . thus , the treatment approach for wounds caused in non - missile penetrating spinal injuries such as a knife , a power drill bit , or even a pen could be different from common missile penetrating injuries5,6,13,17).to our knowledge , there are few reports in the literature of complete obstruction of the va due to penetration of a foreign body through the neural foramen into the spinal canal . herein , the authors report on va dissection and bss caused by penetration of an electric screw driver bit . a 25-year - old machine operator was involved in a violent episode and was stabbed in his right neck with an electric screw driver bit that was thrown by the opponent . on arrival at the emergency department , the electric screw driver bit was placed in the right lateral aspect of the neck at zone i ( fig . the tip of the electric screw driver bit was located at the center of the vertebral canal of c3 ( fig . he was given high - dose methylprednisolone ( bolus dose of 30mg / kg followed by 5.4mg / kg / hour for 23 hours ) according to the protocol for spinal cord injury . an immediate interventional angiography was undertaken without general anesthesia due to the nature of the emergency . the angiography revealed a total occlusion with dissection of the right va at the level of c3 . immediate coil embolization at both proximal and distal ends of the injury site was performed ( fig . an attempt at manual extraction of the electric screw driver bit failed with great resistance . after the patient was moved to the operating room , the electric screw driver bit was removed manually with muscle dissection under general anesthesia . venous blood spilled out and was controlled easily by application of several pieces of gelatin sponge . no postoperative complications such as wound dehiscence , cerebrospinal fluid ( csf ) leakage , or infection were observed . the neurological motor function of the right upper and lower extremities recovered to 3/5 and 4/5 , respectively , with persistent decreased sensory function after one year . fortunately , the patient experienced no neck swelling , auscultation of a neck bruit , or delayed ischemic complications . penetrating injury is the third most frequent cause of spinal cord injury in adults , surpassed only by traffic accidents and falls3,18 ) . stab wounds are associated with lesser surrounding tissue injury than gunshot wounds because the former delivers less energy than missile injuries9 ) . although vascular injury is the most common sequel of penetrating neck trauma , va injury is rare because it is well protected by the transverse foramen4,10 ) . therefore , penetrating injury of the va is mostly caused by gunshot wounds which deliver large kinetic energy , depending upon the bullet 's mass and speed12 ) . in this article , we report a rare case of va penetration by an electric screw driver bit with spinal cord insult , consequently presenting as bss . moreover , surgical exploration of the va can cause additional damage to the spine and surrounding tissues . therefore , it may be reasonable to embolize an occluded artery , because the unilateral ligation of the va rarely results in brainstem ischemia11,16 ) . there are a few reports regarding the treatment of traumatic va injury such as the arteriovenous fistulas and pseudoaneurysms2 ) . emergent surgical exploration is necessary for patients with hard signs of vascular injury , such as hemodynamic instability , hemorrhage exsanguinations , or expanding hematoma15 ) . patients that are hemodynamically stable and who are without respiratory compromise should undergo further diagnostic imaging evaluation15 ) . as presented in this case , endovascular techniques were a safe and effective method of treatment and were not associated with significant morbidity or mortality1 ) . airway management , intubation methods , and surgical positions can be points of debate between anesthesiologists and surgeons9 ) . if a lacerated va can be successfully obliterated , a penetrating electric screw driver bit may be extracted without general anesthesia . nevertheless , the authors recommend that surgeons should be prepared for conversion to open surgery and extraction should be performed with the support of a surgical team . we initially tried to extract the electric screw driver bit manually without general anesthesia in the intervention theater after va embolization . however , the electric screw driver bit was positioned firmly in the neural foramen , and the patient complained of severe pain when the electric screw driver bit was being pulled out . in addition , there was more important rationale that justified surgical exploration for extraction of the electric screw driver bit . on extraction of the electric screw driver bit , the authors describe a rare case of penetrating cervical injury caused by an electric screw driver bit with accompanying va penetration and bss . vogt - koyanagi - harada disease ( vkh ) is a granulomatous inflammatory disorder affecting the eyes , auditory system , meninges , and skin [ 1 , 2 ] . it is one of the most common causes of uveitis in asia and accounts for approximately 16% of all uveitis cases in china . clinically , vkh is divided into four stages : prodromal , acute , convalescent , and chronic recurrent . exudative retinal detachment is a common cause of visual function impairment among vkh patients , but secondary alterations in retinal morphology and function also occur [ 5 , 6 ] . due to advancements in retinal imaging , such as fundus autofluorescence ( faf ) [ 7 , 8 ] and optical coherence tomography ( oct ) [ 9 , 10 ] , important clinical insight into the pathophysiology of the retina can be achieved noninvasively . recent studies report a significant association of the inner segment ( is)/os junction and the cone outer segment tips ( cost ) line in various retinal diseases with visual function [ 1113 ] . however , the majority of previous studies have used best corrected visual acuity ( bcva ) as a measure of visual function . recently , microperimetry was found to provide a more comprehensive assessment of macular sensitivity and functional changes in the macula [ 14 , 15 ] . we investigated the characteristics of late - stage vkh via oct , especially photoreceptor microstructure integrity and its potential correlation to visual function . patients with late - stage vkh ( defined as 12 months from disease onset ) who visited the uveitis clinic at fudan university eye and ent hospital ( shanghai , china ) between april 2011 and march 2013 were enrolled in this cross - sectional study . the diagnosis of vkh was made according to the revised diagnostic criteria proposed by the international nomenclature committee . the inclusion criteria were treatment with high - dose corticosteroid therapy ( iv methylprednisolone at 1 g / d for 3 d ) followed by a slow tapering of the drug dose over a 6-month period , undergoing a thorough examination , including oct and microperimetry , and providing the required information at the 12-month follow - up , including sex , age at vkh onset , bcva at initial presentation , current bcva , interval from the onset of vkh symptoms to the initiation of high - dose corticosteroid treatment , and current ocular inflammatory status . exclusion criteria were inadequate mydriasis ( < 7 mm ) , media opacities ( caused by cataract or other ) , and glaucoma ( defined as characteristic optic nerve damage or typical visual field defects accompanied by compatible optic nerve damage ) . this study was conducted in accordance with the tenets of the declaration of helsinki and was approved by the local ethics committee of the fudan university eye and ent hospital . 1.5.12.0 , heidelberg engineering , heidelberg , germany ) using the volume scan mode ( 1024 25 ; 25 lines , 1024 a - scans per line , each line comprising 30 averaged scans obtained using eye tracking ) , which covered a 30 30 degree area centered on the fovea . microperimetry covered the central 12 of the macula ; therefore , only nine scans at the central macula ( firstly the scan across the central fovea was found and then subsequent four scans in both the superior and inferior directions were picked ) , which covers about 12 of the central macula , were selected for further analysis . changes in the is / os junction , the cost line , and the rpe layer were graded by an experienced ophthalmologist ( chunhui jiang ) , who was masked for the clinical and other findings of the patients . grading was performed using a scale from 1 to 3 based on a standardized protocol ( table 1 ) . also if one part of the outer nuclear layer ( onl ) was thinner than the other , or all onl was obviously much thinner than normal , then it was considered as thinning of onl . based on a 42 staircase strategy on a set of 45 goldmann iii stimulating spots covering the central 12 of the fundus , all tests were performed using an automated pattern in which a 1 circle was the target . the intensity of each stimulus was varied according to a 1-step ( 0.1log ) scale from 020 decibels ( db ) for an equivalent duration of 200 ms per step . the software automatically calculated the mean sensitivity , which was defined as the average of all 45 scores . all analyses were performed using a statistical software package ( spss for windows , ver . differences in mp and bcva between groups of late - stage vkh eyes with different oct grading were examined using the mixed linear model test . differences in each prognostic factor ( sex , age of onset , initial bcva , and interval from onset to treatment ) were analyzed using generalized estimation equations test . the durations between patients who were still using systemic corticosteroids alone or in combination with an immunosuppressant or not at 12 months were analyzed using mann - whitney u test . during the study period , 34 vkh patients with a follow - up of longer than 12 months visited our clinic . twenty - nine patients ( 15 males and 14 females , 58 eyes ) who met the inclusion criteria were enrolled in the study . the mean follow - up time was 12.2 months ( range 1214 months ) . the mean age at onset was 34.24 10.67 years ( range 1265 years ) ; mean bcva at onset was 0.96 0.56 ( logmar , range 2.000 ) ; mean bcva at 12 months was 0.07 0.16 ( logmar , range 0.40 to 0.08 logmar ) ; and mean retinal sensitivity was 17.05 2.35 db ( range 9.920 db ) . at 12 months , 16 of the 29 patients were still using systemic corticosteroids alone or in combination with an immunosuppressant . among them , 10 patients were tapering down and using low - dose prednisone . another six patients were receiving corticosteroids and cyclosporine prescribed at 3 mg / kg / d and were tapering , guided by the severity of ocular inflammation . the oct findings in the late - stage vkh eyes were localized to the outer retina . the findings included thickening of the rpe layer ( 28/58 eyes ) ; breakage or disappearance of the cost line ( 51/58 eyes ) and/or the inner segment / outer segment ( is / os ) junction ( 28/58 eyes ) ; and thinning of the outer nuclear layer ( onl ) ( 17/58 eyes ) . the grading of the rpe , cost line , and is / os conjunction is listed in table 2 . notably , all eyes displaying intact cost lines also displayed intact is / os junctions and normal rpe layers . however , 23 of the 30 eyes displaying intact is / os junctions displayed fragmented ( grade 1 , 3 eyes ; grade 2 , 20 eyes ) cost lines . in 18 of the 51 eyes displaying fragmented cost lines ( grade 1 or 2 ) and in 16 of the 28 eyes displaying fragmented is / os junctions ( grade 1 or 2 ) , the central fovea remained intact ( figures 1(a)1(c ) ) . our analysis revealed a strong correlation between retinal sensitivity / bcva and the oct findings of the cost line , is / os junction , and rpe layer ( all p 0.01 , table 2 ) ( figure 2 ) . for clarification , the patients were categorized according to the cost line and is / os junction characteristics , which were graded as intact ( + ) or not ( ) ( table 3 ) . the three groups had similar gender / age distributions ( table 3 ) but different bcva and retinal sensitivities . the is / os+/cost+ group displayed the best retinal sensitivity and bcva ; furthermore , only this group displayed relatively normal retinal sensitivity ( table 3 ) . the oct findings for the cost line and is / os junction displayed clear correlations to the interval from symptom onset to the initiation of high - dose corticosteroid treatment and usage of systemic corticosteroids and/or immunosuppressant at 12 months after onset ( table 3 ) . the is / os+/cost+ group exhibited the smallest interval from symptom onset to treatment and no usage of systemic corticosteroids and/or immunosuppressant . in contrast , 11/23 and 21/28 patients in the is / os+/cost and is / os/cost groups were using systemic corticosteroids alone or in combination with an immunosuppressant . patients who were using systemic corticosteroids alone or in combination with an immunosuppressant at 12 months had longer durations than those without ( using versus not using , median 30 versus 9 , p < 0.0001 by the mann - whitney u test ) . changes of the outer retina and rpe in late - stage vkh patients , including thickening of the rpe layer , disruption or loss of the cost line and is / os junction , and thinning of the onl , were clearly demonstrated by sd - oct . further analysis revealed a close correlation between these oct findings , which also correlated to the bcva , retinal sensitivity , and clinical characteristics . better oct findings correlated to better vision , prompt treatment at disease onset , and less incidence of systemic corticosteroids and/or immunosuppressant at 12 months after onset . clinical findings of late vkh patients vary greatly . da silva et al . recently reported that , in late - stage vkh eyes ( > 6 months past disease onset ) , grading by color fundus photos ( which was mainly based on diffuse fundus depigmentation or subretinal fibrosis ) correlated well with full - field electroretinogram ( erg ) results . however , not all vkh patients had diffuse fundus depigmentation or subretinal fibrosis ; therefore , an objective method with higher resolution might be useful in the study of vkh patients with less fundus change . oct is routinely used in the diagnosis and follow - up of many retinal diseases [ 913 ] . reported defects in the is / os junction and multifocal thickening of the rpe in late - stage vkh patients . however , the use of oct to identify retinal morphological changes in late - stage vkh patients has not been extensively studied [ 5 , 7 , 8 , 18 ] . this study comprised a relatively large series of late - stage vkh patients , in whom oct clearly demonstrated retinal changes and rpe . in addition to defects in the is / os junction and thickening of the rpe , thinning of onl and a high prevalence ( 51/58 ) of defects in the cost line were found , thereby indicating that oct is useful in the observation of late - stage vkh eyes . all eyes displaying intact cost lines displayed normal is / os junctions or rpe layers , whereas those displaying intact is / os junctions or normal rpe layers displayed disrupted cost lines . it has been reported in central serous chorioretinopathy or macular hole patients that the cost line is later to recover than are the is / os line and other characteristics . notably , patients displaying cost line and is / os junction defects or loss at the perifovea exhibited intact structures at the central fovea . the mechanism underlying this finding is not fully understood , but the high density of photoreceptors at the fovea may play an important role . alternatively , the cones at the fovea display a different configuration and arrangement than cones at other regions of the fundus , which might also explain these findings . whether these structures were preserved or were first to regenerate , studying the mechanism behind this characteristic might improve our understanding of vkh and of the neuroprotection or neuroregeneration of the retina . our study also demonstrated a strong correlation between the cost line and/or is / os junction integrity and visual function in late - stage vkh patients . a former paper reported a similar correlation in patients with macular holes [ 12 , 20 ] . however , bcva was employed as the measure of visual function in previous studies . this time , microperimetry , which provides a quantitative measurement of retinal sensitivity at the macula , , we found that only the is / os+/cost+ eyes exhibited normal results ( is / os+/cost+ group : 19.56 0.36 db ; normal group : 19.1 0.9 db ) . these results suggest that the presence or reappearance of an intact cost line might serve as a more sensitive indicator of photoreceptor functional status than that of the is / os junction . in accordance with yokota 's report of a higher cone density in eyes displaying intact cost lines than in eyes displaying disrupted cost lines , our finding supported fujita and itoh 's theory that the recovery of the cost line , but not the is / os junction , suggests enhanced restoration of the photoreceptors [ 11 , 12 ] . on the other hand , it might also suggest that retinal sensitivity measured by microperimeter appeared to be able to provide a comprehensive and accurate evaluation of macular function in late - stage vkh patients and might do the same in patients with other retinal diseases . the oct findings were also related to the mean interval from symptom onset to the initiation of systemic steroid therapy and using of systemic corticosteroids and/or immunosuppressant at 12 months after onset ( table 3 ) . therefore , the interval from symptom onset to the initiation of systemic steroid therapy is an important prognostic factor for patients with late - stage vkh . chee reported that early treatment ( < 2 weeks ) of high - dose corticosteroids was associated with better visual results in vkh patients . our is / os+/cost group displayed a relatively normal bcva and an average treatment interval of 20.22 13.12 days . in contrast , only the cost+/is / os+ group exhibited normal retinal sensitivity , normal bcva , and treatment interval of 6.71 0.76 days . this result suggests that more prompt treatment might be required to achieve complete restoration of macular structure and function . the importance of prompt treatment was also suggested by the correlation with less incidence of systemic corticosteroids and/or immunosuppressant at 12 months after onset . the strengths of our study include its well - defined population who underwent standard long - term follow - up . a relatively large sample size was achieved by using sophisticated statistics like mixed linear model and others . these methods correct the dependence of both eyes from the same patients , so both eyes of the patients could be included . the primary outcome measures used in this study ( oct findings , microperimetry , retinal sensitivity , and bcva ) are largely objective measures that are easily quantifiable . the study 's retrospective design is a limitation , as is the fact that longitudinal assessment of disease activity and visual function could not be assessed due to the use of a cross - sectional study design . the mechanisms underlying particular findings , such as the preservation of the retinal structure at the fovea , are not fully understood . further follow - up study might be able to tell us more about how these vkh patients recovered from their acute episode and how the functional and anatomic recovery were correlated . in summary , our study demonstrated a significant correlation between the oct findings and visual function in late - stage vkh patients , as determined via bcva and microperimetry . the integrity of the cost line was more sensitive than that of the is / os junction . moreover , prompt initiation of high - dose corticosteroid treatment was associated with improved prognosis for visual function , retinal structure , and less incidence of systemic corticosteroids and/or immunosuppressant at 12 months .

there are few reports in the literature of complete obstruction of the vertebral artery ( va ) due to an electric screw driver bit penetration through the neural foramen into the spinal canal with brown - squard syndrome ( bss ) . a 25-year - old man was admitted to the emergency department with a penetrated neck injury by an electric screw driver bit after a struggle . the patient presented the clinical features of bss . computed tomography scan revealed that the electric screw driver bit penetrated through the right neural foramen at the level of c3 - 4 , and it caused an injury to the right half of the spinal cord . emergent angiography revealed va dissection , which was managed by immediate coil embolization at both proximal and distal ends of the injury site . after occlusion of the va , the electric screw driver bit was extracted under general anesthesia . bleeding was minimal and controlled without difficulties . no postoperative complications , such as wound dehiscence , csf leakage , or infection , were noted . endovascular approaches for occlusion of vertebral artery lesions are safe and effective methods of treatment . purpose . to characterize the optical coherence tomography ( oct ) findings in late - stage vogt - koyanagi - harada ( vkh ) disease and its correlation with visual function . methods . the records of patients with late - stage vkh disease ( defined as 12 months from disease onset ) were retrospectively reviewed . the analysis focused on the oct findings and microperimetry , in addition to the possible correlation between morphology and functional findings . results . twenty - nine patients ( 58 eyes ) were included . mean age at onset was 34.24 10.67 years . the oct revealed that the outer retina and retinal pigment epithelium ( rpe ) were mainly affected . these effects included rpe thickening and breakage or disappearance of the cone outer segment tip ( cost ) line and/or inner segment / outer segment ( is / os ) junction . the cost line and is / os results were related to macular function and the interval between symptom onset and initiation of high - dose corticosteroid treatment ( all p < 0.01 ) . eyes with intact cost lines demonstrated intact is / os and normal rpe layers as well as better visual function and normal retinal sensitivity . conclusions . the oct findings are strongly correlated with macular function , as well as other clinical findings in late - stage vkh . with respect to the cost line and retinal sensitivity especially , the oct and microperimetry findings may be useful for evaluating later - stage vkh .

over the past decade , protein separation techniques and peptide analysis by mass spectrometry have greatly improved , and qualitative and quantitative proteome comparisons have become powerful tools for defining the composition of complex proteomes and the functions of protein complexes.(1 ) current ion trap mass spectrometers , in particular , are very sensitive and have fast analysis times that enable the identification of hundreds of proteins in a single lc - ms / ms analysis . however , obtaining comprehensive protein profiles from very complex samples , such as biological fluids or mammalian cell or tissue lysates , remains challenging due to the large number of proteins present in the sample over wide concentration ranges . to improve proteome coverage , samples typically are fractionated at either the protein level or peptide level , or both . regardless of the initial fractionation methods used , the final step in most strategies involves analysis of tryptic peptides by lc - ms / ms . however , undersampling will occur , that is , only a subset of the peptides will be identified , if the complexity of these tryptic digests exceeds the analytical capacity of the mass spectrometer ( e.g. , when more peptides elute from the hplc column per unit time than can be analyzed , or low - abundance peptides are below the instrument detection limit ) . a common observation when complex proteomes are analyzed is that the lists of proteins identified are often quite variable , which has raised concerns about the general value of proteomics for most applications , if it actually does have poor reproducibility . typically , the identifications of abundant proteins are reproducible , while most of the variability is observed in lower - abundance proteins . one strategy of achieving more comprehensive proteome coverage actually exploits this variability by simply performing repetitive lc - ms / ms analyses on each sample . other strategies for increasing the number of proteins identified include prefractionating the proteome based on protein physical properties , improving separation of the peptide mixture at the hplc separation step , modifying the esi interface to enhance ionization , or adding an ion mobility spectrometry ( faims ) interface before the ion trap.(19 ) proteome coverage also can be improved at the data analysis stage by using high - mass - accuracy data(20 ) or improved data - process algorithms . other strategies include a two - step method that reanalyzes the same sample with an inclusion list ( eliminating redundant precursor selection in the second analysis ) and a replay run that analyzes a sample twice from a single injection with targeted analysis of undersampled features in the replay run.(26 ) although each of these methods can achieve increased sensitivity , it is difficult to assess the relative importance of strategies that have not been directly compared in a single study due to variations in samples , study design , mass spectrometry platforms , data - analysis strategies used , and other laboratory - to - laboratory variations . consequently , a number of direct comparisons of alternative analysis strategies have been conducted to identify the best analysis approaches for specific types of samples such as serum and cell or tissue lysates . factors that should be considered when comparing alternative analysis strategies are the amount of total protein required for the analysis and the total amount of mass spectrometry instrument time required by each method . it is now well - established that repetitive lc - ms / ms analyses , longer hplc gradients , or additional fractionation via either more fractions or more modes of separation of complex proteomes will usually confer some benefit in terms of depth of analysis . the key factor is to determine the most time- and resource - effective strategies for achieving a high depth of analysis , as these factors directly affect sample throughput and proteome analysis cost . in this study , we compared the effects of two commonly used strategies : gelc - ms / ms with repetitive lc - ms / ms analysis and additional protein prefractionation and assessed their effects on depth of analysis and reproducibility using cancer cell lysates . neither strategy requires expensive special hardware or reagents , and both have been integrated into diverse analyses of complex proteomes by multiple laboratories . because of its simplicity , repetitive analysis often has been used to increase protein identifications after different fractionation schemes , most of which involve two dimensions of separation at either the peptide or protein levels , such as mud - pit or gelc . utilizing a cell extract of the human metastatic melanoma cell line 1205lu,(31 ) the gelc - ms / ms analysis with and without repetitive injections was compared and these data sets were compared to a 3-d method that included the prefractionation of the cell lysate using microscale solution isoelectrofocusing ( microsol ief ) prior to the sds - page separation . to balance the total number of lc - ms / ms runs and total instrument time between the repetitive 2-d method and the 3-d method , the 20 fractions from the sds - page fractionation each were analyzed four times and the cumulative result was compared with the 80-fraction proteome analysis using the 3-d approach . other factors such as hplc gradient , mass spectrometry method , and data processing were kept uniform to demonstrate clearly the relative effects of repetitive analyses versus an additional prefractionation step at the protein level . these comparisons showed excellent reproducibility between the methods , and substantially improved proteome coverage using the 3-d method compared with the repetitive analysis of fractions from the 2-d method . rpmi-1640 cell culture medium , sypro ruby stain , and nupage precast gels were from invitrogen corporation , carlsbad , ca . ultra pure urea , thiourea , dtt , and chaps were from ge healthcare , ltd . , giles , u.k . the bradford protein assay kit was from thermo fisher scientific , waltham , ma , and sequencing - grade porcine trypsin was from promega corporation , madison , wi . the human melanoma 1205lu cell line was maintained in rpmi-1640 medium supplemented with 10% fetal calf serum . cells were harvested on ice by scraping in cold phosphate - buffered saline ( pbs ) with inhibitors ( 0.3 mm pmsf , 2 mm sodium orthovanadate , 50 mm sodium fluoride , 1 g / ml leupeptin , 1 g / ml pepstatin ) when 7090% confluence was reached . the tissue culture plates then were washed three times in cold pbs with inhibitors ; the washes were collected , combined with the initial scraped cells , collected by centrifugation , and the cell pellet was quick frozen in liquid nitrogen followed by storage at 80 c . cell lysates were prepared using 800 l of lysis buffer ( 25 mm tris , ph 8.0 , 8 m urea , 2 m thiourea , 4% chaps , 1 mm dtt ) per cell pellet collected from a 15-cm tissue culture dish . the lysate was sonicated with a probe sonicator on ice with a duty cycle setting of 50% and an output level < 5% . insoluble material was removed by centrifugation at 100 000 g for 60 min and was estimated to be < 1% of the total protein based on staining intensity of colloidal coomassie sds gels . the supernatant was reduced using dtt and alkylated with n , n - dimethylacrylamide ( dma ) , as previously described.(11 ) total protein in the supernatant was measured using the bradford method . cell lysates ( 1.5 mg ) were separated into four ph ranges by microsol ief using a zoom ief fractionator ( invitrogen corporation , carlsbad , ca ) . for these separations , the fractionator contained five immobiline gel disks , each sustaining a ph of 3.0 , 5.4 , 6.2 , 7.0 , and 12 , respectively . these disks delineated four separation chambers and the cell lysate in a sample buffer consisting of 8 m urea , 2 m thiourea , 4% chaps , 1% dtt , 1% ph 37 zoom focusing buffer , and 1% ph 712 zoom focusing buffer was loaded into the middle two separation chambers . the outer chambers were loaded with sample buffer only and standard electrode solutions were used . samples were focused at a constant 750 v and a maximum of 1 ma for 3 h. the solution in each sample chamber was removed and measured . each chamber was rinsed with a small volume of sample buffer so that , when combined with the original solution removed from that chamber , the final volume was 700 l . proteins trapped in the immobiline gel disks were recovered using two sequential 175 l extractions for 30 min each using 10 mm tris , ph 8 , 1% sds for 30 min . the cell lysate and microsol ief fractions were initially analytically separated on 10% nupage minigels ( with mes running buffer ) using standard separation conditions . preparative sds - page was run using unfractionated cell lysate and the four microsol ief fractions in an analogous manner to the analytical run , except the separation distance was limited to 40 mm to minimize required numbers of fractions and gel volume per fraction . typically , six lanes two lanes from the gelc ( 2-d ) method containing 60 g each of cell lysate and one lane each from the 3-d method were sliced into 4 mm 1 mm 1 mm thick gel pieces . two adjacent pieces in each lane were combined in a digestion well and were digested overnight with trypsin . corresponding digestions from the duplicate 2-d method lanes were combined , generating a total of 20 digestions for the 2-d method . trypsin digestions were injected into a 75 m i.d . 15 cm picofrit ( new objective , inc . , woburn , ma ) column packed with 5 m magic c18 resin and peptides were separated by nano - hplc ( eksigent technologies , dublin , ca ) interfaced with a ltq linear ion trap mass spectrometer ( thermo fisher scientific , waltham , ma ) . for each analysis , 8 l of trypsin digest was loaded onto the column using solvent a ( 0.1% formic acid in milli - q [ millipore corporation , billerica , ma ] water ) . peptides were subsequently eluted using the following gradient conditions with 0.1% formic acid in acetonitrile as solvent b : 128% b over 42 min , 2850% b over 25.5 min , 5080% b over 5 min , and 80% b for 5 min . to minimize carryover twenty 2-d samples were analyzed first , followed by another replicated injection of the same 2-d samples . then , 80 3-d samples were analyzed , followed by the third and fourth replicated injections of the 2-d samples . the ltq mass spectrometer was operated with dynamic exclusion enabled for 30 s , full scans from 4002000 m / z , and data - dependent ms / ms analysis on the six most intense ions . 28 , rev . 13 , university of washington , seattle , wa ) in bioworks ( ver . 3.1 , thermo fisher scientific , waltham , ma ) , and the human uniref 100 ( ver . may , 2007 ) protein database , which was downloaded from the protein information resource at georgetown university . to generate the decoy database , the protein amino acid sequence for each database entry was reversed and the entire reversed database was appended in front of the original forward sequences . the data were searched against the combined forward / reverse database using partial trypsin specificity with a 2.5 da precursor mass tolerance and 1 da fragment ion mass tolerance . consensus protein lists were generated by dtaselect ( ver 1.9 , licensed from scripps research institute , la jolla , ca ) after applying the following filters : full tryptic boundaries , xcorr 1.8 ( z = 1 ) , 2.1 ( z = 2 ) , 3.25 ( z = 3 ) , cn 0.05 . custom software was then used to ensure that each unique peptide sequence was used only once in assembling the protein list . to identify common and unique proteins found by 2-d and 3-d methods , protein and peptide data were put into a relational database ( mysql ) and matched using custom software . the strategy used to compare ( 1 ) the conventional gelc - ms / ms method ( protein separation by sds gel + reverse - phase peptide separation ) , ( 2 ) gelc - ms / ms with repetitive injections , and ( 3 ) a 3-d method that used microsol ief fractionation prior to the sds - page step ( protein ief + sds gel + reverse - phase peptide separation ) is summarized in figure 1 . to directly compare the relative impact on proteome coverage of repetitive injections versus an additional protein separation step , equal amounts of the same cell lysate were fractionated and an equal number of lc - ms / ms analyses were used . a cell lysate of 1205lu cell line was processed in such a manner that each common step was comparable . the 3-d method consisted of microsol ief , sds - page , and lc - ms / ms . subsequently , the final minimum consensus protein lists from the three data sets were compared to evaluate the ability of enhancing sensitivity of repetitive analysis and additional fractionation strategies . microsol ief initially was developed to prefractionate complex proteomes prior to narrow ph range 2-d gels(33 ) and was subsequently used prior to 1-d sds - page as part of a 4-d strategy for fractionating serum or plasma.(11 ) in this study , we evaluated its utility for prefractionating cell lysates prior to gelc - ms / ms , since this separation mode is orthogonal to sds - page and does not suffer from many of the limitations of 2-d gels . because the dynamic range of protein concentrations in cell lysates is substantially less than in plasma or serum , the microsol ief separation was designed to yield four final fractions rather than the larger number of fractions typically used for plasma . the immobiline membrane disks between chambers were extracted because , as shown previously , proteins with pi values close to the ph of the disk remain in the disk but can be recovered in high yield . extracts from disks with extreme ph values ( 3.0 and 12 ) showed only trace amount of proteins , as few proteins have pi s near these values . extractions from disks located between separation chambers contained a mixture of proteins unique to that disk and proteins present in adjacent chambers as previously observed . when similar samples were separated by microsol ief and analyzed on 2-d gels , comparisons of solution fractions and membrane fractions showed that most proteins recovered from membrane disks were either unique to the membrane or had pi s slightly lower than the ph of the membrane ( data not shown ) . since the goal in this experiment was to fractionate the entire proteome into a small number of fractions at the microsol ief step , disk extracts were combined with the fraction to its right to maximize recovery ( figure 2 ) . the extraction from the ph 12 disk was discarded as it contained a negligible amount of protein . thus , after microsol ief fractionation , the cancer cell proteome was divided into four fractions of roughly similar complexity and with a simpler protein content . separation of the melanoma 1205lu cell extract by microsol ief and pooling to produce four fractions . equal portions of each fraction and separation membrane disk extract were analyzed by sds - page to evaluate separation and relative amounts of total protein in each fraction . the solution recovered from individual chambers was pooled with the adjacent membrane disk extract on the low ph side of the pool , as shown at the bottom of the gel , to produce four fractions . the extract from the ph 12 membrane disk had negligible protein and was not used further . for the conventional gelc - ms / ms method , 60 g of the original cell lysate , which was close to a maximum load while avoiding band distortion , was loaded into each of two lanes of a nupage gel ( figure 3a ) . proteins were separated until the tracking dye migrated 40 mm . after staining the gel with colloidal coomassie , the 40-mm lane was divided into 20 equal fractions and digested with trypsin . digests from corresponding positions in the two replicate lanes were combined to yield sufficient volume for four 8 l injections . sds - page separation of melanoma 1205lu cell lysate and microsol fractions for proteome analysis . samples were electrophoresed until the tracking dye migrated 4 cm ; gels were stained with colloidal coomassie and individual lanes were cut into 20 equal - sized slices , as shown . ( a ) for the 2-d method , the unfractionated lysate of the 1205lu cells was separated in two lanes ( 60 g / lane ) . ( b ) microsol ief fractions derived from 120 g of cell lysate were separated for the 3-d method . for the 3-d method , the amount of each fraction loaded onto the preparative gel was the protein recovered from 120 g of total cell lysate ( figure 3b ) . hence , the total amount of protein in the four gel lanes in figure 3b should be close to the amount of protein in the two lanes in figure 3a , provided sample losses during the microsol ief procedure were low , as has been previously demonstrated . as noted above , the gel lanes from the microsol fractions were cut and digested in the same manner as the gel slices from figure 3a , except in this case there were no duplicate gel lanes to be combined . hplc and mass spectrometer performance were carefully monitored to ensure consistent performance through these experiments . to further minimize effects of minor instrument performance variations , samples were injected in an order described in materials and methods . consistent performance of the autoinjector was monitored by weighing each sample vial before and after injection . there was no significant difference in the injection amount between the two methods ( t - test p > 0.05 ) . all data were consistently analyzed and filtered as described in materials and methods , which resulted in estimated false - positive rates ( fpr ) calculated by dividing reverse - hit peptide counts by forward - hit peptide counts of 1.6% and 1.4% for 2-d and 3-d data , respectively . figure 4 shows the nonredundant peptide and protein counts for the 3-d data set as well as differing numbers of replicates for the 2-d sample set . similar numbers of peptide and protein identifications were obtained for all individual 2-d data sets ( data not shown ) . as expected , the total number of nonredundant peptides increased moderately as additional replicate data sets were combined , and the incremental increase diminished as each new replicate was added . it was evident from the curve shown in figure 4a that the data set resulting from combining four replicate runs was approaching a plateau . parallel trends were observed for the peptide data sets defined by proteins identified by 3 , 2 , or 1 peptide(s ) . a total of 25 641 nonredundant peptides were identified after combining all four replicates of the 2-d analysis , which was 26% less than the 32 216 peptides identified by the 3-d method . ( a ) nonredundant peptide counts from a single 2-d analysis , combined data from increasing numbers of replicate analyses , and the 3-d method . ( b ) corresponding nonredundant protein counts for the same data sets as shown in panel a. the total number of lc - ms / ms runs that each data set contains is shown at the bottom of the figure . protein counts showed a similar trend as the peptide counts ( figure 4b ) , that is , adding a second replicate increased the number of nonredundant proteins more markedly than adding a third and fourth replicate . for example , the number of proteins identified by two or more peptides increased by 594 , 305 , and 162 when two , three , and four replicates were combined . the most appropriate comparison of the 3-d and 2-d methods is between the 3-d data set and four replicates of the 2-d samples because they both involve a total of 80 lc - ms / ms runs using equal amounts of initial cell lysate . when protein identifications based on two or more peptides were counted , the 3-d method identified 3486 proteins compared with 2850 proteins for the 2-d method . these 636 additional proteins ( 22.3% increase ) indicate a clear advantage of the 3-d method compared with the 2-d / replicate run method , even when equal amounts of mass spectrometer time are utilized . the proteomes produced by the 2-d / four repetitive - run and the 3-d methods were compared using the lists of proteins identified by two or more peptides . this comparison was facilitated by the fact that both data sets were searched using the same database and that dtaselect lists all protein names identified in the search . if a protein identification carried one protein name that could be found in the other data set , we deduced that both data sets had identified the same protein . this comparison at the protein name level showed extensive overlap between the two data sets even at this first level comparison ( figure 5 ) . specifically , the two data sets shared 2555 proteins , which corresponded to 90% of the smaller 2-d / repetitive - run proteome . the 295 proteins that appeared to be unique to the less in - depth 2-d / replicate run data set were further investigated to determine the basis for this apparent lack of reproducibility between in - depth analyses of the same biological sample . further analysis showed that 184 of these proteins were present in the 3-d data set as single - hit proteins . a majority ( 135 ) of these 184 proteins were two - peptide proteins in the 2-d data , indicating that the 3-d method identified these proteins by one less peptide . when the unfiltered data for the 3-d proteome was examined using randomly selected proteins in this group , we found that a substantial number of these proteins had been identified by the same second peptide as in the 2-d data set , but one or more of the filtering parameters was slightly lower than the cutoff values selected for global data filtering . as a result , these moderate changes in scoring parameters reduced the two- or three - hit protein identification in the 2-d data set to a 1-hit protein in the 3-d data set . we also further evaluated the 111 proteins found exclusively in the 2-d method . more than three - quarters ( 88 ) of them were two - peptide proteins . among these identifications , the 11 proteins identified by more than three unique peptide sequences were a particular concern , as the number of peptide identifications suggested that these proteins were not near the detection threshold and should have been identified in the more in - depth 3-d proteome . we found that the majority of the peptide sequences associated with these 11 proteins in the 2-d data set also existed in the 3-d data set , but they were assigned to highly homologous proteins ( supplemental table 1 ) . although a large number of common sequences for these proteins were observed in both data sets , a small number of unique sequences led the dtaselect program to assign these peptides to different proteins for each data set . of the 385 peptides assigned to the 111 proteins unique to the 2-d / replicate run data set , 226 peptides were present in the filtered 3-d data the two methods identified 2555 common proteins with two or more peptides per protein ( 90% of the total proteins identified in the smaller 2-d / replicate - run data set ) . of the 295 apparently unique proteins in the 2-d method , 184 proteins were identified in the 3-d data set by one peptide . the pie charts in the lower panels show the number of peptide hits for the 2-d method for the proteins that were not directly identified in the 3-d data set ( 111 proteins or 3.9% of the proteins in the 2-d data set ) and those identified by a single peptide in the 3-d data set ( 183 proteins or 6.5% of the proteins in the 2-d data set ) . these proteins , which were identified by two or three peptides in the 2-d / replicate data set , were identified by a single peptide in the 3-d data set . in each case , a second peptide was detected in the 3-d data set but failed to pass the data filter cutoff due to slightly lower values for xcorr or cn ( bolded values in table ) . the filtering cutoff values used were xcorr 1.8 ( + 1 ) ; 2.1 ( + 2 ) ; 3.25 ( + 3 ) , cn 0.05 . the bold values indicate they are below cutoff value . - indicates not found . to a first approximation , those proteins identified in the 2-d / repetitive - runs data set by only two or three peptides can be regarded as likely low - abundance proteins in the sample analyzed , since sequence coverage is usually a rough indicator of protein abundance level . the relative depth of coverage for these putative low - abundance proteins was compared in the 2-d / repetitive - run and the 3-d methods . as shown in figure 6 , for those proteins identified in both data sets , the majority showed greater sequence coverage in the 3-d data set . that is , of the 491 common proteins identified by two peptides in the 2-d data , the 3-d method found more peptides for 317 proteins ( 64.6% ) and the remaining proteins in this group were identified with an equal number of peptides . furthermore , in 114 cases , the 3-d method found at least three additional unique peptides , indicating a substantially greater depth of analysis . of the 394 common proteins identified by the 2-d method with three peptides , 325 proteins ( 82.5% ) were identified by an equal or larger number of peptides in the 3-d method . these data clearly indicate that , in most cases , the 3-d method had the ability to detect more peptides for low - abundance proteins than did the 2-d method . comparison of the number of peptides identified in the 2-d / repetitive and 3-d methods . ( a ) among proteins common to both data sets , 491 proteins were identified by two peptides in the 2-d / repetitive data set . the 3-d method found an equal number of peptides for 174 proteins and more peptides for 317 proteins . ( b ) among 394 proteins identified with three peptides by the 2-d method , the 3-d method found one less peptide for 69 proteins ( i.e. , + 1 for 2-d ) , an equal number of peptides for 99 proteins , and more peptides for 226 proteins . in this study , we systematically evaluated the relative merits of repetitive lc - ms / ms runs compared with introduction of an additional protein level separation step for increasing proteome coverage of cancer cell lysates . factors that affect apparent reproducibility between proteome analyses performed on the same sample also were evaluated . the basic analysis platform used for repetitive analyses was the commonly utilized gelc - ms / ms method , which can be considered to be a 2-d proteomics method as it involves two dimensions of separation , that is , protein separation using sds - page and reverse - phase hplc separation of tryptic peptides . this method was compared to a 3-d method consisting of solution ief at the protein level followed by the gelc - ms / ms method . it is important when comparing alternative analysis platforms to consider the total number of lc - ms / ms runs per proteome , because improved proteome coverage typically can be achieved by lengthening the hplc gradient or by repeating lc - ms / ms analysis of complex samples . similarly , many separation modes prior to lc - ms / ms can be at least incrementally improved by simply increasing the number of fractions collected , provided that the resolution of the separation method exceeds the initial fraction size used . but in some cases , increases in protein coverage may be too small to be considered advantageous when total analysis time per proteome is considered . hence , evaluation of the merits of greater depth of analysis , particularly small improvements , must be constantly weighted relative to overall throughput . furthermore , total mass spectrometer instrument time frequently is the limiting resource , and when fractionation prior to the lc - ms / ms step is used , it is the rate - limiting step in proteome analysis throughput . hence , the most meaningful comparisons are those where the total mass spectrometer analysis time per proteome is held constant . in this study , we used a consistent gradient time and 80 lc - ms / ms runs for both the 3-d method and the 2-d / repetitive - run method ( four repeat injections ) . similarly , all other experimental variables were held as constant as possible , including use of replicate aliquots of a single cell lysate preparation , gel separation lengths , gel volumes per trypsin digestion reaction , instrument tuning , and data - analysis methods . our goal was to determine , quantitatively , which method represents the more efficient utilization of mass spectrometer time when analyzing complex proteomes . robust proteome analysis methods should be reproducible in addition to identifying the majority of proteins present in a biological sample . one major cause of variations in proteins identified in replicate analyses of the same proteome is undersampling in the mass spectrometer , as discussed above . therefore , high proteome coverage should be linked to good reproducibility of proteome analysis results because extensive proteome coverage will occur only if undersampling is minimized . a second factor that will contribute to poor reproducibility between proteome protein lists is use of data filtering conditions that result in high false - peptide and protein - identification rates , since false positives usually are random . hence , data filtering stringency is another tradeoff that must be considered when selecting a proteome analysis strategy . while low stringency filters contribute to noise and low reproducibility , excessively stringent filters will greatly diminish the number of protein identifications and hence the value of the experiment . in the current study , data filters were used that yielded peptide false - positive rates between 1 and 2% as estimated using a decoy reverse database , thereby minimizing apparent poor reproducibility between data sets . this level of stringency results in very few false positives for proteins identified by two or more peptides and , while there are some false positives within the one - hit protein list , a majority of these identifications are correct . the repetitive analyses of 2-d data showed increased peptide and protein counts ( figure 4 ) indicative of undersampling in the basic gelc - ms / ms method used here . the overall gain from four repetitive analyses of proteins identified by two or more peptides was 1061 ( 59% ) compared to the initial single analysis . a similar increase ( 61% ) was observed in repetitive mudpit using nine analyses.(10 ) as expected , the greatest positive impact on proteome coverage was use of a second replicate run , which increased the number of proteins identified by two or more peptides by 33% while doubling instrument time . in contrast , adding a third and fourth replicate only increased protein coverage by 13% and 6% , respectively . these data indicate that performing a second analysis of each fraction when using gelc - ms / ms would be a positive tradeoff between instrument time and protein coverage . however , further doubling of instrument time by performing four repetitive runs is unlikely to represent optimal use of instrument time for most types of experiments . of course , an alternative to performing a repetitive analysis of gel fractions would be to obtain more slices per gel lane . in analogous experiments where gel lanes were divided into 40 or 60 fractions , we observed increases in the number of proteins identified that were similar to those obtained in this study for duplicate and triplicate analyses of the 20 fractions per gel lane ( data not shown ) . although producing more fractions per gel lane increases the number of in - gel digestions , the overall increase in total analysis time for a proteome is minor . hence , we generally prefer to use more fractions per lane rather than to replicate analyses when greater depth of analysis is desired using gelc - ms / ms experiments . longer gels and larger numbers of fractions per lane were not used in the current study because we wanted to keep total mass spectrometer time per proteome ( approximately 160 h per proteome ) within practical limits while simultaneously matching gel lengths , gel volumes , and other parameters . that is , extrapolating from other 2-d and 3-d experiments that we have performed , we expect that the total number of proteins for each data set ( 2-d , 2-d / repetitive runs , and 3-d ) would have increased moderately if we would have used 40 or 60 slices per gel lane for all samples . but use of 40 or 60 fractions per gel lane would have increased total instrument time to about 320 and 480 h per proteome , which represents an impractically low throughput for most studies . interestingly , as we increase the number of fractions per gel lane to 40 or 60 fractions , the incremental increases in new proteins identified diminish , analogous to the diminishing benefits of adding each additional replicate in the repetitive - run approach ( figure 4a ) . although similar trends are observed for these two approaches , the mechanisms for increasing protein coverage are quite different . that is , using a larger number of gel fractions increases protein separation and simplifies the mixture of proteins present in each fraction , while repetitive runs exploit subtle variations in peptide separations in replicate hplc runs and subtle variations in data - dependent selection of low - level ions for ms / ms fragmentation and analysis . the 3-d method clearly provided superior protein and peptide coverage compared with the 2-d / repetitive method , which indicates that adding an additional protein separation step represents a more efficient use of mass spectrometer instrument time . this method identified 3486 proteins with two or more peptides , which is 22% more than the 2-d / repetitive method that used equal instrument time . at the peptide level , the 3-d method identified 30 385 high - confidence , nonredundant peptides , which is nearly 2.5 times more than that found in a single survey using the 2-d method ( 12 160 ) and 28% more than the cumulative count in four repetitive analyses ( 23 648 ) . furthermore , more unique peptides were found for most low - abundance proteins in the 3-d method data compared with the cumulative 2-d method data ( figure 6 ) . it is not surprising that adding solution ief as an additional orthogonal protein separation step to a gelc - ms / ms method is an efficient strategy for increasing proteome coverage and sequence coverage of lower - abundance proteins . microsol ief separates the proteins that would normally be in a single gel slice into four gel slices ( see figure 3 ) . , the simpler samples should decrease ion suppression effects and reduce dynamic range within each digest . finally , in some cases , improved scores for ms2 spectra in the 3-d method probably resulted from a lower probability of interfering ions being isolated with the target ion for fragmentation . while the repetitive analysis strategy also improved proteome coverage , it had neither a built - in mechanism to reduce repeated sampling of abundant ions between replicates , nor could it explore the ions below the ms2 triggering threshold . an alterative technique that has sometimes been used to improve replicate runs is to scan different mass ranges in each replicate . however , pilot experiments suggested that this approach was less productive than the simple repetitive analysis method used here . one frequent criticism of proteomics methods is that the proteins identified on repeat analyses often are not very reproducible . a recent study suggested good reproducibility was achievable across 27 laboratories on a simple 20-protein mixture after uniform data processing was used.(35 ) but this simple sample of abundant proteins at the same concentration does not reflect real biological complexity . hence , in the current study , we compared the reproducibility between different analysis methods using a very complex sample of biological interest , that is , a human cancer cell lysate . among four replicate analyses of the 2-d samples , this indicated at least 76% of the proteins observed in one analysis were reproducibly detected despite significant undersampling . more importantly , at least 90% of the proteins observed in the 2-d / four - replicate data set based on two or more peptides directly matched to a corresponding protein 3-d data set , and most of the apparent mismatches were caused by trivial data analysis issues . a more rigorous comparison of the two comprehensive data sets showed that greater than 96% of the proteins identified in the 2-d / repetitive - run proteome were actually observed within the complete 3-d data set . one reason for the initially apparent , lower reproducibility when protein names were compared was slight variations in peptides scores together with use of rigid data filter cutoff values ( see above and figure 5 ) . that is , the 10% of proteins that were apparently unique to the 2-d / repetitive - run data set included 184 proteins ( 6.4% of the 2-d / repetitive protein list ) that were identified by a single peptide in the 3-d data set . reasons why these proteins were only identified by a single peptide in the 3-d data set include run - to - run variations in automated selection of low - abundance signals for ms / ms and run - to - run variations in sequest scores coupled with use of rigid data filters . a second contributing factor to the initially apparent , lower reproducibility at the protein list level is database redundancy and limitations of current software for consistently producing consensus protein lists from identified peptides . of the 111 proteins apparently unique to the 2-d / repetitive - run data set and not identified by a single - hit protein in the 3-d data set , most were highly homologous to proteins identified in the 3-d data set ( see results and supplemental table 1 ) . among the 385 peptides belonging to the 111 unique proteins in the 2-d data , although these 111 unique proteins comprised 4% of all proteins identified , the 159 unique peptides were only 0.7% of all 2-d filtered peptides . this illustrates that very small variations in identified peptides can have a proportionally higher apparent since we used each unique peptide a single time during assembly of consensus protein lists , the common sequences were assigned to the protein with the most unique sequences . consequently , for a group of proteins that have high sequence identity , one or two unique peptides could determine which protein in the protein family emerged in the final consensus protein list . this illustrates that better software tools are needed for identifying and displaying putative unique proteins within protein families . similarly , improved databases with uniform names or other labels that clearly indicate membership within a protein family would be beneficial . in conclusion , additional prefractionation with microsol ief substantially increased proteome coverage and sequence coverage compared with a gelc - ms / ms - repetitive run method that utilized an equal amount of mass spectrometer time . furthermore , the reproducibility of protein lists between the two methods was quite high because undersampling during data acquisition had been minimized . most of the apparent differences in protein identifications were due to limitations of current sequence databases and protein naming conventions , as well as software limitations for filtering database search results and building consensus protein lists . there is increasing evidence of a clear genetic link between the phenotypic characteristics of dogs and adverse drug reactions . the fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions , prediction of the behaviour of drugs and discovery of new drug targets for study . the introduction of a new parasiticide in the 1980s revealed a pre - existing mutation in dogs that predisposes the animals to potentially fatal neurotoxicosis . the drug , ivermectin , exerts its antiparasitic action by potentiating ligand - gated chloride ion channels in the peripheral nervous system of several invertebrate phyla . the mdr1 - 1 mutation in the multidrug resistance gene ( mdr-1 ) causes ivermectin intoxication in carriers . this mutation results in altered expression of p - glycoprotein and altered behaviour of the drug in collies and other related dog breeds . the mdr-1 and cyp2d15 genes have been studied in the uruguayan cimarron dog breed ; however , no mutations were identified in the 36 uruguayan cimarron animals analysed in these studies . several recent pharmacogenetic findings have been shown to be clinically relevant for patients of veterinary clinics , and the dog population is one of the most suitable models for examination of population genetics . clinically , collies and other related dog breeds have been observed to be more susceptible to the effects of ivermectin on the central nervous system ( cns ) . the clinical signs of these effects include tremors , salivation , coma , depression and ataxia . moreover , very small doses ( 1/100 - 1/200 standard ) cause acute and severe reactions in some , but not all collie breeds . approximately 75% of collies in the united states , france and australia have a mutant allele for the expression of modified p - glycoprotein . furthermore , the affected breeds have a similar lineage that includes other sheepdog breeds , such as old english sheepdogs , australian shepherds , shelties , english shepherds , border collies , german shepherds , longhaired whippets and silken windhounds . breeds that suffer from a deletion of the mdr-1 gene are more likely to experience adverse reactions in response to low doses of the drug . the cyp2b6 enzyme system ( cyp2b6 is a member of the cytochrome p450 group of enzymes ) is encoded by the cyp2b6 gene in humans and cyp2b11 in dogs . this class of enzymes is responsible for the metabolism of a wide variety of drugs . while the risk of drug - drug interactions involving human cyp2b enzymes appears to be low due to minimal involvement in drug oxidation and low hepatic expression , canine cyp2b11 has exhibited surprisingly high levels of activity in vitro toward drugs that are used in dogs , such as benzodiazepines . the cyp1a2 enzyme is a member of cytochrome p450 that is encoded by the cyp1a2 gene . cytochrome p450 ( cyp ) is a superfamily of enzymes that plays an important role in the oxidative metabolism of a wide variety of xenobiotics and endogenous compounds . additionally , this gene is constitutively expressed in human and dog livers and involved in the metabolism of many drugs , including caffeine , phenacetin , teophylline and tracing drugs . single nucleotide polymorphisms ( snps ) have been identified as deficiencies in the canine cyp1a2 gene . these deficiencies have been shown to significantly alter the pharmacokinetic behaviour of two drugs and associated with large inter - individual differences in the kinetic behaviour of a third drug . the resulting deficiency of cyp1a2 has been found to cause significant kinetic variations in ac3933 , ym-64277 , and a few other drugs in beagles . however , the significance of the effects of genetic polymorphisms of other canine cyps have not yet been fully explored . in contrast , snp genetic markers are considered to be the most recent generation of molecular markers . any of the four nucleotides may be present at any position in the genome ; therefore , it must be assumed that each snp has four alleles . this is theoretically possible , but in practice , the majority of snp variants have only two alleles , the original sequence and a single mutated version . this is because of the way in which they appear and are distributed in a population . this study was conducted to gain further insight into the characteristics of a breed or population and identify the snp genetic polymorphisms associated with the three genes ( mdr-1 , cyp1a2 and cyp2b11 ) involved in the metabolism of drugs used for medical treatments in several animals belonging to four different canine breeds . as several authors have noted , further studies are required to understand the regulatory effects of the polymorphisms and their potential clinical relevance . the rationale was to identify genetic polymorphisms in the genes that encode proteins and enzymes involved in drug transport , metabolism and action that can predict the usefulness of a particular drug to increase the numbers of responders and decrease the numbers of subjects affected by adverse drug reactions . the future of pharmacogenetics and its veterinary applications will have lasting effects on clinical decisions made in the future . the goal of drug therapy is to maximise the therapeutic effects while minimising the adverse effects associated with drugs and drug interactions . a total of 106 different animals belonging to four different canine breeds were studied ; 25 uruguayan cimarrons , 23 border collies , 29 labrador retrievers and 29 german shepherds . for analysis , blood was extracted under aseptic conditions from dogs while they were with their owners in their houses . dna was extracted from the blood samples using a dneasy tissue kit ( qiagen , the netherlands ) according to the manufacturer 's instructions , after which the dna quality and purity were evaluated using a nanodrop nd1000 spectrophotometer . twenty - six snps belonging to three different genes , mdr-1 ( 5 snp ) , cyp1a2 ( 20 snp ) and cyp2b11 ( one snp ) , were investigated according to the results obtained by several authors , with reference to the snp cluster report of the dbsnp ( ncbi ) genbank ( fig . 1 and table 1 ) . samples were sent out to geneseek ( neogen , usa ) for snp testing . a total of 5,512 sequences were analysed in the 26 loci ( two sequences for each locus in the 106 animals ) . the resulting sequences were then analysed using the blast programme to search for the most similar sequences in genbank ( national center for biotechnology information , usa ) . snp polymorphisms were analysed using genetix , arlequin , multivariate descriptive statistical analyses ( correspondence analyses ) and the snpstat programmes . this analysis allows investigation of a qualitative variable ( breed ) in terms of other qualitative variables ( alleles ) and the classical term of inertia can be assimilated into diversity . this method is conceptually similar to principal component analysis , but applies to categorical rather than continuous data . in a manner similar to principal component analysis , this method provides a means of displaying or summarising a set of data in three - dimensional graphical form . the analysis is conducted from a contingency table according to breed in which the rows are composed of the dependent variable ( breed ) and the columns are the set of explanatory variables ( alleles ) . the results allow acquisition of the relative importance ( inertias ) of both breeds and alleles . a graphical representation is created through a system of points in euclidean space , and the degree of their robustness is verified . the snpstat programme has been designed to conduct genetic association studies using snps and to analyse moderate numbers of snps ( 26 dnps in this paper ) . this programme provides allele and genotype frequencies , a test for hardy - weinberg equilibrium , analysis of the association with a response variable based on linear or logistic regression , analysis of interactions , linkage disequilibrium statistics , haplotype frequency estimation , analysis of the associations of haplotypes with the response and analysis of interactions ( haplotypes - covariate ) . the cumulative frequency is also known to facilitate selection of the threshold cut - off point with which rare haplotypes are grouped for further analysis . the association analyses of haplotypes are similar to those of genotypes in that logistic regression results are shown as either ors and 95% cis or as linear regression results with differences in means and 95% cis . the most frequent haplotype is automatically selected as the reference category and rare haplotypes are pooled together in a group . the 26 snps examined in the 106 animals exhibited different frequencies in each breed ( table 2 ) . an exact test for hardy - weinberg equilibrium was performed , and it was confirmed in the uruguayan cimarron population . indeed , this population was in equilibrium for all loci ( snp12 , snp14 , snp15 , snp16 and snp18 0.05 < p < 0.10 ) and was therefore used as the control population . as shown in table 2 , snp1 , snp2 , snp3 , snp4 and snp 5 exhibited allelic fixation in the uruguayan cimarron and labrador retriever breeds . all of the obtained fis values were negative , indicating a lack of inbreeding in the examined animals . 2 . the cimarron population ( population 1 ) exhibited a greater genetic distance with respect to the border collie ( population 2 ) , labrador retriever ( population 3 ) and german shepherd ( population 4 ) breeds . correspondence analysis indicated that the border collie breed exhibited 60% inertia , which was the greatest inertia observed in all four breeds , while the uruguayan cimarron breed exhibited the least ( 8% ) . the first axis , which explained 65.68% of the total inertia , clearly differentiated the cimarron breed from the other breeds . axis 2 explained only 10.42% of the total inertia , and the third axis , which explained 23.90% , did not clearly discriminate any of the breeds . the uruguayan cimarron breed exhibited the greatest genetic difference with respect to the other studied breeds . the haplotypes for all individuals were analysed using the snpstat programme , which enabled estimation of the haplotype frequencies . although the number of potential combinations of haplotypes can be very high ( 2 ) , the most frequent haplotypes ( those with frequencies greater than 1% ) included 12 in the cimarron population , 19 in the border collie population , 22 in the labrador retriever population and 18 in the german shepherd population . these haplotypes were compared to those of the control population , while those that exhibited frequencies below 1% are included in the rare haplotypes section . the programme selected the most frequent haplotype as the reference , while rare haplotypes were pooled together into a group . in each breed , 34% of the individuals exhibited haplotype number 1 , while 21% exhibited haplotype number 2 and the remaining haplotypes had frequencies below 10% . border collies exhibited haplotype number 1 at 32% , while the remaining haplotypes occurred at frequencies below 10% . the labrador retriever breed exhibited haplotype number 1 at 36% , while the german shepherd breed exhibited haplotype 1 at 16% and haplotype number 2 at 13% . the remaining haplotypes exhibited frequencies below 10% . analysis of the associations of each snp were performed for the binary response variable , and logistic regression analysis provided a summary of the genotype frequencies , proportions , odds ratios and 95% confidence intervals . we used the null hypothesis of similar effects in all studied dog breeds to determine if significant differences existed among breeds . an example of locus snp22 is shown in table 5 , in which the snp marker exhibited a significant difference from the control population ( p < 0.0001 ) . in this case , an association with the effects on the cyp1a2 gene was shown . according to the obtained results , the following 10 of the 26 snps exhibited significant differences and were associated with drug sensitivity : snp6 , snp12 , snp15 , snp16 , snp20 , snp21 , snp22 , snp24 and snp25 of the cyp1a2 gene , and snp26 of the cyp2b11 gene . all of these snps are potential candidates for studies of drug sensitivity in these breeds . the results obtained in this study revealed that three breeds ( border collie , labrador retrievers and german shepherd ) exhibited several candidate alleles useful for investigation of genes that control drug sensitivity . the correspondence analysis indicated that the border collie breed exhibited greater genetic variability and reduced inbreeding characteristics ( -0.52382 fis ; fis values range from -1 to + 1 ) . in contrast , the uruguayan cimarron breed exhibited less genetic variability and fewer inbreeding characteristics ( -0.27243 fis value ) and was also free of inbreeding problems . snps have been used as markers for diagnoses of specific features that are abundant in the genome , genetically stable and easily analysed . the underlying principle is based on identifying associations between a gene or genes that affect a variable ( e.g. , drug resistance ) and the snp markers . if the application of a statistical programme ( i.e. , snpstat in the present study ) reveals significant differences between genotypic classes , it can be concluded that there is an association between that marker and the studied characteristic . our results indicated that the border collie , labrador retriever and german shepherd breeds exhibited significant differences from the uruguayan cimarron breed in 10 snps of the cyp1a2 and cyp2b11 genes . different studies have examined the frequencies of several single nucleotide substitutions of canine genes that are associated with product resistance and drug sensitivity to survey other purebred populations that might be genetically at risk . our results are concordant with those obtained by other authors who have examined other canine breeds . the cyp1a2 gene of the beagle breed has been examined using the csnp method and a non - functional allele that indicated that interindividual differences in the pharmacokinetics of cyp1a2 substrates should be examined in this breed was discovered . additionally , one snp identified in cyp1a2 causes a protein deletion and deficiencies in toxicological evaluations . most common diseases involve complex genetic traits , and multiple genetic and environmental components contribute to susceptibility . it has been proposed that common genetic variants , including single nucleotide polymorphisms ( snps ) , influence the susceptibilities to common disease . this proposal has begun to be tested in numerous studies of the associations between the genetic variations in these common dna polymorphisms and the variations in disease susceptibilities . our preliminary results indicate that several polymorphisms were present in the studied genes and that these polymorphic loci are potential candidates for investigations of drug resistance . the analysis of these genetic marker snp candidates might be very useful before subjecting animals to drugs and other medical treatments because they are easily identified , and the methodology is simple and inexpensive . the results presented herein will contribute to efficient and reliable studies of drug candidates in dogs . this technology can detect animals that might be at risk and verify their genotypic characteristics to personalise drug therapy at the individual level rather than the population level . with advances that provide clear genetic responses , we can identify new drug targets and more precisely apply drug therapy to minimise adverse effects and maximise therapeutic benefits . academic and clinical research efforts in pharmacogenetics and pharmacogenomics might be beneficial to the entirety of veterinary medicine in the coming years .

in - depth , reproducible coverage of complex proteomes is challenging because the complexity of tryptic digests subjected to lc - ms / ms analysis frequently exceeds mass spectrometer analytical capacity , which results in undersampling of data . in this study , we used cancer cell lysates to systematically compare the commonly used gelc - ms / ms ( 1-d protein + 1-d peptide separation ) method using four repetitive injections ( 2-d / repetitive ) with a 3-d method that included solution isoelectric focusing and involved an equal number of lc - ms / ms runs . the 3-d method detected substantially more unique peptides and proteins , including higher numbers of unique peptides from low - abundance proteins , demonstrating that additional fractionation at the protein level is more effective than repetitive analyses at overcoming lc - ms / ms undersampling . importantly , more than 90% of the 2-d / repetitive protein identifications were found in the 3-d method data in a direct protein level comparison , and the reproducibility between data sets increased to greater than 96% when factors such as database redundancy and use of rigid scoring thresholds were considered . hence , high reproducibility of complex proteomes , such as human cancer cell lysates , readily can be achieved when using multidimensional separation methods with good depth of analysis . the fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions . specific screening tests can predict which animals express modified proteins or genetic sequences responsible for adverse effects associated with a drug . among the genetic variations that have been investigated in dogs , the multidrug resistance gene ( mdr ) is the best studied . however , other genes such as cyp1a2 and cyp2b11 control the protein syntheses involved in the metabolism of many drugs . in the present study , the mdr-1 , cyp1a2 and cyp2b11 genes were examined to identify snp polymorphisms associated with these genes in the following four canine breeds : uruguayan cimarron , border collie , labrador retriever and german shepherd . the results revealed that several snps of the cyp1a2 and cyp2b11 genes are potential targets for drug sensitivity investigations .

the emergency department ( ed ) is known to be one of the most congested units in any hospital that faces greater pressure in terms of patient load and health care resources as compared to other departments of the health care system . studies across various countries reported that quality of care decreases when the ed is overcrowded . overcrowding can result in delayed treatment , long patient waiting time and stay , overburdened working staff , patient elopement , high medical error rate , low productivity and poor patient outcomes . an efficient patient flow system serves critical patient quickly minimizing unnecessary delay in treatment . on the other hand , a patient arriving in the ed encounters repeated waits as he / she progresses in different stages , which may last for hours or even days . waiting time has been often cited as the most important cause of patients dissatisfaction in the ed . cooke cited reduction of waits as the most important area for improvement in ed . delays in the process have been associated with adverse outcome and increased violence in eds . waiting time in turn depends on multiple factors including volume of patients and workload on existing staff . if the outflow of patients from ed ( either by means of transfer out or by discharge ) is obstructed , this upstream bottleneck will also cause delays in the treatment . the present study was conducted to assess the patient flow system by assessing the arrival time pattern and waiting time distribution of patient in the ed of a tertiary health care institute of india . by understanding flow trends , hospital administrators can streamline processes to minimize wait times , improve efficiency , reduce overcrowding in the emergency out patient department ( eopd ) and in turn improve patients satisfaction . it was a short term cross sectional descriptive study conducted in may , 2011 in ed of a tertiary level medical , research and health care institution of north india . the institute caters to medical care needs of around 370 million populations of 7 states of india . in 2010 - 11 , the institute catered to a yearly load of around 16,57,200 out - patients and 64,969 inpatients , whereas the ed of the institute attended to 52,894 out - patients and 32,563 inpatients . a data collection tool to gather the required information was developed and pilot tested . during the study period , the investigator was stationed in the ed from 8.00 to 18.00 h. the data regarding waiting time ( the length of time , when patient is waiting idle in the ed for delivery of the service , he requires ) and inter - arrival gap ( time gap between arrivals of two consecutive patients in the ed ) distribution was collected . however , the data regarding arrival time ( the time that the patient is first recognized , as requesting service in the ed ) was obtained from the emergency records . the investigator approached patients / attendants presented in the eopd during the data collection hours and asked them about their background characteristics and time of entry in the eopd . they were further asked to document the waiting period for a service by asking a question for what service the patient is waiting for and from how much time ? the consent for undertaking the study was obtained from in - charge , ed of the institute . the ed block of the institute had a treatment area of approximately 23,088 square feet , which included 2 halls for patients with medical emergencies known as emergency medical opd and one hall for patients with surgical emergencies known as emergency surgical opd . the ed has one main entrance for patients and two more inlets from within the hospital , one from the main hospital and one from advance trauma center . there is a cabin for enquiry adjoining main entrance where two receptionists along with one assistant public relation officer respond to the queries of the patients 24 7 , along with other assigned works . there is a registration counter manned by one medical record technician , who register the patients , issued the gate passes to the patients attendants and mark no payment stamp on the recommendation form of patients after checking their eligibility . a fee clerk receives the hospital charges in a cabin adjoining to the registration counter . the fee counter runs from 8.00 to 20.00 , after which that the collection of fees is done on the registration counter . there is a radiology room ; attendant 's waiting hall , laboratories , chemist shop , blood bank , toilets ( male and female ) and senior medical officers ( smo 's ) room [ figure 1 ] . ground floor plan of emergency block of study institute in north india . a : hall a emergency medical out patient department ( emopd ) ; b : hall b emopd ; c : emergency surgical out patient department ; d : waiting hall ; e : chemist shop ; f : laboratory ; g : ultrasonography room ; h : x - ray room ; i : reception ; j : corridors ; k : waiting area for patients the overall signage system was not adequate to guide the patients and their relatives . there was no designated parking space for the vehicles of patients or staff who came to ed . a deputy medical superintendent assisted by five smo look after the ed services . at present , four posts of smo were lying vacant and only one smo ( ad - hoc ) was in a position . the administration of the emergency complex was looked after by the smo , such as dealing with medico - legal cases , providing poor free services to deserving patients and supply life - saving medicines and consumable items to the poor patients and supervise patient management in case of a disaster . in the eopd , medical staff works in 3 shifts / day viz . from 8.00 - 14.00 h , 14.00 - 20.00 h and 20.00 - 8.00 h. it was observed that majority ( 70% ) of the patients who visited the eopd was male and around 28% were in the age group of 45 - 59 years followed by 24.7% patients from the age group of 15 to 29 years . majority ( 85% ) of the patients who arrived in eopd were in the conscious state of mind and around 64% were referred from other health care facility before coming to eopd [ table 1 ] . arrival time pattern of patients showed that around 26.3% patients came during the 9.00 - 12.00 h. peak hour of the presentation was 10.01 - 11.00 with 11% of the total presentations . the maximum inter - arrival gap ( 46 min ) was observed during 6.01 - 7.00 h [ figure 2 ] . background characteristics of study population arrival time pattern of patients in the emergency out patient department of study institute it was observed that maximum patients ( 29.6% ) were waiting under observation after preliminary diagnosis by the physician and their median waiting time was 16 h. the next highest category of patients ( 16.4% ) was those who were waiting for diagnostics tests and their results with the median waiting time as 1 h. the number of patients , who had completed the treatment process and waiting for doctors decision regarding discharge from eopd was also substantial ( 14.6% ) with the median waiting time of 2 h. maximum median waiting time ( 38 h ) was recorded for patients waiting for their turn for operative procedure after the decision to operate . it was observed that waiting of around 71% of patients in eopd was attributed to factors within ed itself , whereas waiting of 26% patients was attributed to the reasons outside ed but within hospital [ figure 3 ] . waiting time distribution of patients for various services in the emergency out patient department of study institute ; a : waiting time attributed to reasons within the emergency department ( ed ) complex ; b : waiting time attributed to reasons outside the ed but within hospital ; c : waiting time attributed to reasons outside hospital with a steep increase in life - style diseases and road traffic accidents , the demand for emergency medical health care has been increasing . however , resource constraints in terms of scarce health manpower lead to overcrowding in hospitals , which in turn compromises the quality of care . most of the tertiary care hospitals of the india are facing problem with patient flow system . such problem is even more apparent in ed , which are mostly overcrowded , over - utilized , inappropriately staffed and often lack coordination of care . overcrowded eds and poorly managed patient flow results in excessively long waits for patients , increase in the risk of inappropriate care and poor quality of services . there has been a steady rise in ed attendance in the institute over the years both in absolute terms as well as daily averages . this is in conformity with an another study , conducted in a ed of a tertiary care hospital in new delhi in 2003 , which concluded that during the last 3 - 4 years there had been a steady rise in the ed attendance , both in absolute terms as well as daily averages . the higher use of the ed in the tertiary care hospitals can also be attributed to many reasons such as increase in the illness and chronic diseases , changes in demographic and epidemiological trends of diseases , increase in acute injuries , changes in people 's perception about emergency need for health problems , better quality of health services rendered and growing population etc . this is supported by the data on accidental deaths , which had shown a rapidly increasing trend during the decade 2000 - 2010 . moreover , the patient 's preference toward a tertiary care institute was also documented in a study earlier done in the same institute wherein more than two - third patients were satisfied with hospital services and wish to avail them over secondary level care . the provision of low cost tertiary level care treatment in study institute compared with the catastrophic cost of treatment in private hospitals might be a reason for choosing health care service in the institute . lack of tertiary care hospitals of such repute in neighboring states also lead to high rush of referral cases in the institute . at present , eopd of study institute was looking after around 154 patients at a time , which was around 5 times of its original capacity . to accommodate this heavy load of patients , additional patients were put on patient trolleys . as per the guidelines of australasian college of emergency medicine , the area of ed under study was adequate only for around 29,000 yearly patient load against present 53,000 . due to shortage of space , even the corridors of the eopd were full with patients on trolleys with a very little space left for movement . another study conducted in the same institute backs up this finding that patients were being treated in the non - treatment areas of the eopd . had also concluded that frequency of medication errors in ed increased with the crowding in ed . a study by goel et al . in same study institute also suggested that heavy load of patients and attendants in the non - treatment areas of eds may be a potential source of transmission of deadly infections . other studies have concluded that plague infection spread rapidly through infected patient to other patients in an overcrowded emergency ward of a tertiary care institute . the maximum patients in ed in our study from neurosurgery department are indicative of the fact that accidents mostly on road are increasing the volume of patients in ed . chan et al . in their study concluded that total census of major patients in ed is major reason behind wait in ed . the present study revealed that majority of the patients using the ed services were males . these results were easy to attribute to our culture where males are more exposed to hazards due to outdoor nature of their work as opposed to females . a little less than one - third of the patients who visited the ed were from the productive age group of 15 - 29 years closely followed by age group of 45 - 59 years . somewhat similar using pattern were shown in an another study carried out in barbados which shows that around 36% who used the ed services were from the age group of 21 to 45 years and around 26% were above 50 years of age . increase in the number of vehicles in chandigarh and its neighboring states resulting in steep upward trend in the number of road accidents might lead to increase in admission of males of the productive age group . the peak arrival hours were at 9.00 - 12.00 h in which around one fourth of the patients arrived in the eopd . the finding in the present study were in consonance with another study done in a tertiary care hospital of barbados , where only 10% of daily census entered in the ed during night . another study done in turkey also showed that the ed visit during the night had decreased substantially when compare to day time visits . the results of another study conducted in saudi arabia are contrary to this finding in which around 46% patients attended emergency during night . the reason for low arrival of patient in ed during night time in the study , can be attributed to the fact that public transport facility are shut down after 21.00 h and hefty charges are made by the private taxi operators at night . moreover , since no on call ambulance is provided by study institute , arranging one 's own vehicle might not be an easier option in the night . moreover most of the patients arriving in ed of study institute belong to lower strata of society who prefer to wait until morning rather than taking the patient to the hospital during night time . even though there was highly random arrival patterns observed in the study , yet surprisingly , the staffing pattern was somewhat similar for all 24 h spans . overloading of patient due to high arrival rate in peak hours and inappropriate staffing pattern may result in clogging of patients . the guidelines adopted by american academy of emergency medicine states that 1 physician is required per 2.5 patients / hour and the nurse , patient ratio should not exceed 1:3 . whereas , in the eopd of the study institute , around 10 - 11 resident doctors and 12 - 14 nurses cater to the average daily load of around 150 patients , which is much lower than the requirements . the volume of patients attending an ed was observed to be a major determinant of the waiting time , if resources are fixed . the resources in the department need to be matched to the workload on an hour by hour basis , despite the inherent variation in the workload . our study depicts that the factors influencing the patient flow do not lie solely in ed , although waiting of at least three fourths of all the patients can be explained by the reasons lying in the ed complex itself . similar to our study , schull and shanks cited unavailability of alternative levels of care in the community , delay in diagnostics and patients held in ed awaiting admission are the main causes of wait in ed . derlet and richards in their study concluded that the most important cause of ed overcrowding was insufficient inpatient capacity for ed patients , who required hospital admission . unavailability of inpatient beds not only delay those requiring bed but create a log jam effect , leading to unavailability of space and consequently delay of other patients who can be discharged from the ed . due to un - specified observation area in eopd in study area , most of the under observation chauhan et al . in their study also suggested formulating a strong admission and discharging policy in the ed to regulate the patient turn out rate . fast decisions on life - and - death cases are critical in ed . as a result , doctors face great pressures to over test and over treat . the fear of missing something often leads to extra blood tests and imaging scans . in the ed complex of the study institute , only routine diagnostic tests ( x - ray , hematology , biochemistry , ultrasonography and echocardiogram ) were performed whereas patients have to visit the concerned departments for specific tests . this finding is supported by the fact that around 12.3% patients in the eopd were found waiting for specialized tests with the median waiting time of 12 h. in the eopd , the blood samples for laboratory test were taken by already overworked resident doctors . another study done on the ed of study institute has also observed extreme rush in laboratories present within the premises of ed . other studies have also shown similar trend wherein , unnecessary tests conducted in laboratory complex increases the rush of non - patients . cooke also concluded that waiting for results of tests was one of the four commonest reasons for patient waiting in the ed . a review on consultants in ed of tertiary hospitals had suggested that average response time of consultants ranges from 30 to 45 min , depending on the current needs of the patient . in our study , this might be due to high proportion of junior resident doctors in the ed , higher rush of patients needed super specialty consultations and lack of standard operating procedures for consultations . there is ample evidence that lack of timely consultation and coordination in emergency team is a leading cause of ed overcrowding and poor case management . in our study , a small fraction ( 1% ) of patients were not aware about their reason of wait which can be possibly sketched by the fact that people feel hesitant in enquiring about the treatment from their care givers . frank et al . has also concluded that insufficient information provided on waiting time , was a cause of people 's perception of waiting time being extended . one of the main limitations of the study is small sample size and short duration of study . for the services availed by the patients in the absence of investigator , the investigator had to rely on the statement of the patients / attendants for time taken during the service . there might be a possibility of recall bias , where patient may forget about certain type of service rendered . there was a possibility of some incorrect data provided by patients or over exaggeration of waiting time . to tackle the random inflow of patients , it was recommended that experienced staffing in the ed should be matched with temporal arrival pattern of patients . further , benchmark should be formulated for various services ( waiting time , service time ) with annual audit mechanism . a laboratory technician should be posted in eopd area for blood sample collection to ease out the already overworked resident doctors . the communication with patients in ed should be improved , where waiting is inevitable , health education and promotion should be introduced . one of the main limitations of the study is small sample size and short duration of study . for the services availed by the patients in the absence of investigator , the investigator had to rely on the statement of the patients / attendants for time taken during the service . there might be a possibility of recall bias , where patient may forget about certain type of service rendered . there was a possibility of some incorrect data provided by patients or over exaggeration of waiting time . to tackle the random inflow of patients , it was recommended that experienced staffing in the ed should be matched with temporal arrival pattern of patients . further , benchmark should be formulated for various services ( waiting time , service time ) with annual audit mechanism . a laboratory technician should be posted in eopd area for blood sample collection to ease out the already overworked resident doctors . the communication with patients in ed should be improved , where waiting is inevitable , health education and promotion should be introduced . based on the above study it can be concluded that identifying operational factors that influence patient flow in ed help hospital administration to device suitable strategy for improvements in the functioning of ed . why is this topic importantthe policy makers faces a challenge of overcrowded emergency departments ( eds ) and an ill - managed patient flow in tertiary care institutes , especially in developing countries , which result in excessively long waits for patients . through this study , we tried to analyze the patient flow system by assessing the arrival and waiting time distribution of patients in the emergency out patient department ( eopd ) . this study will inform the hospital administrators and policy makers in effectively design a system so as to reduce waiting times and hence overcrowding in ed . this will in - turn benefit the patient by increasing their satisfaction rate.what does this study attempt to show ? the paper provides insight to some of operational factors and solutions , which are easily replicated across other countries.what are the key findings ? arrival time pattern of patients in the eopd was highly random with peak hour of presentation was 10.01 - 11.00 with 11% of the total presentationsthe primary waiting areas of patients included patients under observation overcrowding resulting from poor patient flow and excess waits can result in delayed treatment , long patient waiting time and stay , overburdened working staff , patient elopement , high medical error rate , low throughput and poor patient outcomes . why is this topic important the policy makers faces a challenge of overcrowded emergency departments ( eds ) and an ill - managed patient flow in tertiary care institutes , especially in developing countries , which result in excessively long waits for patients . through this study , we tried to analyze the patient flow system by assessing the arrival and waiting time distribution of patients in the emergency out patient department ( eopd ) . this study will inform the hospital administrators and policy makers in effectively design a system so as to reduce waiting times and hence overcrowding in ed . the paper provides insight to some of operational factors and solutions , which are easily replicated across other countries . arrival time pattern of patients in the eopd was highly random with peak hour of presentation was 10.01 - 11.00 with 11% of the total presentationsthe primary waiting areas of patients included patients under observation arrival time pattern of patients in the eopd was highly random with peak hour of presentation was 10.01 - 11.00 with 11% of the total presentations the primary waiting areas of patients included patients under observation ( 29.6% ) ; waiting for routine diagnostic tests ( 16.4% ) and waiting for discharge ( 14.6% ) . around 71% overcrowding resulting from poor patient flow and excess waits can result in delayed treatment , long patient waiting time and stay , overburdened working staff , patient elopement , high medical error rate , low throughput and poor patient outcomes . why is this topic importantthe policy makers faces a challenge of overcrowded emergency departments ( eds ) and an ill - managed patient flow in tertiary care institutes , especially in developing countries , which result in excessively long waits for patients . through this study , we tried to analyze the patient flow system by assessing the arrival and waiting time distribution of patients in the emergency out patient department ( eopd ) . this study will inform the hospital administrators and policy makers in effectively design a system so as to reduce waiting times and hence overcrowding in ed . this will in - turn benefit the patient by increasing their satisfaction rate.what does this study attempt to show ? the paper provides insight to some of operational factors and solutions , which are easily replicated across other countries.what are the key findings ? arrival time pattern of patients in the eopd was highly random with peak hour of presentation was 10.01 - 11.00 with 11% of the total presentationsthe primary waiting areas of patients included patients under observation overcrowding resulting from poor patient flow and excess waits can result in delayed treatment , long patient waiting time and stay , overburdened working staff , patient elopement , high medical error rate , low throughput and poor patient outcomes . why is this topic important the policy makers faces a challenge of overcrowded emergency departments ( eds ) and an ill - managed patient flow in tertiary care institutes , especially in developing countries , which result in excessively long waits for patients . through this study , we tried to analyze the patient flow system by assessing the arrival and waiting time distribution of patients in the emergency out patient department ( eopd ) . this study will inform the hospital administrators and policy makers in effectively design a system so as to reduce waiting times and hence overcrowding in ed . the paper provides insight to some of operational factors and solutions , which are easily replicated across other countries . arrival time pattern of patients in the eopd was highly random with peak hour of presentation was 10.01 - 11.00 with 11% of the total presentationsthe primary waiting areas of patients included patients under observation arrival time pattern of patients in the eopd was highly random with peak hour of presentation was 10.01 - 11.00 with 11% of the total presentations the primary waiting areas of patients included patients under observation overcrowding resulting from poor patient flow and excess waits can result in delayed treatment , long patient waiting time and stay , overburdened working staff , patient elopement , high medical error rate , low throughput and poor patient outcomes . primary intracystic squamous cell carcinoma ( scc ) in the breast is an extremely rare neoplasm . primary sccs in the breast are quite rare , although scc mixed with ductal carcinoma is more common . it can arise from metaplastic epithelium associated with other primary malignancy of the breast , metastasis from primary elsewhere in the body or an extension of malignancy from the skin covering . a 45-year - old female presented with a lump in the right breast , which she had noticed since three weeks . her menstrual cycles were regular and she had two children . clinical examination revealed a firm cystic lump of 12 10 cm in the upper inner and central quadrant of the right breast . the skin over the right breast appeared stretched , with a slight retraction of the nipple . pre - operatively , the patient was relegated to clinical stage iii ( t3n0m0 ) . ultrasonographic examination of the right breast showed an irregular - shaped hypoechoic lesion measuring 12 6 cm with an internal anechoic area measuring 6 3 cm , suggesting a cystic malignant tumor . fine needle aspiration from the lump in the right breast yielded 10 ml of pale yellow serous fluid . wet - fixed and air - dried smears were prepared from the centrifuged aspirated fluid and were stained with papanicolaou ( pap ) stain and may - grnwald - giemsa ( mgg ) stain , respectively . the smears studied were cellular and showed malignant squamous cells predominantly in singles and occasional syncytial groups , and some cells were spindle shaped . the malignant squamous cells had rounded borders with hyperchromatic enlarged nucleus and coarse irregular granular chromatin . cytoplasm of the squamous cells showed a variable degree of keratinisation [ figure 2 ] . cytology smears showing malignant squamous cells in singles with numerous cyst macrophages ( mgg , 400 ) cytology smear showing malignant squamous cells in singles , demonstrating cytoplasmic keratinisation ( pap , 400 ) to exclude metastatic scc , a search for remote primary scc included chest radiograph , cystoscopy , colposcopy , oesophagogastroscopy , laryngoscopy and cervical pap smear , which did not reveal any extramammary cancer . the criteria include : ( 1 ) no other neoplastic element , such as ductal or mesenchymal ones , are present in the tumor , ( 2 ) the tumor is independent of adjacent cutaneous structures and ( 3 ) no other distant epidermoid tumor exists in the patient . later , simple mastectomy with axillary clearance was performed and the specimen was sent for histological examination . the cut - section of the mastectomy specimen showed a large cystic tumor measuring 8 3 cm , containing yellow serous fluid . the cystic cavity was lined by dysplastic squamous epithelium with an infiltrating tumor , showing malignant squamous cells in broad sheets , groups and whorls , with keratin - pearl formation and intercellular bridges . immunostaining for estrogen / progesterone receptor and her-2/neu oncoprotein were negative and that for cytokeratin was positive . the smears studied were cellular and showed malignant squamous cells predominantly in singles and occasional syncytial groups , and some cells were spindle shaped . the malignant squamous cells had rounded borders with hyperchromatic enlarged nucleus and coarse irregular granular chromatin . cytoplasm of the squamous cells showed a variable degree of keratinisation [ figure 2 ] . cytology smears showing malignant squamous cells in singles with numerous cyst macrophages ( mgg , 400 ) cytology smear showing malignant squamous cells in singles , demonstrating cytoplasmic keratinisation ( pap , 400 ) to exclude metastatic scc , a search for remote primary scc included chest radiograph , cystoscopy , colposcopy , oesophagogastroscopy , laryngoscopy and cervical pap smear , which did not reveal any extramammary cancer . the criteria include : ( 1 ) no other neoplastic element , such as ductal or mesenchymal ones , are present in the tumor , ( 2 ) the tumor is independent of adjacent cutaneous structures and ( 3 ) no other distant epidermoid tumor exists in the patient . later , simple mastectomy with axillary clearance was performed and the specimen was sent for histological examination . the cut - section of the mastectomy specimen showed a large cystic tumor measuring 8 3 cm , containing yellow serous fluid . multiple sections studied showed cystic breast tumor . the cystic cavity was lined by dysplastic squamous epithelium with an infiltrating tumor , showing malignant squamous cells in broad sheets , groups and whorls , with keratin - pearl formation and intercellular bridges . immunostaining for estrogen / progesterone receptor and her-2/neu oncoprotein were negative and that for cytokeratin was positive . pure scc and adenosquamous carcinoma have been listed under metaplastic breast carcinomas in the world health organization classification . the japanese breast cancer society has defined scc of the breast as a special type of cancer , in which malignant cells are arranged in broad sheets and whorls with keratin formation or intercellular bridge . the incidence of scc reported in the western countries is 0.13.6% and in japan , it is 0.17% . the age group affected is between 32 and 65 years , with an increased tendency of left - sided involvement . the characteristics of scc of the breast generally reported are a large - sized tumor , rapidly growing with a central cyst formed by necrosis . this is unlikely in our case because there was little necrosis elsewhere in the tumor . the possible mode of origin of scc is epidermoid cyst of the breast , chronic abscess and complete metaplasia of glandular breast tissue . lymph node involvement is reported to be less frequent than might be expected , given the larger tumor size . the tumor cells are negative for vimentin , estrogen and progesterone and are diffusely positive for high molecular weight cytokeratin and c - erbb-2 . squamous cells in fine needle aspiration cytology ( fnac ) of breast lesions can be found in various benign lesions , like epidermoid cyst , subareolar abscess , fibroadenoma , infracted papillomas , spindle cell metaplasia , cystic sarcoma phyllodes , pseudosarcoma and malignant breast tumors or metastatic malignancy . the benign breast conditions with abundant squamous cells may sometimes mimic malignant squamous lesion and vice versa . in general benign squamous cells are bland looking and are often associated with anucleated squames , and no tumor , cell cannibalism . the malignant squamous cells are more pleomorphic , mitotically active and dyskeratotic and , sometimes , bizarre - shaped cells can be seen . the differential diagnosis of malignant squamous cells in fnac of the breast includes primary scc and metastatic scc of the breast . careful assessment of cytological features of squamous cells and the background appearance appears to be critical for arriving at a correct diagnosis . however , in the case of intracystic scc , numerous foamy macrophages coexist with malignant squamous cells . hormonal therapy is not indicated as most cases of scc are negative for hormonal receptors . scc of the breast is reported to be resistant to both radiotherapy and standard chemotherapy performed for invasive ductal carcinoma . in our case , radical mastectomy with axillary clearance was performed with postoperative adjuvant therapy , as given for common types of breast cancers . there was no evidence of recurrence of the tumor after four years of treatment . in conclusion , the presence of malignant squamous cells in fine needle aspiration of the breast suggests primary scc or metastatic scc . the finding of a pure scc necessitates more accurate work - up to exclude skin lesion or metastasis . the primary scc should not be confused with a much more largely manifested metaplastic change in other usual breast cancers . although the presence of numerous cyst macrophages in the background of breast fnac smears suggests a benign lesion , when present with malignant squamous cells , they suggest intracystic scc .

background : emergency department ( ed ) of tertiary health care institute in india is mostly overcrowded , over utilized and inappropriately staffed . the challenges of overcrowded eds and ill - managed patient flow and admission processes result in excessively long waits for patients.aim:the objective of the present study was to analyze the patient flow system by assessing the arrival and waiting time distribution of patients in an emergency out patient department ( eopd).materials and methods : this short cross - sectional descriptive study was conducted in the eopd of a tertiary level health care institution in north india in the month of may , 2011 . the data was obtained from 591 patients , who were present in the eopd during the month of may , 2011 . the waiting time , inter arrival time between two consecutive patients were calculated in addition to the daily census data ( discharge rate , admission rate and transfer out rates etc . ) of the emergency.results:arrival time pattern of patients in the eopd was highly stochastic with the peak arrival hours to be 9.00 - 12.00 h in which around 26.3% patients arrived in the eopd . the primary waiting areas of patients included patients under observation ( 29.6% ) ; waiting for routine diagnostic tests ( 16.4% ) and waiting for discharge ( 14.6% ) . around 71% patients were waiting due to reasons within emergency complex.conclusion:the patient flow of the ed could only be addressed by multifaceted , multidisciplinary and hospital wide approach . primary intracystic squamous cell carcinoma ( scc ) of the breast is an extremely rare entity and has a low incidence in comparison with other breast cancers . we report a rare case of primary intracystic scc in a 45-year - old woman who presented with a cystic lump in the right breast . cytological smears of the fluid aspirated from the breast tumor revealed malignant squamous cells dispersed in single and occasional groups along with numerous cyst macrophages , suggesting cystic scc . histological study of the mastectomy specimen confirmed the diagnosis of primary intracystic scc . although the presence of abundant foamy macrophages in the background of fine needle aspiration cytology smears of the breast suggest benign breast lesion , when associated with malignant squamous cells , these suggest cystic primary scc or metastatic scc . the primary scc should not be confused with metaplastic change in other breast carcinomas .

translating empirically validated treatments into routine clinical practice is an essential activity that requires a multitude of factors to be addressed . for any given disorder , some of these factors include the type of patients being treated , the setting in which treatment is delivered ( including the funding arrangements and costs incurred ) , the attitudes and perceptions of staff about the treatment , and the other psychological and physical disorders , diseases , and disabilities that may co - occur with the condition being treated . these various factors mean that the translation will first involve a thoughtful adaptation to the given setting that takes into account local issues , current practices , staff attitudes , and funding arrangements . secondly , translation will involve evaluation of the effectiveness of the resulting treatment plus an empirical consideration of factors believed to influence outcome so that treatment can be further improved [ 2 , 3 ] . the present paper reports an attempt to translate a cognitive - behavioral program for depression to an inpatient hospital setting . there is sound evidence supporting cognitive - behavioral therapy ( cbt ) in the treatment of depression , but little direction about adaptation to inpatient settings . first , people with depression who require inpatient admissions often do so at a time of crisis or extreme symptom severity , and the aim is to quickly and safely return them to levels of independent function . for instance , inpatient treatment may be appropriate if there is a risk of suicide , but a goal will be to reduce symptoms rapidly , so the need for a restricted environment is removed swiftly . within these constraints , in addition , individuals with depression who do not require hospitalization may find an optimal treatment format to be brief , weekly sessions . however , for individuals who are in a hospital , the logistics of organizing a more time - intensive program are less ( especially since the patients might otherwise not be occupied for much of the day ) , and hospital staff are already present . thus , an intensive day - long format of cbt becomes not only a viable option , but also a potentially good use of time . third , given the constant turnover within a hospital , it is harder to mount disorder - specific treatments because the patients will possess various degrees of comorbidity and might not fit neatly into diagnostic categories , yet they may benefit from a cbt program . for reasons such as these , perth clinic developed an intensive cbt program suitable for inpatients . it is a closed - group program that is open to various diagnostic groups ( but mainly depression and anxiety ) during office hours for a two - week period . the first question to answer regarded the degree to which the outcomes reported in the published literature regarding group cbt for depression would generalize to this different format . the second focus of the present study concerned variables related to outcome . in a hospital setting , many factors are uncontrollable . for instance , the degree and type of comorbidity is a potentially important moderator of treatment success . in addition , success may be moderated by the frequency of depression ( i.e. , whether the current presentation is a single or a recurrent episode ) or the severity of the depression . according to beutler , prognosis is attenuated by patient complexity / chronicity , and by an absence of patient distress . facilitating social support enhances the likelihood of good outcome among patients with complex / chronic problems . therefore , to assist with future treatment planning , it was necessary to consider the degree to which patient - related variables affected outcome . in conducting an evaluation of the current program , there were a variety of constraints imposed by the context . chiefly , it was not possible to randomly assign patients to a control condition , and therefore , a variety of methods were used to evaluate the size of the treatment effect and to address some alternative explanations of a pre / posttreatment change . to increase confidence in the presumption that any pre / postchanges were associated with the provision of treatment , outcomes were compared against inpatients who did not receive the cbt program and those who began the treatment but did not complete . the former provide an indication of the extent of improvement without the program , but due to nonrandom assignment to conditions , it is possible that patients with poorer outcomes were not encouraged to enroll in the cbt program . this problem is partly addressed by examining the patients who enrolled in but did not complete the cbt program . ideally , these patient groups will be similar at admission to hospital , but patients who complete the cbt program will have superior outcomes . another way to address the causal role played by the cbt program is to examine the specificity of treatment changes . since cbt is aimed at changing emotions , mood - related symptoms should change more during the cbt program than at other points in the inpatient admission . finally , to evaluate the size of the treatment change , published treatment data were used to generate benchmark pre- and posttreatment data . one study that could serve as a benchmark for patients with depression was reported by peterson and halstead . they examined the effectiveness of group cbt for depressed ( i.e. , major depressive disorder single - episode and recurrent , dysthymic disorder , depressive disorder not otherwise specified , and adjustment disorder with depressed mood ) outpatients . their study found a reduction from a pretreatment mean of 23.1 on the beck depression inventory ( bdi ) to 14.4 by posttreatment . another relevant benchmark can be derived from studies that have conducted group psychotherapy to treat depression and used the bdi as an outcome measure . reviewed these studies found 35 such studies from which they calculated an overall pretreatment mean on the bdi of 23.9 and a posttreatment mean of 12.3 . this review incorporated a variety of treatments ; these values represent an acceptable benchmark because most of the studies included were versions of cbt . excluding those studies that were not cbt , the values improved the estimate of the treatment effect somewhat ( bdi before treatment of 25.5 to 11.6 at after treatment ) , for clients with a mean age of 36 and a mean number of 20 therapy hours . a final benchmark was an outpatient evaluation of group cbt for depressed patients treated within diagnostically heterogenous groups . their outcomes for the bdi revealed a decline from 26.9 ( sd = 8.9 ) to 16.5 ( sd = 11.3 ) . the effect size ( cohen 's d ) of this change was very large ( i.e. , 1.2 ) with 59% of patients demonstrating a clinically significant change after treatment . thus , the aim of the present study was to examine the effectiveness for inpatients with depression treated with a cbt program modified to run intensively over a two - week period . the total patient population comprised an archival dataset of 998 consecutive inpatient admissions diagnosed with a major depressive disorder from july 1996 to march 1999 , who spent at least one day in hospital and who completed a bdi at admission to hospital . patients were diagnosed using a clinical interview according to dsm - iv by their treating psychiatrist . the decision to admit the person as an inpatient was made by the psychiatrist and was made when it was apparent that the required treatment could not be provided optimally while the patient was in the community . the total patient group had a mean age of 43.2 ( sd = 14.4 ) , and 73% were female . of the total sample , 46% were married , 26% were separated or divorced , 17% were widowed , and the remainder were never married . the depressive disorders were classified as a single episode among 52% of cases and recurrent among the remaining 48% ; the depression was the primary diagnosis among 82% of patients and secondary to another diagnosis among the remainder . for those with a primary diagnosis of depression , the most common recorded comorbid disorders were anxiety disorders ( 18% ) , substance use ( 12% ) , and personality disorders ( 5% ) , and 58% of patients had at least one medical condition coded on axis iii . patients spent an average of 12.3 days in hospital ( sd = 8.9 ; range of 266 days ) . of the total patient group , 225 began the cbt program during their inpatient stay or immediately upon discharge , and 201 ( 89% ) completed the program . patients had provided consent for their data to be used for research and evaluation purposes , and the university 's human research and ethics committee approved the analysis of the deidentified data . in addition to medications managed by their treating psychiatrist and the cbt program , a variety of problem - focused open group inpatient programs are available , including an acute care program , a substance abuse program , and an interpersonal therapy program . thus , while patients may have had exposure to other treatments before or after the cbt program , they were not engaged in any other treatment ( except concurrent psychopharmacotherapy ) at the time . the type of pharmacotherapy at admission , during the inpatient stay or at discharge , was not collected , but almost all inpatients would be on one type of medication . perth clinic 's cbt program is a closed group for up to eight patients , conducted over a period of ten working days [ 5 , 10 ] . the program begins with problem identification and goal setting , leading to psychoeducation and cognitive therapy . the cognitive therapy draws upon the work of both beck and ellis and harper and involves self - monitoring and the identification and challenging of irrational beliefs . behavioral interventions such as anxiety management and stress reduction are taught ( e.g. , relaxation , breathing control , etc . ) , and patients engage in pleasant events scheduling and behavioral assignments to practise newly developed skills . in the latter part of treatment , the focus shifts to self - esteem , assertion , and communication training and concludes with a relapse prevention module . the structure allows sufficient time and flexibility for the discussion of both group and individual issues . there is also a supporters ' session , where each participant is invited to attend with a supporter . the aim of this session is to increase awareness of psychological disorders , the nature of treatment , and helpful caring behaviors . patients could complete the cbt program as an inpatient , as a daypatient ( immediately following discharge ) , or being discharged from inpatient status at some point within the program . the average time between admission and initiation of the cbt program was 4.9 days ( sd = 7.4 ) . all patients received the questionnaires at admission , discharge , and where applicable , at the beginning and end of the cbt program . questionnaires were sent to all patients who completed the cbt program at a six - week followup . staff - rated measures were administered at admission and discharge by ward staff and at the beginning and end of the cbt program by the treating therapists ( clinical psychologists and occupational therapists ) . the bdi is a 21-item self - report scale designed to measure the level of depression among clinical and non clinical populations and is widely used in research on depression . in addition , patients completed the locus of control of behaviour scale ( lcb ) to assess their sense of control over themselves and their lives . the rosenberg self - esteem scale ( rses ) was administered to measure general self - concept and consists of ten items using a four - point likert - type response format . clinic staff rated patients ' general level of psychiatric symptoms using the health of a nation outcome scales ( honos ) [ 16 , 17 ] and global assessment of function ( gaf ) . the honos is a 12-item clinician - rated scale that is comprehensive in coverage , clinically relevant , and quick to administer . honos was introduced to the clinic in the middle of 1997 , and therefore data are not available for one year of the study . first , the change in symptoms from admission , pre - cbt , post - cbt , to 6-week followup was examined . the bdi scores improved somewhat from admission ( m = 30.3 ) to pre - cbt ( m = 24.7 ) , f(1,152 ) = 60.76 , = .29 , p < .001 , markedly from pre- to post - cbt ( m 12.2 ) , f(1,152 ) = 299.90 , = .66 , p < .001 , and remained stable until followup ( m = 12.2 ) , f(1,152 ) = 0.003 , = .00 , p = ns . to compare the effectiveness of the present form of therapy with the results typically obtained in research trials , a 95% confidence interval was placed around the difference between pre- and posttreatment bdi scores and compared against the benchmark values identified in the introduction . thus , the 95% confidence interval around the difference of 12.5 between the pre- and posttreatment bdi scores extended from 11.1 to 13.9 . the mean difference of 11.6 for studies of group psychotherapy for depression was within this confidence interval ( as was the 13.9 difference if these studies were limited to those using cbt ) , supporting that the conclusion that the difference obtained in the present investigation was comparable to that found with group treatment for depression generally . another way to examine the overall effectiveness was to examine the clinical significance of the outcomes . calculating a reliable change index and using the normative data on the bdi described by robinson et al . , 60.2% of patients demonstrated clinically significant improvement after treatment . comparing the present results with those of in the depressed outpatient sample , it is apparent that inpatients were more severe at admission than the outpatient population ( i.e. , m = 30.3 versus 26.9 ) , but their scores had declined to less severe ( i.e. , m = 24.7 ) by the start of the cbt program . this is consistent with a treatment model within which acutely unwell patients are first stabilised and then gains consolidated in a cbt program . the number of patients who demonstrated a clinically significant improvement from pre- to post - cbt ( i.e. , 60.2% ) was similar to that found in an outpatient setting ( i.e. , 59% ) . these changes in outcome were also observable on other clinician - rated and self - reported measures . in terms of the staff - rated gaf , the scores rose from admission ( m = 49.3 ) to pre - cbt ( m = 56.9 ) , f(1,120 ) = 32.00 , = .21 , p < .001 , and continued to increase from pre- to post - cbt ( m = 69.1 ) , f(1,120 ) = 219.65 , = .65 , p < .001 . patients ' lcb scores did not increase from admission ( m = 47.9 ) to pre - cbt ( m = 47.2 ) , f(1,151 ) = 1.24 , = .01 , p = ns , became substantially more by post - cbt ( m = 38.2 ) , f(1,151 ) = 144.14 , = .49 , p < .001 , but it became marginally more external again by the 6-week followup ( m = 40.0 ) , f(1,151 ) = 6.51 , = .04 , p < .05 . the patients ' self - esteem scores did not improve from admission ( m = 23.5 ) to pre - cbt ( m = 23.6 ) , f(1,102 ) = 0.03 , = .000 , p = ns , but they became more internal from pre to post - cbt ( m = 28.7 ) , f(1,102 ) = 106.87 , = .51 , p < .001 , and remained stable at the 6-week followup ( m = 28.9 ) , f(1,102 ) = 0.22 , = .002 , p = ns . although the changes during the cbt program are comparable to those in published studies , one possible interpretation of the present findings is that the patients would have improved to the same degree due to their hospital stay . to partly address this concern , the patients who completed the cbt program were compared at admission and discharge to hospital with those who did not begin the cbt program on the one hand and those who withdrew from the program on the other . there were significant differences between the groups across these two time intervals , f(2,602 ) = 3.74 , = .01 , p < .05 . follow - up tests revealed no significant differences between groups at admission , but the patients who had completed cbt were less depressed ( m = 13.1 ) than those who never began cbt ( m = 16.6 ) , t(611 ) = 2.56 , p < .05 , who in turn were not different from those who had dropped out of cbt ( m = 16.9 ) , t(516 ) = 0.10 , p = ns . thus , despite the absence of differences at admission , those who completed the cbt program were the least depressed at discharge . to address the concern that the observed improvement may be due to the general effects of hospitalization rather than the specific effects of cbt , the profile of symptom change from admission to pre - cbt and then from pre- to post - cbt was examined . assuming a degree of symptom specificity of treatments , because cbt is focussed on symptoms of depression and anxiety , the changes in these domains should be greater during cbt , whereas the earlier phase in an inpatient admission will be focussed on acute symptom management . to examine these issues , the changes from admission to pre - cbt and then to post - cbt in individual honos items what is apparent from figure 1 is that the inpatient stay prior to entry into cbt is associated with small changes in anxiety and depression , but substantial improvements in memory / orientation , activities of daily living , self - harm , aggression , and substance problems , whereas the cbt program is associated with the greatest changes in emotional symptoms . thus , the symptoms targeted by cbt change following the treatment , but domains that are not the focus of treatment shift little during the cbt program . given the data showing that symptoms of depression improved during the time patients were in the cbt program , the effect on outcome of various clinical variables was examined . of the variables examined there was no difference in bdi scores between patients from pre- to post - cbt for whom the depression was a single episode or a recurrent episode , f(1,146 ) = 2.37 , = .02 , p = ns , or for whom the depression was judged to be primary or secondary to another disorder , f(1,151 ) = 0.15 , = .001 , p = ns . considering those patients for whom depression was the primary disorder , there was a hint that the total number of secondary diagnoses might be associated with a poorer outcome , f(1,97 ) = 2.98 , = .03 , p = .09 , but there was no indication of poorer outcomes for patients with personality disorders , f(1,151 ) = 0.19 , = .001 , p = ns , affective disorders , f(1,151 ) = 0.15 , = .001 , p = ns , and neurotic disorders , f(1,151 ) = 0.83 , = .01 , p = ns . although there was no difference in bdi scores before and after cbt between patients with more secondary medical conditions , f(6,146 ) = 1.67 , = .06 , p = ns , there was a hint that patients with more secondary substance use problems might have worse outcomes , f(3,149 ) = 2.44 , = .05 , p = .07 . thus , there was no strong evidence in the present sample that presentations of major depression that were complicated by other axis i , ii , or iii were any more resistant to a cbt program than the less complicated presentations . however , there was one clinical variable that was strongly related to outcome and that was severity of depression at admission . using a trecile split to divide patients into three equal - sized groups based on admission bdi scores , it was apparent that the change from admission to pre - cbt , f(2,150 ) = 7.20 , = .09 , p < .01 , and from pre- to post - cbt , f(2,150 ) = 16.73 , = .18 , p < .001 , was systematically related to severity . from figure 2 , it is apparent that the greatest changes were observed in those with the most severe problems , but that once cbt was finished , patients maintained the level of symptoms after treatment for the next six weeks , f(2,150 ) = 0.31 , = .004 , p = ns . the present study examined the effectiveness for patients with depression of a cbt program adapted to an inpatient psychiatric clinic . the intensive program brought about reductions in depression ratings compared to those found in the literature more generally , but importantly , it was able to achieve these gains within a two - week period , rather than the more usual 1620 weeks of treatment . the speed of the treatment gains is important because patients who require inpatient treatment are often distressed and may represent a threat to their own safety . therefore , a relatively rapid treatment means that a safer environment can be provided , but the period of time when they are removed from their typical routines and support is minimized . thus , cbt for depression can be adapted to an intensive program suitable for delivery within the model of care found in inpatient settings . however , within inpatient settings , multiple interventions co - occur . for the present study , this means that it is not possible to use the data to conclude that the cbt program caused the symptom changes . however , this is an empirical issue that has already been answered numerous times in controlled efficacy studies , and the present question is related to the effectiveness of cbt in a particular context . in particular , it is also clear that packaging a cbt program as part of an inpatient treatment program can deliver comparable treatment outcomes to spaced treatments , but it did not achieve these outcomes at the expense of longevity of treatment gains . the gains observed were stable over the six - week followup , and there was no evidence that the more severe patients were any more likely to return to problematic levels following the termination of the program . from a clinical perspective , a common issue raised among practitioners is that the treatments developed and then demonstrated in efficacy studies will not generalize to real - world clinical settings . in our experience , common reasons given for the lack of generalization include the greater severity of patients outside efficacy studies , the higher occurrence of comorbidity ( especially substance use and personality disorders ) since these patients can rarely be prohibited from receiving treatment in the way that they may be appropriately excluded from an efficacy study , and the setting itself . therefore , it was curious to see that in the present sample , the only variable that was associated with differential outcomes was symptom severity such that the greatest change was observed in the patients who were most depressed . given that other studies have found that comorbidity and type of depression have an impact on outcome , it is intriguing that the present study failed to find these differences . one possible reason for the lack of a difference is the intensity of the program . there may be greater opportunity for the comorbid conditions to interfere with treatment when an intervention extends to months , whereas in an intense cbt program that fills most of the day , there may be less opportunity for these associated conditions to manifest themselves . for instance , a person with a substance use problem may be able to maintain harm - free use or abstinence for a couple of weeks while in treatment , whereas over a few months , this might be more difficult . without random assignment to intensive or spaced treatment sessions , it is not possible to offer any more than this as a speculative suggestion , but the present data would encourage investigation of the benefits and costs of alternative forms of delivery . in considering the results , there are a number of limitations that need to be borne in mind . first , the hospital is a private clinic where the patients are insured , and therefore , the extent of generalization to the public sector is not clear . it is possible that even though comorbid conditions are present , the severity or types of comorbidity that may be present in public hospitals ( e.g. , a greater frequency of psychotic disorders ) may mean that particular presentations were not represented in the current sample . second , by its nature , the present investigation was a retrospective examination of effectiveness and not a randomized controlled trial . therefore , there are a variety of issues that make it hard to conclude that the cbt program caused the observed effects . it is possible that the symptom changes that are observed during the cbt program may not be attributable to the program , but due to patient selection and other factors associated with hospital care ( e.g. , concurrent pharmacotherapy ) . while these can not be ruled out , attempts were made to examine the degree to which these variables may have affected the data . the difference between inpatients who began the cbt program and those who did not suggests that the cbt program is providing benefits over and above treatment as usual . the benefits by discharge for people who began cbt and finished it , relative to those who dropped out , address the issue that staff selected cbt patients who were likely to improve regardless of the content of treatment , because those who dropped out were not different at admission or discharge from those who never began cbt . in addition , the apparent specificity of the treatment gains that occurred during cbt speaks against arguments about the general effect of being in hospital as causing the reductions in symptoms . however , while it remains possible that the treatment did not cause the observed outcomes , given that cbt has been demonstrated to be an efficacious treatment for depression and the current treatment effects were comparable to those observed in the efficacy studies , it is not unreasonable to presume that the application of an empirically validated treatment , albeit in a modified form , was largely responsible for the observed improvements . third , the present study did not set out to establish the efficacy of cbt , but to examine the effectiveness within a clinical setting and relied on the patients ' self - reports and the judgments of the treating clinicians . although the internal validity of the ratings is less than those carried out by a person blind to the treatment , the external validity of the ratings ( when conducted by clinicians who have spent two weeks with the patient ) is greater . nonetheless , there is a possibility for bias as patients may have tried to please therapists , and the staff may have overestimated the change to a greater degree in the cbt program than in the hospital more generally . finally , it is important to note that in contrast to efficacy studies , in a hospital context , it is not possible to exclude patients from a treatment with a bdi below a cut - off ( e.g. , ) , that is , the decision to treat a particular patient will involve a consideration of the self - reported severity of the depression , but other factors will also be taken into account , and therefore , the sample contains individuals who might not meet the criteria for inclusion in an efficacy study , applying such a criterion ( by excluding them from the analyses ) if anything strengthens the conclusions of the present study , since the pre- to post - cbt scores change from 28.4 to 13.2 ; increasing the absolute treatment difference from 12.5 bdi points when all patients are included to 15.2 points . despite these limitations , it appears reasonable to conclude that cbt can be adapted to an inpatient psychiatric clinic , and the effectiveness of such a program is comparable to that observed in more homogenous patient samples found in efficacy studies . however , it is not clear from the present study the degree to which the apparent lack of comorbidity and type of depression to interfere with treatment outcomes is associated with the intensity of the treatment , the setting of the therapy , and the temporal location in the overall treatment program when cbt was delivered . future research could investigate these questions and in so doing assist in clarifying the most effective way to deliver treatment in different settings to different patient presentations . another future research question could relate to the ability to deliver cbt to homogeneous patient groups . the patients in the present study were all diagnosed with depression , but they were treated within a group of patients where not all suffered from depression . the clinic provides a cbt program and makes it available to patients who might benefit from the program , and therefore a mixture of other primary diagnoses . although it was not possible to evaluate the outcomes of these other patients ( because the numbers of each diagnostic group were small , the outcome measures were not specific to the conditions , and there was not a sufficient literature on the disorders with the measures used to benchmark against the present data ) , it is clear that at least for people with depression , they can be treated alongside patients with other conditions without degradation of their treatment outcomes . the therapy staff observes that the mixed patient groups , rather than being a hindrance , are an assistance to therapy , since patients can assist one another in helping each other with their different problems . the degree to which this clinical observation can be replicated in a controlled study , it should be possible to start to isolate the factors that are beneficial about treating heterogenous patient groups . the present study can not identify the extent to which incorporating cbt into an inpatient program can reduce the overall length of hospital stay . the length of stay at the present clinic is relatively short , when compared to other private psychiatric clinics in australia which treat similar patient groups ( moira munro , personal communication , 27 , october 2011 ) , but different to public psychiatric hospitals ( which tend to treat proportionally more people with schizophrenia ) . one possible explanation is the reliance on adjunctive psychotherapy , and we have shown elsewhere that the addition of psychotherapy enhances the outcomes following an inpatient stay . however , the duration of hospital stay varies markedly across health care systems and countries , and therefore , this question is probably better addressed using multilevel modelling , where hospitals are nested with relevant units ( e.g. , healthcare system , nation , etc . ) . in conclusion , the present study found that an evidence - based treatment for depression could be modified to an inpatient treatment setting without substantial loss of effectiveness . clinically , this is important because people are often admitted as inpatients when suicide risk is high , and therefore , data consistent with the view that adding cbt to the treatment is available at such times is associated with a rapid reduction in symptoms . for a suicidal patient , a reduction in symptoms is going to be associated with a lowered risk of harm , and therefore , the sooner the reduction can be achieved , the sooner a less intensive treatment regime can the implemented . in addition , some concerns in generalizing from efficacy studies to clinical effectiveness that arise due to complexity of patients ( e.g. , comorbidity ) were addressed , and no strong evidence was found that these issues compromised outcomes in the present study . retinal vein occlusion ( rvo ) is an obstruction of the retinal venous system , it has an abrupt onset and is an important cause of visual morbidity . retinal vein occlusions constitute the second most common cause of retinal vascular disease after diabetic retinopathy , with a prevalence of between 1% and 2% in persons older than 40 years of age [ 25 ] . in the wisconsin beaver dam eye study , 12% of eyes that developed severe visual impairment ( best corrected visual acuity 20/200 ) during a 15-year followup were due to rvo . although the exact etiology of rvo is not known , it is likely to follow a thrombotic event , possibly caused by external compression or disease of the vein wall [ 1 , 7 ] . rvo can be categorized as branch retinal vein occlusion ( brvo ) , if the obstruction is located in one of the branches of the central vein ; or central retinal vein occlusion ( crvo ) , if it is located in the central vein , at the level of the optic nerve . brvo encompasses a heterogeneous group of disorders with different clinical aspects and presents with dilated and tortuous retinal venous system in a particular quadrant or hemisphere of the retina and is often associated with macular edema . crvo is presented with hemorrhagic changes in all four quadrants of the retina and dilated and tortuous veins . in both brvo and crvo , cotton wool spots , disc edema , and neovascularization the incidence of rvo has been estimated to be 0.12%/year in adults aged 45 years for brvo and 0.04%/year in adults aged 45 years for crvo in caucasian populations [ 6 , 11 ] . however , the canadian incidence of visual impairment ( vi ) due to macular edema ( me ) secondary to rvo is unknown . this study aims to determine the annual incidence of vi and characteristics of patients with me secondary to brvo and crvo in a real - world canadian setting . records from a longitudinal population - based database of more than 170,000 patients in 53 family practice clinics in southwestern ontario , canada were analyzed between january 1 , 2008 and december 31 , 2009 . these records contained chart - abstracted information such as visit diagnosis , medications , and consultation notes . in order to compare characteristics and comorbidities of patients with diabetic macular edema ( dme ) to those of the general population , a control cohort was constructed by matching age and gender for all patients in the database > 18 years by clinic location . initial extractions of control cohort and rvo patients with me , ( defined as retinal thickening within 500 m of the macular center ) and vi ( defined as best corrected visual acuity 20/40 in the rvo eye ) , were accomplished utilizing international classification of disease codes ( icd9/icd10 ) , reviewing patient charts for text entries of symptoms that supported a diagnosis of rvo and concomitant comorbidity , and reviewing patient treatment records unique to rvo including consultation notes and hospital discharge summaries . data included in this study comprised patient characteristics and demographics , cardiovascular comorbidity and events , and medication coverage . the swo database has recorded patient level data on the clinical diagnoses at each visit , symptoms corroborating the diagnoses , clinical data , prescribed treatments including lifestyle interventions and medications , physician visits , hospitalizations , and diagnostic / laboratory test results , allowing for the conduct of patient level analyses , since 2000 . data from the 53 practices participating in the swo database cohort are routinely updated on a quarterly basis with immediate reconciliation at the point of care . all practices included in the swo database are part of a family practice research network involved in various audit and clinical research activities . practices have consented to centralized accrual of clinical data from the patient record ( uwo irb 09572 ) . each patient 's ontario health insurance plan ( ohip ) number is assigned a unique patient identification number in the swo database . to protect the privacy of patient 's medical information , a 128-bit ssl certificate is installed on the production swo web server . the industry standard data protection method ensures the security of data during transmission across the internet . validation studies of the swo database confirming the quality and completeness of the recorded data show good agreement between estimates of the prevalence of cardiovascular risk factors obtained from the swo database and other published estimates [ 1215 ] . moreover , there is a correlation between the swo database and national data ( i.e. , ims , brogan pharmastat ) on the utilization of prescription medication ( personal communication , petrella ; kamino , ims ) . starting from the index date ( the date of the first diagnosis of rvo during the study period until december 2009 ) , all subjects meeting inclusion / exclusion criteria were analyzed to understand the demographics and treatment patterns of care . for treated patients , the treatment choices were characterized , and the treatment pattern was related to clinical characteristics of patients , including type of drug coverage ( public , private , out of pocket ) . for continuous variables , the mean , standard deviation , median , minimum and maximum values were estimated . for categorical variables , the number and percentage of each category within an assessment was calculated for non - missing data . 73 of 47,166 patients over 40 years of age with new diagnosis of rvo and a control cohort of 76,077 patients were extracted for this analysis . please refer to table 1 for data on demographics for the control and rvo cohorts . examination of recorded episodes during the observation period by gender and age revealed the following : only 8% of patients with a new diagnosis of rvo during this time were males between the ages of 4059 years , compared with 43% of females in that age group . ninety - two percent of males with new diagnoses of rvo were over the age of 60 , compared to 54% of females . a higher percentage of caucasian ( 81% ) and aboriginal ( 11% ) ethnic groups were affected by rvo compared to their respective groupings ( 78% and 9% ) within the control cohort . please refer to table 2 for data on disease characteristics for the control and rvo cohorts . more rvo patients were overweight ( 23% ) or obese ( 13% ) compared to the control cohort . more rvo patients had hypertension ( 68% versus 14% ) or dyslipidemia ( 16% versus 10% ) than control cohort ( p < 0.05 ) . one quarter of rvo patients had a history of vascular disease , primarily mi and stroke . fifteen percent of patients with rvo suffer from chronic kidney disease . please refer to table 3 for data on incidence and type of rvo within the rvo cohort . seventy - three patients over 40 years old with a new diagnosis of rvo were identified from 47,166 patients over 40 years old . fifty - three patients had brvo , and 20 patients had crvo as interpreted from consultation notes . the annual incidence of vi due to me secondary to brvo and crvo was found to be 0.056% , 95% ci ( 0.0110.072 ) and 0.021% , 95% ci ( 0.0080.081 ) , respectively . as shown in table 4 , majority of patients had public drug coverage . macular edema ( me ) is a complication of rvo that can lead to blindness . this was the first study to assess the incidence of vi due to me secondary to brvo and crvo and to describe the disease characteristics of patients with rvo in a canadian setting . a few other studies have assessed the incidence of rvo . in the australian blue mountains eye study , the 5-year incidence of any rvo was 1.0% , and the 10-year incidence was 1.6% ; in the us , the 5-year incidence was 0.8% , and the 15-year incidence was 2.3% ; and in japan , the 9-year incidence was reported at 2.0% . the incidence of brvo appears generally higher than the incidence of crvo . in all these studies , as in ours , the small number of individuals that developed incident rvo makes it difficult to know with accuracy the ratio of incident brvo to incident crvo and to use these estimates as a basis for comparison . more males ( 92% ) over 60 years versus 54% females had the diagnosis of rvo . rvo is rarely seen in individuals younger than 50 , but may affect up to 5% of individuals over the age of 80as noted by laouri et al . in a review of the literature on the burden of retinal vein occlusion . consistent with previous literature , we found that patients with rvo were more likely to present hypertension , dyslipidemia , or vascular diseases compared with general population [ 4 , 1719 ] . further , our findings concur with previous studies that found risk factors more common in patients with brvo . as observed by lattanzio et al . , given the close association of rvo with systemic vascular disease , new patients with rvo should be evaluated for hypertension , diabetes , and lipid abnormalities , as it may be the presentation of significant vascular morbidity . in the case of younger patients , who may be otherwise healthy , the pathogenesis and risk factors are still poorly understood and additional evaluation of coagulation disorders and a history of thromboses may be necessary [ 8 , 20 ] . this retrospective study was conducted utilizing icd9/icd10 disease codes and reviewing patient charges and treatment records , including consultation notes and hospital discharge summaries . brvo and crvo are easily detected using standard ophthalmological diagnostic tools and techniques , so while the diagnosis and classification of rvo are valid , difficulties in interpreting data contained in consultation notes , as well as data concerning number of episodes requiring consultation and treatment during the observation period , may have resulted in some inconsistencies in data capture . this paper presents a description of the characteristics of patients with vi due to me secondary to brvo and crvo in a real - world canadian setting . consistent with findings in other studies , rvo in this patient population was associated with several vascular comorbidities . the annual incidence of vi due to me secondary to brvo and crvo was estimated to be 0.056% and 0.021% , respectively .

the effectiveness among inpatients with depression of a modified cognitive behavior therapy ( cbt ) program was examined . a group of 300 inpatient admissions with a primary diagnosis of depression attending a private psychiatric clinic were assessed at the beginning and end of a two - week cbt program . the effectiveness of the treatment was demonstrated by improvements on the beck depression inventory ( bdi ) , the health of the nation outcome scales , locus of control of behaviour scale , and the global assessment of function . the changes on the bdi for patients with depression were benchmarked against estimates generated from published studies . the degree of change in a two - week period for inpatients with depression was similar to that observed in efficacy studies of cbt that typically run over a more extended time . implications for integrating cbt with inpatient services are discussed . retinal vein occlusion ( rvo ) is an obstruction of the retinal venous system , and macular edema ( me ) is a complication of rvo that can lead to blindness . the canadian incidence of visual impairment ( vi ) due to me secondary to rvo is unknown . this observational , retrospective study used records from the southwestern ontario database to observe the annual incidence , demographics , and comorbidity characteristics of patients with vi due to me secondary to rvo . from 47,166 patients , 73 with rvo ( > 40 years old ) were identified : 53 with branch retinal vein occlusion ( brvo ) , 20 with central retinal vein occlusion ( crvo ) . the annual incidence of vi ( visual acuity < 20/40 in snellen equivalent ) due to me secondary to brvo was ( mean ( 95%ci ) ) 0.056% ( 0.0110.072 ) , and to crvo was 0.021% ( 0.0080.081 ) . furthermore , a greater proportion of rvo patients had hypertension ( 68% versus 14% ) or dyslipidemia ( 16% versus 10% ) , when compared to a healthy control cohort of 76,077 subjects ( p < 0.05 ) . this study presents a description of the characteristics of patients with vi due to me secondary to rvo in a real - world canadian setting . the results demonstrate that brvo was more frequent than crvo , and that rvo in this patient population was associated with several vascular comorbidities .

it has been well documented in medical literature that diabetes management during ramadan fasting poses challenges to the treating physician ; and pre - ramadan diabetes assessment , evaluations , and diabetes education are important for successful management.1 this is because unplanned diabetes management during ramadan fasting may lead to hypoglycemia . hence , in the past few decades , efforts have been made by various researchers to manage diabetes during ramadan fasting without the risk of hypoglycemia . these include alteration / reduction of the dosages for oral hypoglycemic agents ( ohas ) and insulins , and shifting patients from ohas and insulins to metformin or other agents such as dipeptidyl peptidase-4 ( dpp-4 ) inhibitors . the increase in hypoglycemic events during the month of ramadan has been previously reported in the literature . the epidemiology of diabetes and ramadan ( epidiar ) study has reported a 7.5-fold increase in risk of hypoglycemia in patients with type 2 diabetes.2 however , this risk can be reduced by extensive diabetes education and pre - ramadan medication adjustments.1 hence , ramadan itself is not a risk , but poor education and use of medications which cause hypoglycemia with high dosages carries a risk . hypoglycemia itself has an adverse effect on the quality of life , is an obstacle while managing diabetes ( to control glycemia ) , and is associated with poor compliance to medication and treatment . it has also been well documented that skipping meals and a reduced food intake are the main causes for the hypoglycemia during ramadan fasting . hence , diabetes education plays a central role , and diabetic patients should be given education and counseling before ramadan fasting.1,3,4 in general , severe hypoglycemia carries a risk of morbidities , with major cardiovascular events such as stroke , myocardial ischemia / failure , and ventricular arrhythmias.5 for these reasons , allowing patients to fast / fasting during ramadan without risk of hypoglycemia is a personal / patient - centered decision . a study conducted in saudi arabia by aziz1 on 1,046 patients has demonstrated that ramadan fasting itself does not pose a risk to human metabolism or health , but conversely has beneficial health effects on physiological parameters ( eg , an opportunity to lose weight ) and on chronic disease prevention . this study has demonstrated that this goal can be achieved only by optimal pre - ramadan assessment and diabetes education.1 similar observations were reported in other studies as well.68 however , in 2003 , laarijani et al9 demonstrated a slight decrease in fasting serum glucose among healthy subjects during ramadan fasting . a similar finding has also been demonstrated by aziz1 in the ramadan study , with the lowest prevalence of hypoglycemia ( 4.58% ) . contrary to these facts , different studies conducted in the past during ramadan fasting have demonstrated high prevalence of hypoglycemia during ramadan fasting ( up to 21.7%).1018 however , these studies were mostly observational in nature , and patients were not selected before ramadan for extensive diabetes self - management education ( dsme ) , counseling , assessment for hba1c / creatinine , and alteration of therapy . hence in other words , it can be concluded , in general , that blood glucose levels fall during ramadan fasting in diabetic and nondiabetic subjects , and prevention of hypoglycemia with medication adjustments / alterations are the basic strategies to manage diabetes during ramadan fasting . with this literature background , the current review focuses on a class of medications which does not cause hypoglycemia , both in general and during ramadan fasting . one of them is dpp-4 inhibitors , and the drug available in the market is vildagliptin . we will focus on the pathophysiology of type 2 diabetes , dpp-4 inhibitors , and the role of vildagliptin during ramadan fasting . dpp-4 inhibitors are the new oral antidiabetic agents ( including vildagliptin sitagliptin , saxagliptin , linagliptin , alogliptin and other agents as well which are under extensive research ) . these agents / drugs reduce serum glucose concentrations and improve the glycemic control by augmenting the effects of incretins ; hence this strategy is also called incretin based therapy for diabetes management . under normal physiological state , the gut , in response to meals , releases hormones called incretins , for example , glp-1 ( glucagon like peptide-1 ) and gip ( gastric inhibitory polypeptide ) , which augment biosynthesis and secretion of insulin ( known as incretin effect ) as well as slow gastric emptying as well.1924 normally , these incretin hormones are degraded within minutes after their release by the enzyme dpp-4 . as the dpp-4 inhibitors , contrary to this normal physiology , in diabetic patients the balance between insulin secretion and hepatic glucose production is dysregulated . in type 1 diabetic subjects , there is absolute insulin deficiency due to autoimmunity against -cells with destruction of these cells , as compared with type 2 diabetic subjects who exhibit relative insulin deficiency with insulin resistance.25,26 furthermore , absolute or relative hyperglucagonemia , due to deficiency of incretin hormones in diabetic state , is a hallmark of both type 1 and type 2 diabetic subjects.27,28 in other words , in type 2 diabetic subjects , there is insulin resistance and hyperglucagonemia . due to these pathophysiological states , lipolysis and ketosis may worsen the metabolic state leading to diabetic ketoacidosis ( dka ) during prolonged fasting , with absolute or relative insulin deficiency.29,30 insulin resistance can be reversed by metformin , and hyperglucagonemia by incretin based therapy . additionally , vildagliptin has also been shown to improve -cell function in type 2 diabetics , apart from enhancing incretin effect.31 other research studies have demonstrated efficacy of vildagliptin to lower hba1c and improve glycemic control as well.3236 hence , reversion of diminished incretin effect is also essential to manage diabetes effectively , both in general and during ramadan fasting , while preventing hypoglycemia at the same time . one of the interesting phenomena of dpp-4 inhibitor or vildagliptin is that it is blood glucose - dependent and does not cause hypoglycemia when given as monotherapy , which is again a therapeutic advantage of dpp-4 inhibitors / vildagliptin during ramadan fasting . in this section the literature for the safety and efficacy of vildagliptin during ramadan fasting will be reviewed . sulfonylureas ( sus ) and oral hypoglycemic agents ( ohas ) are still widely used by general practitioners as oral antidiabetic agents , both in general and during ramadan fasting , because of their ability to effectively reduce hba1c and their low cost.37,38 however , they carry a higher risk of severe hypoglycemia , especially if dose is not reduced during ramadan fasting , and special precautions are required , together with individual considerations , especially in older age group.1,39 hence , highly variable rates of hypoglycemia have been reported in published research trials with sus / ohas ( 3%40% ) during ramadan fasting . however , this risk was significantly reduced when vildagliptin was prescribed during ramadan fasting , and this has also been recently reported in indo - pakistani populations from the uk and in cohorts of uk south asian muslim patients in the vector ( vildagliptin experience compared to gliclazide observed during ramadan ) study.4042 furthermore , in general , these agents have also been demonstrated to be safer in older age group.43 a recent observational study which combined metformin and vildagliptin therapy together with or without ohas during ramadan fasting has demonstrated advantage of reduced hypoglycemia incidence.44 they have shown one case of severe hypoglycemia in the arm treated with oha compared to the vildagliptin group , which showed no hypoglycemia event . the hypoglycemia events were 12 times more in the group treated with oha as compared to the vildagliptin group . other studies have reported similar results when comparing ohas with vildagliptin.45 another study conducted on vildagliptin and sus or ohas during ramadan fasting has reported higher incidence of hypoglycemia during ramadan fasting in the group treated with su and metformin vs vildagliptin plus metformin ( 26 episodes vs 19 episodes ) ; and also reported hba1c reduction in the vildagliptin group , however , with insignificant p - values.46 a study conducted by aziz1 has also concluded that the patient group prescribed with dpp-4 inhibitors such as sitagliptin / vildagliptin did not show any episodes of hypoglycemia during ramadan fasting . a recent review published in switzerland and france has studied worldwide the role of dpp-4 inhibitors ( including vildagliptin ) during ramadan fasting , and has come to the conclusion that the anti - diabetic agents dpp-4 inhibitors could be a more safer option while managing type-2 diabetes during ramadan fasting , with a very low risk of hypoglycemia.47 another prospective , noninterventional study published in france to assess real life rate of hypoglycemia during ramadan fasting in patients with type 2 diabetes and their ongoing dual therapy of metformin vildagliptin or metformin sulfonylurea has shown that hypoglycemia as an adverse events ( aes ) was higher in su group as compared to vildagliptin group ( 17.9% vs 7.5% ; p=0.025 ) , and better compliance was seen with vildagliptin group.48 the virtue ( vildagliptin experience compared with sulphonylureas observed during ramadan ) study , which recruited 1,333 patients from 10 different countries worldwide , has demonstrated significantly fewer hypoglycemia events as compared with su therapy ( 5.4% vs 19.8% , respectively ; p<0.001 ) . additionally , good glycemic and weight control and better tolerance were observed in vildagliptin - treated patients.49 the steadfast ( study evaluating vildagliptin compared to gliclazide in patients with type 2 diabetes fasting during ramadan ) study , a multicenter , double - blind , and randomized trial , which recruited 557 type 2 diabetic patients has demonstrated significantly lower hypoglycemia prevalence as compared to sus ( 3.0% vs 7.0% , respectively ; p=0.039).50 similar results have been reported in the muslim populations of india.51 in summary , vildagliptin has been proven to be effective , well tolerated , and associated with low incidence of hypoglycemia in recent clinical trials . this is true especially in high risk population such as elderly and those with renal impairment or those who require insulin based therapy with metformin and dpp-4 inhibitors.44,5256 despite the islamic rule of exemption , most of the diabetic patients essentially fast during ramadan ; and this fact should be considered while managing diabetes during ramadan fasting.1,2 drugs such as dpp-4 inhibitors / vildagliptin should be selected with other diabetes medications as these are not associated with high risk of hypoglycemia . it has been observed that general practitioners have limited knowledge of diabetes management during ramadan fasting . furthermore , survey results have shown that 53% of patients fasted against medical advice.57 however , this can be prevented effectively by extensive dsme and ramadan - focused diabetes management ; ramadan education and awareness in diabetes ( read ) program and similar studies have demonstrated promising and good results in terms of minimizing hypoglycemia risk during ramadan fasting.1,58,59 effective diabetes education for patients empowerment and motivation with self - care awareness involves health care professional teams , families , the community , and religious authorities as well.60,61 additionally , newer antidiabetic agents , such as vildagliptin ( dpp-4 inhibitor ) , that are associated with lower risk of hypoglycemia , are considered to be one of the safer options while managing diabetes during ramadan fasting , and have also shown higher treatment adherence as compared to other medications.62 pseudoprogression ( pspd ) and pseudoresponse ( psr ) following anticancer therapy are major areas of controversy in the management of high - grade glioma . in the era of temozolomide , discrimination of pspd and true progression of high - grade glioma after concurrent radiochemotherapy ( crt ) with conventional magnetic resonance imaging ( mri ) novel imaging modalities such as mr spectroscopy , positron emission tomography ( pet ) , and perfusion mri are showing promising results but have yet to be validated in prospective studies . we previously reported the significance of overexpression of p53 for predicting development of pspd10 ) . in addition to the method of interpretation for early radiologic deterioration , when to determine this is also a critical issue because it affects the clinician 's decision making regarding whether to switch to salvage therapy for recurrent disease or to continue planned adjuvant therapy . as pspd was termed " early necrosis " in early reports , many series determined pspd at 4 weeks after treatment . conversely , wen et al.28 ) proposed new response assessment in neuro - oncology ( rano ) working group criteria for high - grade glioma . an increasing non - enhancing component in t2-weighted and fluid attenuated inversion recovery ( flair ) mr images was considered to be progression for patients receiving anti - angiogenic therapy with the new criteria . they also suggested that progression can be true progression only in the cases of recurrence outside the radiation field or in pathologically confirmed cases , if radiological progression is detected within 12 weeks when pspd is most prevalent . however , further validation would be necessary because this suggestion is not based on a clinical study that demonstrates comparable survival outcome between early true progression ( 4 weeks after treatment ; etpd ) and late true progression ( progression between 4 and 12 weeks after treatment ; ltpd ) . if the survival difference between etpd and ltpd are significant , early progressive findings should be treated based on the assumption that the lesion would be the recurrent tumor . psr is the concept that represents temporary radiological improvement after using an antiangiogenic agent such as bevacizumab while having progression in effect . phase ii trials using bevacizumab and cpt-11 in recurrent malignant glioma have shown a high response rate of 63% , but this encouraging result did not lead to a significantly improved overall survival ( os)11,27 ) . this phenomenon has been proposed to represent the effect of bevacizumab normalizing the permeability of leaky endothelium that causes edema , showing a steroid - like feature . in the present study , we evaluated pspd following radiation therapy combined with concurrent temozolomide ( tmz ) and also assessed psr following anti - angiogenic therapy for patients with recurrent disease using criteria of the rano working group . all data from patients diagnosed with world health organization ( who ) grade 3 or 4 glioma from march 2005 to february 2011 were retrieved from the archives of the pathologic reports . the clinical and pathologic data of relevant patients were obtained in accordance with the protocol approved by the institutional review board . fifty - five of sixty - two patients with high - grade glioma who received radiotherapy ( rt ) with concurrent and adjuvant tmz were included in the present study . gross total resection was performed in 16 ( 29% ) patients , subtotal or near total resection was performed in 25 ( 45% ) patients , and stereotactic biopsy was performed in 14 ( 26% ) patients . the median dose used for radiation therapy was 61.2 gy ( range , 59.4 - 61.2 gy ) with daily fractions of 1.8 - 2.0 gy . forty - five patients received a total dose of 61.2 gy in 34 fractions , nine patients received 60 gy in 30 fractions , and one patient received 59.4 gy in 33 fractions . the median time from surgery to the initiation of crt was 26 days ( range of 11 - 77 days ) . tmz concomitant with postoperative rt was administered at 75 mg / m a day . for adjuvant tmz , 150 - 200 mg / m was administered daily for 5 days , every 28 days . in terms of salvage therapy following recurrence , the surgical resection was performed for the patients who had a small progressive lesion involving non - eloquent area , and gamma knife surgery was reserved for the cases with small lesions and medically inoperable condition . bevacizumab with or without cpt-11 was considered as a primary option for salvage therapy in all inoperable cases showing true progression . the patients who had previous episode of intracranial hemorrhage or who were not be able to afford bevacizumab received nimustine ( acnu)/cisplatin ( cddp ) or procarbazine , lomustine , and vincristine ( pcv ) chemotherapy , or metronomic tmz . mri with gadolinium enhancement and t2/flair was performed 1 month after crt and every 3 months thereafter . when a progressive finding was present , mri was performed at 2-month intervals . progression was defined as a more than 25% increase in the sum of the products of perpendicular diameters as in the macdonald criteria , an increase in the non - enhancing lesion in recurrent cancer with bevacizumab15 ) . etpd and ltpd were defined as true progression found at first and second post - treatment mri with the exception of pspd , respectively . psr was scored when progressive enhancement or a non - enhancing t2-weighted lesion was shown right after response to bevacizumab , or after discontinuation of the drug due to toxicity . in some cases , cerebral blood flow ( cbf ) , cerebral blood volume ( cbv ) values using perfusion mri , and apparent diffusion coefficient ( adc ) maps the kaplan - meier method was used for the survival analysis to compare os between etpd and ltpd . all data from patients diagnosed with world health organization ( who ) grade 3 or 4 glioma from march 2005 to february 2011 were retrieved from the archives of the pathologic reports . the clinical and pathologic data of relevant patients were obtained in accordance with the protocol approved by the institutional review board . fifty - five of sixty - two patients with high - grade glioma who received radiotherapy ( rt ) with concurrent and adjuvant tmz were included in the present study . gross total resection was performed in 16 ( 29% ) patients , subtotal or near total resection was performed in 25 ( 45% ) patients , and stereotactic biopsy was performed in 14 ( 26% ) patients . the median dose used for radiation therapy was 61.2 gy ( range , 59.4 - 61.2 gy ) with daily fractions of 1.8 - 2.0 gy . forty - five patients received a total dose of 61.2 gy in 34 fractions , nine patients received 60 gy in 30 fractions , and one patient received 59.4 gy in 33 fractions . the median time from surgery to the initiation of crt was 26 days ( range of 11 - 77 days ) . tmz concomitant with postoperative rt was administered at 75 mg / m a day . for adjuvant tmz , 150 - 200 mg / m was administered daily for 5 days , every 28 days . in terms of salvage therapy following recurrence , the surgical resection was performed for the patients who had a small progressive lesion involving non - eloquent area , and gamma knife surgery was reserved for the cases with small lesions and medically inoperable condition . bevacizumab with or without cpt-11 was considered as a primary option for salvage therapy in all inoperable cases showing true progression . the patients who had previous episode of intracranial hemorrhage or who were not be able to afford bevacizumab received nimustine ( acnu)/cisplatin ( cddp ) or procarbazine , lomustine , and vincristine ( pcv ) chemotherapy , or metronomic tmz . mri with gadolinium enhancement and t2/flair was performed 1 month after crt and every 3 months thereafter . when a progressive finding was present , mri was performed at 2-month intervals . progression was defined as a more than 25% increase in the sum of the products of perpendicular diameters as in the macdonald criteria , an increase in the non - enhancing lesion in recurrent cancer with bevacizumab15 ) . etpd and ltpd were defined as true progression found at first and second post - treatment mri with the exception of pspd , respectively . psr was scored when progressive enhancement or a non - enhancing t2-weighted lesion was shown right after response to bevacizumab , or after discontinuation of the drug due to toxicity . in some cases , cerebral blood flow ( cbf ) , cerebral blood volume ( cbv ) values using perfusion mri , and apparent diffusion coefficient ( adc ) maps the kaplan - meier method was used for the survival analysis to compare os between etpd and ltpd . all patients were treated with rt combined with concurrent and adjuvant tmz after surgical resection or stereotactic biopsy . nine ( 24% ) of thirty - seven patients with disease progression during all treatment period and 1 case of pspd were treated with bevacizumab with or without cpt-11 . surgical resection , gamma knife surgery , acnu / cddp , pcv chemotherapy , or metronomic tmz were also used as an option for the salvage therapy . in 13 of 55 patients cbv , adc , or pet were undertaken , and it was insufficient number to analyze the efficiency of the techniques . the exams only provided additional information and were not absolute means of diagnosis . at the first follow - up after crt , 21 patients ( 38% ) showed radiologic progression . and at second post - crt mri , 16 ( 29% ) of them persistently showed progression and considered as etpd . five ( 9% ) of those patients showed progression after the first mri taken at post - treatment 4 weeks and showed improvement afterwards without specific salvage treatment with a decreasing or stable dose of dexamethasone . these groups of patients were finally categorized as pspd . and at second post - crt mri , 7 showed new progression that was not seen in previous mri ( ltpd ) . the follow - up time was defined as the period between surgery ( including stereotactic biopsy ) and the last follow - up day or death . the estimated median survival was 25.6 months ( range of 3.3 - 89.7 months ) . the os rates at 1 and 2 years were 84.8% and 59.2% , respectively . when divided into who grade iii and iv gliomas , os at 2 years were 71.4% and 53.3% , respectively ( p=0.125 ) . 1 , the os of the etpd group vs. ltpd group was 57% vs. 86% at 1 year , and oss were 30% and 51% at 2 years , respectively . we also performed subgroup analysis in 33 patients having available data for o-6 methylguanine - dna methyltransferase ( mgmt ) methylation status . although there was no statistically significant os difference between the patients with methylated mgmt and those with unmethylated mgmt ( p=0.178 ) , it showed trend toward better survival with mgmt methylation ( fig . 2 ) . in multivariate analysis using covariates ( recursive partitioning analysis , who grade , p53 status , mgmt methylation status and extent of resection ) , none of the factors including etpd and ltpd was related to os . the median survivals of treatment with or without bevacizumab , were 10 and 6 months , respectively ( p=0.835 ) . although 5 ( 50% ) of 10 patients showed a radiological response following salvage treatment with bevacizumab , the response was durable in one case and the stable state persisted for 11 months . for one case , the mri scan showed decreased high t2 signal intensity 1 month after bevacizumab and cpt-11 , the lesion showed progression at the subsequent follow - up , and we considered this case to be pspd . for three other cases , radiologic improvements were shown within 3 months and bevacizumab was continued until thromboembolic events in two cases and cerebral infarction in one case . all showed rapid regrowth of the tumor immediately after discontinuation of bevacizumab on mri ( fig . all patients were treated with rt combined with concurrent and adjuvant tmz after surgical resection or stereotactic biopsy . nine ( 24% ) of thirty - seven patients with disease progression during all treatment period and 1 case of pspd were treated with bevacizumab with or without cpt-11 . surgical resection , gamma knife surgery , acnu / cddp , pcv chemotherapy , or metronomic tmz were also used as an option for the salvage therapy . in 13 of 55 patients cbv , adc , or pet were undertaken , and it was insufficient number to analyze the efficiency of the techniques . at the first follow - up after crt , 21 patients ( 38% ) showed radiologic progression . and at second post - crt mri , 16 ( 29% ) of them persistently showed progression and considered as etpd . five ( 9% ) of those patients showed progression after the first mri taken at post - treatment 4 weeks and showed improvement afterwards without specific salvage treatment with a decreasing or stable dose of dexamethasone . these groups of patients were finally categorized as pspd . and at second post - crt mri , 7 showed new progression that was not seen in previous mri ( ltpd ) . the follow - up time was defined as the period between surgery ( including stereotactic biopsy ) and the last follow - up day or death . the estimated median survival was 25.6 months ( range of 3.3 - 89.7 months ) . the os rates at 1 and 2 years were 84.8% and 59.2% , respectively . when divided into who grade iii and iv gliomas , os at 2 years were 71.4% and 53.3% , respectively ( p=0.125 ) . 1 , the os of the etpd group vs. ltpd group was 57% vs. 86% at 1 year , and oss were 30% and 51% at 2 years , respectively . no statistically significant difference was found between etpd and ltpd groups ( p=0.595 ) . we also performed subgroup analysis in 33 patients having available data for o-6 methylguanine - dna methyltransferase ( mgmt ) methylation status . although there was no statistically significant os difference between the patients with methylated mgmt and those with unmethylated mgmt ( p=0.178 ) , it showed trend toward better survival with mgmt methylation ( fig . using covariates ( recursive partitioning analysis , who grade , p53 status , mgmt methylation status and extent of resection ) , none of the factors including etpd and ltpd was related to os . the median survivals of treatment with or without bevacizumab , were 10 and 6 months , respectively ( p=0.835 ) . although 5 ( 50% ) of 10 patients showed a radiological response following salvage treatment with bevacizumab , the response was durable in one case and the stable state persisted for 11 months . for one case , the mri scan showed decreased high t2 signal intensity 1 month after bevacizumab and cpt-11 , the lesion showed progression at the subsequent follow - up , and we considered this case to be pspd . for three other cases , radiologic improvements were shown within 3 months and bevacizumab was continued until thromboembolic events in two cases and cerebral infarction in one case . all showed rapid regrowth of the tumor immediately after discontinuation of bevacizumab on mri ( fig . the primary goal of this study was to evaluate the survival difference between etpd and ltpd . thus , we could not identify a statistically significant survival difference between the two groups , indicating that it would be safe to continue planned adjuvant tmz rather than treatment for recurrence when radiologic progression appears in the first post - treatment mri . this unique treatment strategy dealing with radiologic progression for high - grade glioma comes from the absence of a diagnostic modality providing high accuracy and resemblance between in - field recurrence and treatment response as pspd or radiation necrosis . the mechanism of pspd which is exclusive phenomenon in high - grade glioma , is not clarified , but might be related to the nature of tumor itself . blood tumor barrier of glioma has high expression of p - glycoprotein , a 170-kda drug efflux pump protein , which is the main component of blood brain barrier ( bbb ) and inhibits penetration23 ) . generally , glioblastoma has compromised barrier that gadolinium can penetrate and shows enhancement . bbb breakage after rt has been well documented but the mechanism is unclear . in vitro study showed that rt induces early transient increase in paracellular permeability while maintaining the tight junction of capillary endothelial cells6 ) . an experimental study revealed that higher exposures of tmz in brain tumor can also attribute to breakdown of the bbb20 ) . increased permeability of blood tumor barrier caused by rt and tmz would result in contrast enhancement due to leakage , which presents as pspd . rano criteria were suggested to overcome the limitation of macdonald criteria and drew considerable attentions . although it provides innovative assessment in the treatment of malignant glioma , yet several limitations were pointed out as discriminating true or false lesions in non - enhancing lesions , too simple tumor size measurement , and not reflecting any of new imaging techniques14 ) . despite current criteria as macdonald or rano , purely objective judgement of treatment response only with imaging increased cbv / cbf and a decreased adc value do not imply progressive disease at all times , however , the decisions are made from these novel imaging modalities for most of the confusing cases . latest imaging techniques as dynamic susceptibility contrast mri perfusion , diffusion - weighted mri , mri / pet scans using biomarker , and thallium single photon emission ct are introduced but no comparison under prospective studies have been performed2,13,29 ) . thus , disparity sometimes in clinical judgment between oncologists and radiologists makes further treatment planning more complex and often needs to be discussed by a multidisciplinary tumor board . for example , one patient who was considered to show pspd in our last study was confirmed to show true progression by surgery . in this case , cbv was not increased and the tumor demonstrated stabilization after 4 weeks at the second post - treatment mri , which could be considered to be pspd . recently , vogelbaum et al.26 ) suggested that retrospective assessments of serial images to distinguish pspd form true progression and when the finding is ambiguous , the response should be called indeterminate even with rano criteria . this shows despite the latest techniques and reorganization of the criteria they do not guarantee more reliable diagnosis or appropriate further treatment plan therefore validation should be needed . retrospective comparative analysis among various criteria has been performed placing emphasis on the nonenhancing tumor8 ) . the major component that we adopted from rano was the timing of decision making as well as imaging criteria . appropriate timing is crucial in avoiding the misdiagnosis that would lead to discontinuation or initiation of the effective therapy . also in canadian recommendations pspd can not be diagnosed until 12 weeks after the end of rt unless new lesion shows out - of - field5 ) . in the review of sanghera et al.22 ) , timing of defining pspd varied from 2 weeks to 6 months among 8 studies . considering that most of the decisions are made at 4 weeks or 6 months in performed studies , it should be confirmed that observation until 12 weeks is not related to decreased os as in the present study . as our data showed no difference between etpd and ltpd , confirmation after serial observations would be reasonable rather than deciding early from a little information . correlation between mgmt methylation and pspd , and association with improved os has been reported by brandes et al.1 ) . in our study , the survival graph comparing mgmt methylated / unmethylated group showed difference but not statistically significant . may reflect increased bbb permeability , administration of bevacizumab under misdiagnosis of true progression normalizes a leaky vasculature and therefore may show a " response"25 ) . it is highly persuasive but only a hypothesis and needs further examination in the future study . in the present study the regression of in - field recurrence occurred 1 month after administration of the drug , and remained stable disease for 11 months . however , as we had impression of both possibilities , true progression vs. treatment related change , we still can not rule out late pspd in this case . counting such cases as the treatment effect may result in a falsely high response rate to an anti - angiogenic agent . as we can not perform pathologic confirmation in every suspected recurrence , pspd and psr should not be considered separately . regarding psr , conflicting views as mentioned earlier , a strikingly high response rate and progression - free survival ( pfs ) does not result in increased os ; thus , doubt exists regarding whether bevacizumab has an antitumor effect . in a pooled analysis by norden et al.18 ) , a statistically significant increase was found in pfs of patients receiving bevacizumab ; however , os demonstrated no statistically significant difference . in the same context bevacizumab causes adverse effects such as intracranial hemorrhage , thromboembolic events , and proteinuria7 ) . in a phase ii study of bevacizumab and cpt-11 in recurrent malignant glioma , 4 of 32 ( 13% ) patients had a thromboembolic event and two of them died of toxicity / neurologic deterioration27 ) . . one possible advantage of bevacizumab is considered to be reduction of corticosteroid use3 ) , but paradoxically it can cause dependency on bevacizumab , which is one of the most expensive drugs . additionally , bevacizumab can cause vegf - independent mechanisms of progression in the heavily treated patients18 ) . furthermore , as glioblastoma is known to be associated with a high risk of thromboembolism , anti - coagulation would be needed to prevent severe complication19 ) . however , not every patient planning to be administered bevacizumab can receive prophylaxis , and thus a positive effect for survival may be countervailed . this life - threatening toxicity can not be overlooked , and costs and benefits must be considered . one of the limitations of the current study is showing high os than other reported data , and this may be due to censored data , as 9 of the patients were unavailable for follow - up9 ) . when recurrence was suspected , salvage treatment varied among patients because until now there is no standard chemotherapy regimen in the management of high - grade glioma . although small - sized and retrospective in nature , the present study suggests methodology and evidence for the safety of postponed treatment of recurrent high - grade glioma until 12 weeks after termination of crt and shows clinical features of psr , raising a question about the true effect of bevacizumab . initiating a further treatment plan after 12 weeks as recommended by rano does not lead to loss of overall survival . additionally , in recurrent high - grade glioma , bevacizumab must be administered with caution in consideration of pspd and psr . however , mri with gadolinium enhancement alone is not sufficient to characterize tumor response or growth . in addition to clinical correlation with adequate duration of follow - up and histopathologic validation , further studies regarding additional physiologic imaging modalities are required for accurate evaluation of tumor response .

diabetes management during ramadan fasting is challenging to the physician in terms of minimizing the risk of hypoglycemia . as compared to oral hypoglycemic agents ( ohas ) and sulfonylureas ( sus ) , which carry a higher and significant risk of hypoglycemia , newer antidiabetic agents such as dipeptidyl peptidase-4 ( dpp-4 ) inhibitors have demonstrated lower risk of hypoglycemia during ramadan fasting , with better patient compliance . in addition to diabetes education and pre - ramadan assessments , the physician should also consider use of dpp-4 inhibitors ( such as vildagliptin ) during ramadan fasting to minimize the risk of hypoglycemia in type 2 diabetic subjects . severe episodes of hypoglycemia have been demonstrated in recent research and clinical trials with ohas / sus . conversely , these research observations have also demonstrated comparative safety and efficacy with lower risk of hypoglycemia associated with vildagliptin . current research review has collected evidence - based clinical trials and observations for the drug vildagliptin to minimize the risk of hypoglycemia during ramadan fasting , while at the same time focusing the role of diabetes self - management education ( dsme ) , pre - ramadan assessments , and patient care . objectivewe evaluated pseudoprogression ( pspd ) following radiation therapy combined with concurrent temozolomide ( tmz ) , and we assessed pseudoresponse following anti - angiogenic therapy for patients with recurrent disease using the response assessment of the neuro - oncology working group.methodspatients who were pathologically confirmed as having high - grade glioma received radiotherapy with concurrent tmz followed by adjuvant tmz . bevacizumab ( avastin ) with cpt-11 were used as a salvage option for cases of radiologic progression . magnetic resonance imaging ( mri ) was routinely performed 1 month after concurrent radiochemotherapy ( crt ) and every 3 months thereafter . for cases treated with the bevacizumab - containing regimen for progressive disease , mri was performed every 2 months.resultsof 55 patients , 21 ( 38% ) showed radiologic progression within 4 weeks after crt . of these patients , 16 ( 29% ) showed progression at second post - crt mri ( etpd ) and five ( 9% ) showed improvement ( pspd ) . seven of thirty - four initially non - progressed patients showed progression at the second post - crt mri ( ltpd ) . no difference in survival was observed between the etpd and ltpd groups ( p=0.595 ) . five ( 50% ) of ten patients showed a radiological response after salvage bevacizumab therapy . four of those patients exhibited rapid progression immediately after discontinuation of the drug ( drug holiday).conclusiontwelve weeks following treatment could be the optimal timing to determine pspd or true progression . mri with gadolinium enhancement alone is not sufficient to characterize tumor response or growth . clinical correlation with adequate follow - up duration and histopathologic validation may be helpful in discriminating pspd from true progression .

the fontan procedure is used to separate the systemic and pulmonary circulations in patients with various forms of functionally univentricular hearts . in the fontan circulation , systemic venous return is sent to the pulmonary arteries without passage through a ventricle . the fontan procedure can result in various late complications , including central venous hypertension , diminished oxygen delivery , reduced cardiac output , venous thrombosis , and arrhythmia ( 1,2 ) . these complications caused by central venous hypertension lead to parenchymal injury , fibrosis , and cirrhosis of the liver ( 2,3 ) . cardiac cirrhosis is a serious late complication of congenital heart disease and can cause hepatocellular carcinoma ( hcc ) ( 4,5 ) . however , the prevalence and risk factors of cirrhotic changes and hccs have not been clearly identified . furthermore , non - invasive diagnostic tools for hepatic fibrosis and the management of hcc in patients after undergoing the fontan procedure have not yet been clearly established . a 29-year - old woman with a history of a univentricular heart had undergone a fontan operation 10 years of age . she was followed - up at the department of pediatric cardiology in our hospital , and regularly underwent blood examinations without alpha - fetoprotein ( afp ) at 2- or 3-month intervals . a routine follow - up day , she experienced slight abdominal discomfort and received abdominal ultrasonography ( us ) . b - mode conventional us showed a 15 mm hypoechoic mass in liver segment 4 ( s4 ) ( fig . 1a ) , and an 18 mm hyperechoic mass in liver segment 2 ( s2 ) ( fig . furthermore , the liver parenchyma had a coarsened appearance consistent with cirrhosis , and ascites and splenomegaly were observed ( fig . b - mode conventional ultrasonography showed a 15 mm hypoechoic mass in liver segment 4 ( s4 ) ( a ) , the liver parenchyma had a coarsened appearance consistent with cirrhosis , and ascites and splenomegaly were observed ( b , c).ceus using sonazoid showed a homogeneously enhanced mass in the arterial dominant phase ( d ) , a hypoechoic mass relative to the adjacent liver parenchyma during the portal dominant phase ( e ) , and a contrast defect with a clear border in the postvascular phase ( f ) . b - mode conventional ultrasonography showed an 18 mm hyperechoic mass in liver segment 2 ( s2 ) ( a ) ; and ceus showed a homogeneously enhanced mass in the arterial dominant phase ( b ) , isoechoic mass relative to the adjacent liver parenchyma during the portal dominant phase ( c ) , and no defects in the postvascular phase ( d ) . we next performed contrast - enhanced us ( ceus ) using a bolus injection of 0.015 ml / kg sonazoid ( perfluorobutane ; daiichi - sankyo , tokyo , japan ) . the mass lesion in s4 was homogeneously enhanced in the arterial dominant phase ( from 10 to 30 seconds ) ( fig . the lesion became progressively hypoechoic relative to the adjacent liver parenchyma during the portal dominant phase ( from 30 to 120 seconds ) ( fig . 1e ) , and provided a contrast defect with a clear border in the postvascular phase ( 10 minutes later ) ( fig . the mass lesion in s2 was homogeneously enhanced in the arterial dominant phase ( fig . the lesion became isoechoic mass relative to the adjacent liver parenchyma during the portal dominant phase ( fig . 2c ) and provided no defects in the postvascular phase ( 10 minutes later ) ( fig . we subsequently performed magnetic resonance imaging ( mri ) with conventional t1-and t2-weighted imaging ( wi ) before and after contrast media administration , including diffusion imaging . the contrast media used was hepatocyte - specific primovist [ gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid ( gd - eob - dtpa ) ; bayer schering pharma , berlin , germany ] . there was radiographic evidence of liver cirrhosis with portal hypertension , including a nodular surface , a coarse texture , ascites , and splenomegaly on mri ( fig . there was radiographic evidence of liver cirrhosis with portal hypertension , including a nodular surface , a coarse texture , ascites , and splenomegaly on mri ( a , b ) . gd - eob - dtpa mri showed a 15 mm s4 mass with a moderately low intensity in t1-wi ( c ) , moderately high intensity in t2-wi ( d ) , moderately high intensity in diffusion ( e ) , homogeneous arterial enhancement ( b , f ) , and complete washout in the hepatobiliary phase ( g ) . this imaging revealed a mass lesion in s4 with a moderately low intensity in t1-wi ( fig . 3b and f ) , and washout in the portal phase at 70 seconds and interstitial phase at 180 seconds . at 20 minutes ( hepatobiliary phase ) after the contrast uptake the lesion showed washout ( fig . in addition , mri imaging revealed a mass lesion in s2 with a moderately high intensity in t1-wi ( fig . 4a and e ) , an isoenhancement relative to the adjacent liver parenchyma in the portal and interstitial phases , and a high intensity in the hepatobiliary phase ( fig . gd - eob - dtpa mri showed an 18 mm s2 mass with a moderately high intensity in t1-wi ( b ) , moderately low intensity in t2-wi ( c ) , moderately low intensity in diffusion ( d ) , homogeneous arterial enhancement ( a , e ) , and high intensity in the hepatobiliary phase ( f ) . her liver function was well preserved with the child - pugh classification a , and her international normalized ratio was low ( the patient was on warfarin for atrial arrhythmia ) . other lab findings included a high afp level [ normal less than 40 nanograms / milliliter ( ng / ml ) ] , high lens culinaris agglutinin - reactive fraction of afp ( afp - l3 ) ( normal less than 10% ) , low platelet count ( normal greater than 150,000/microliter ) , high hyaluronic acid ( normal less than 50 ng / ml ) , and high type iv collagen 7s ( normal less than 6.0 ng / ml ) . the patient had no other significant medical problems and no other risk factors for cirrhosis , as she expressed negativity for viral and autoimmune markers , no alcoholic consumption , no hepatotoxic drugs ( such as amiodarone ) , and no metabolic factors such as diabetes mellitus , obesity ( her body mass index was 17.7 ) , or fatty liver in us . following the reversal of her warfarin therapy , an ultrasound - guided needle biopsy of the s2 lesion was performed . histological findings revealed that bridging fibrosis in the liver section , and the parenchymal cells did not display cellular or structural atypia ( fig . wbc : white blood cells , rbc : red blood cells , hb : hemoglobin , hct : hematocrit , plt : platelets , pt : prothrombin time , inr : international normalized ratio , tp : total protein , alb : albumin , che : cholinesterase , t.bil : total bilirubin , d.bil : direct bilirubin , ast : aspartate aminotransferase , alt : alanine aminotransferase , ldh : lactate dehydrogenase , alp : alkaline phosphatase , -gtp : -glutamyltranspeptidase , ztt : zinc sulfate turbidity test , ttt : thymol turbidity test , bun : blood urea nitrogen , cr : creatinine , crp : c - reactive protein , icg - r : indocyanine green retention , hbsag : hepatitis b surface antigen , hbsab : hepatitis b surface antibody , hbcab : hepatitis b core antibody , hcv : hepatitis c virus antibody , ana : antinuclear antibodies , ama - m2 : anti - mitochondrial m2 antibody , afp : alpha - fetoprotein , afp - l3 : lens culinaris agglutinin - reactive fraction of afp , cea : carcinoembryonic antigen , ca : carbohydrate antigen a histological specimen was obtained by an ultrasound - guided needle biopsy of the s2 lesion . the parenchymal cells did not display cellular or structural atypia [ hematoxylin and eosin staining ( a ) , and silver impregnation ( b ) with a low - power field ] . the patient underwent a curative operation , involving partial hepatectomy of s4 in the liver . macroscopically , sections of the specimen revealed a yellowish - white encapsulated solid tumor measuring 15 mm in size ( fig . 6a ) . a macroscopic appearance of the resected s4 specimen revealed a yellowish - white encapsulated solid tumor measuring 15 mm in size ( a ) . microscopically , the growth pattern of the s4 liver tumor showed expansive growth with extracapsular invasions [ b , hematoxylin and eosin ( h&e ) staining with a low - power field ] . tumor cells had an increased nuclear / cytoplasmic ratio , polymorphic , and chromatin - rich nuclei , and the pathological diagnosis was poorly differentiated hcc ( c , h&e staining with a high - power field ) . the growth pattern of the liver tumor showed expansive growth with extracapsular invasions ( fig . the tumor cells had an increased nuclear / cytoplasmic ratio , polymorphic , and chromatin - rich nuclei , and the pathological diagnosis was poorly differentiated hcc ( fig . the non - cancerous area of the resected specimen revealed that bridging fibrosis was observed without fat deposition , and the patient was diagnosed with liver cirrhosis ( fig . fibrosis was observed in both the portal and pericellular areas , the sinusoidal structure was maintained , but no signicant inflammation was observed ( fig . the noncancerous area of the resected specimen revealed bridging fibrosis without fat deposition , and the patient was diagnosed with liver cirrhosis [ silver impregnation ( a ) ] . fibrosis was observed in both the portal and pericellular areas , the sinusoidal structure was maintained , and signicant inammation was not seen [ silver impregnation ( b , c ) with a high - power field ] . after discharge from the hospital , the patient was followed - up at 3-month intervals . at the 1-year follow - up , her afp level was 2.7 ng / ml , afp - l3 was less than 0.5% , and the tumor markers remained normal . in the present case , there were no known etiological factors such as hepatitis viral infection , alcoholic liver disease , autoimmune liver diseases , autoimmune hepatitis , primary biliary cirrhosis , metabolic liver diseases [ such as non - alcoholic steatohepatitis ( nash ) ] , or medication with hepatotoxic drugs ( such as amiodarone ) . moreover , significant hepatic inflammation or fat deposition was not seen as a result of processes such as hepatitis viral infection or nash . on histology in the present case , hepatic sinusoidal fibrosis extending from centrilobular areas toward the portal tract without inflammation were observed . schwarz et al . discussed that hepatic fibrosis after the fontan procedure was a mixed disease that affects both the portal and centrilobular areas in liver biopsy and autopsy specimens ( 6,7 ) . sinusoidal fibrosis is believed to result from an increase in central venous pressure transmitted to hepatic cells that surround the central veins , because the extent of cirrhosis is strongly correlated with elevated hepatic venous pressures and a low cardiac index in patients after undergoing the fontan procedure ( 8) . after fontan palliation , a significant liver disease can result in central venous congestion and hypoxia resulting from a low cardiac output , however , little is known regarding fibrogenic mechanisms independent of the inflammation - mediated pathway in congestive liver disease ( cld ) . in cld , mechanotransduction associated with stretching and compression of hepatic stellate cells may be a potent inducer of hepatic fibrosis ( 9,10 ) . using a newly characterized murine cld model , sinusoidal thrombosis and mechanical stretching of adjacent hepatic stellate cells caused by sinusoidal dilatation was shown to induce the release of fibronectin by hepatic stellate cells , and both fibrin and stretching stimulated fibronectin fibril assembly through a 1-integrin and actin - dependent mechanism ( 11 ) . in addition , hepatic complications after the fontan procedure are associated with the duration of follow - up ( 8,12 ) . progression to cirrhosis may even be observed within 10 years after the initial fontan procedure ( 13 ) . in 34 patients with a median follow - up of 11.5 years after the fontan procedure , 30% experienced abnormal transaminases , 61% abnormal -gtp , 32% abnormal bilirubin , and 58% coagulopathy ( 14 ) . as compared to a duration of 0 - 5 years , the odds ratio of hepatic complications was 4.4 for a post - fontan duration of 11 - 15 years and 9.0 for a duration of 16 - 20 years , respectively ( 12 ) . liver cirrhosis is a potential prerequisite for hcc , however , the prevalence and progression of cirrhotic changes in the fontan population have not been clearly identified . non - invasive diagnostic tools for hepatic fibrosis are needed , because a liver biopsy , the golden standard for diagnosing liver cirrhosis , is difficult to perform in fontan patients due to prophylactic anticoagulation . similar to the present case , a radiological assessment of liver fibrosis using various methods such as us , ct , or mri may be useful . as shown in table 2 , a recent pubmed search identified 12 cases of hccs among published reports . the publications described the use of surgical resection , transcatheter arterial chemoembolization , radioembolization , local ablation , or sorafenib therapy ( 15 - 22 ) . in previous reports , two patients were treated with surgical resection ( 20,21 ) . for early stage hcc , surgical resection provides curative treatment with a long - time survival , however , hepatectomy is rarely performed following the fontan procedure because it is difficult to detect early stage hccs . although the present patient did not receive periodic surveillance for hcc , such as us and afp , it is fortunate that early stage hcc was incidentally detected . hcc detected after the onset of symptoms has a poor prognosis ( 5-year survival rate of 0 - 10% ) . in contrast , early stage hccs detected by surveillance can be cured with both surgical resection and liver transplantation ( 5-year disease - free survival rate > 50% ) ( 23 ) . f : female , m : male , nd : not described , tace : transarterial chemoembolization , tae : transarterial embolization thus , surveillance for hcc may be necessary in patients with cld who undergo the fontan procedure because cirrhosis is a high - risk factor for hcc . however , the screening interval for liver disease after the fontan procedure has not yet been established . surveillance is based on an ultrasound examination , and the recommended screening interval is 6 months according to the american association for the study of liver diseases ( aasld ) practice guidelines on the management of hcc ( 24 ) . ( 25 ) , all patients with high - risk risk factors for hcc are advised to undergo periodic surveillance with us and laboratory work ups , including afp and protein induced by vitamin k absence or antagonists - ii ( pivka - ii ) , every 6 months . however , similar to the present case , pivka - ii is not useful in most patients after the fontan procedure because prophylactic antiplatelet with anticoagulation therapies , such as warfarin administration , are necessary to prevent thromboembolic events , which are one of the major causes of morbidity and mortality ( 26 ) . following the fontan procedure , patients face a risk of hcc and require a lifelong follow - up with not only a pediatric cardiologist , but also a hepatologist experienced in the care of patients with liver cirrhosis . we herein described a patient with hcc who was able to safely undergo liver resection following the fontan procedure under a preserved cardiac and hepatic function . this case describes a young non - pregnant cat that presented with uterine prolapse in association with an unusual diffuse , polypoid , fibrosing perimetritis and parametritis . no intra - abdominal complications were seen on ultrasound examination 3 months postsurgery . at the time of writing , uterine prolapse in the cat is relatively rare and usually associated with the periparturient period . inflammatory polypoid perimetritis and parametritis have not previously been documented in cats , and in dogs have only been reported in association with the administration of oestrogenic compounds . the polypoid inflammation affecting the uterus and parametrium may have contributed to increased laxity of the uterine ligaments and predisposed to the development of uterine prolapse . uterine prolapse is rare in the queen , usually occurring after delivery of kittens or abortion . we report uterine prolapse in a non - pregnant queen and in association with an unusual diffuse , polypoid , fibrosis of the perimetrium and parametrium . the perimetrium ( syn uterine tunica serosa ) is equivalent to the visceral peritoneal lining . it is composed of loose connective tissue containing nerves and smooth muscle cells covered by mesothelium . it blends in with the broad ligaments of the uterus , which is also covered by mesothelium and contains a core of connective tissue , the parametrium . while peritonitis in cats is frequently associated with feline infectious peritonitis , specific lesions unique to the perimetrium have not been documented . a stray 6- to 7-month - old domestic shorthair queen , weighing 2.15 kg , was presented to the university veterinary clinic with uterine prolapse after being observed for several days prior to capture . on examination , both uterine horns were prolapsed , everted and swollen , with regions of hyperaemia , particularly towards the tips of the uterine horns . no evidence of placental zoning or tissue necrosis , in particular gangrene , was noted ( figure 1 ) . prolapsed and everted uterus following lavage which revealed swollen , hyperaemic and viable tissue clinical assessment revealed normal appetite and demeanour . the vital parameters were within normal limits ( temperature 37.8c , respiratory rate 40 breaths / min , heart rate 140 beats / min ) . the cat received hydromorphone hydrochloride injection ( hydromorphone ; west - ward pharmaceutical ) at 0.1 mg / kg iv q4h for pain management and to facilitate further examination . initial haematological and blood biochemical evaluations revealed a markedly regenerative anaemia ( packed cell volume 24% [ reference interval ( ri ) 2545% ; erythrocytes 4.64 10/l [ ri 510 10/l ] , reticulocytes 375.84 10 /l [ ri 040 10/l ] ) and a neutrophilic leukocytosis with mild toxic changes ( leukocytes 37.72 10/l [ ri 5.519.5 10/l ] ; neutrophils 25.3 10/l [ ri 2.512.5 10/l ] ) . blood biochemistry showed a mild hyperkalaemia ( 6.0 mmol / l [ ri 3.75.8 mmol / l ] ) and mild hypoglycaemia ( 66 mg / dl [ ri 70150 mg / dl ] ) . oestrogen plasma concentrations were retrospectively evaluated at 25.7 pg / ml ( ri 1540 pg / ml ) . intravenous crystalloid fluid solution ( plasmalyte ; travenol laboratories ) was administered to correct an estimated 8% dehydration deficit and maintenance requirement over 12 h. antibiotic therapy consisting of intravenous cefazolin sodium ( cefazolin for injection ; west - ward pharmaceutical ) at a dose rate of 22 mg / kg iv q8h was also administered . attempts at reducing the uterus were again unsuccessful so an internal ovariectomy and external hysterectomy were performed . the ovarian pedicles were transfixed and ligated prior to transection and before manipulating the uterine body further . the everted uterus was circumferentially ligated distal to the cervix ( approximately 5 cm away from the vulva ) and transected . the remaining uterus was returned to the abdominal cavity . from within the abdominal cavity , the serosa of uterine body was noted to be diffusely thickened by multiple - to - coalescing white villous projections ( figure 2 ) . formalin - fixed perimetrial surface of uterine body revealing diffuse - to - coalescing polypoid lesions there was a small amount of mildly flocculent abdominal fluid present . the cat recovered uneventfully and follow - up examinations in a 6 month period , including ultrasound at 3 months postsurgery , detected no abdominal abnormalities . microscopic examination of the uterine body confirmed the presence of multiple nodular perimetrial proliferations of moderately loose connective tissue forming irregular polyps or plaques blending into the myometrium ( figures 3 and 4 ) . most polyps contained multifocal randomly distributed lymphocyte and plasma cell infiltrations , and regions of neovascularisation . polyps were covered by a combination of normal , attenuated and moderately swollen mesothelial cells . at sites of mesothelial cell loss , the stratum vascularis was moderately oedematous containing both congested medium - to - large size blood vessels and dilated lymphatics . endometrium and uterine epithelium appeared unremarkable with a low glandular content , intact mucosal epithelium and minimal - to - no inflammatory cell infiltrations . the lesion was characterised as a diffuse , chronic , polypoid , fibrotic perimetritis and parametritis . cross - section of uterine body ( u ) revealing multiple polypoid perimetrial projections ( p ) ( haematoxylin and eosin ) polypoid structures blending with myometrium and containing ( a ) foci of angiogenesis and ( b ) regions of active fibroplasia ( haematoxylin and eosin ) uterine prolapse has rarely been documented in cats , with individual case reports citing complete or unilateral uterine prolapse with or without uterine mucosal eversion . the majority have occurred in association with the periparturient period , one of which was proposed to be associated with oestrus . furthermore , a novel lesion inducing extensive perimetrial fibrosis , not previously reported in cats , was noted and could potentially be implicated as the cause . however , other factors have been suggested including uterine atony or excessive abdominal contractions , including tenesmus . for the uterus to prolapse there is a requirement for laxity , stretching or rupture of uterine ligaments , and for patency of the cervix . laxity of the uterine ligaments can occur due to repeated pregnancy , and changes in progesterone , oestrogen and relaxin concentrations near parturition . cervical patency occurs at two time points , oestrus ( with or without ovulation ) and at parturition . based on the absence of any newborn animals , no mammary development and no histopathological features to suggest recent pregnancy , such as placental zoning , recent uterine haemorrhage , inflammation and endometrial regeneration , it is unlikely this animal was in advanced pregnancy or had aborted . it was not possible , however , to establish if this animal had recently been in oestrus as oestrogen levels , which were within normal limits , can vary significantly over short periods of time within the oestrus cycle . while the uterus was large , this latter feature could potentially have contributed to uterine prolapse in at least two ways , namely by inducing ligament laxity and/or acting as a caudal abdominal irritant inducing abdominal contractions or tenesmus . while an active lesion , the extent of mature fibrous tissue would suggest the presence of this lesion in advance of the uterine prolapse . perimetrial inflammation can co - exist as part of peritonitis , and in cats peritonitis is frequently associated with feline infectious peritonitis or primary bacterial septic peritonitis where bacteroides species and fusobacterium species are mainly isolated . however , such lesions do not manifest as polypoid fibrous proliferations . similar proliferative fibrotic lesions focused on the perimetrium have been described in dogs associated with experimental stilboestrol ( synthetic oestrogen ) administration . perimetrial papillae consisting of proliferations of mesothelial cells and subjacent connective tissue were documented in ovariectomised dogs administered stilboestrol over 150350 days . similar to the present case , such lesions were confined to genital organ serosal surfaces , including the broad ligament . such perimetrial lesions were also noted in entire female dogs when administered synthetic oestrogens in association with other lesions , including oestrogen - induced renal fibrosis . proposed mechanisms of regional fibrous proliferations suggested involvement of stromal oestrogen receptors . while the cat develops oestrogen - associated pathology , similar perimetrial pathology has not been documented in association with oestrogen . the ovaries were not submitted for histopathological evaluation to eliminate ovarian pathology definitively ( cystic follicular disease , ovarian neoplasia ) as a potential source of hyperoestrogenism . cats in the tropics are likely to cycle continuously and it is recognised that some unmated cats may appear to be in persistent oestrous , owing to a relatively short interoestrous period and thus remain in a physiological hyperoestrogenic state . it was also unlikely this cat had access to exogenous oestrogenic compounds . in humans , more generalised intra - abdominal , fibrosing lesions of peritoneal surfaces ( intra - abdominal fibromatosis ) are described in mesenteric , retroperitoneal and pelvic locations . sclerosing peritonitis has been noted after surgery , following long - term continuous peritoneal dialysis and also as a side effect of utilising beta - blockers . the pathogenesis of these lesions is thought to be the result of a delayed healing response due to loss of mesothelial cells or continued serosal irritation . however , the appearance is not described as polypoid and ranges through a spectrum of gross changes in the appearance of the visceral peritoneum , from opacification of the serosa ( disorganised collagen fibres and stromal expansion ) through to tanned peritoneum ( hyalinised collagen , fibrin and vessel sclerosis ) , which can progress to a stage where the viscera are palpably stiffened ( mural fibrosis ) . the unique perimetritis and parametritis observed in this case highlights an additional potential cause of uterine prolapse and warrants that , in cases of uterine prolapse in non - pregnant animals , perimetrial and parametrial tissue should be evaluated for varying degrees of inflammation .

a 29-year - old woman who underwent the fontan procedure at 10 years of age had an incidental finding of liver masses on abdominal ultrasonography . subsequent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid magnetic resonance imaging showed a 15 mm hypervascular mass with washout in the hepatobiliary phase in liver segment 4 ( s4 ) , and an 18 mm hypervascular mass without washout in the hepatobiliary phase in liver segment 2 ( s2 ) . the s2 liver mass was pathologically diagnosed to be a regenerative nodule by an ultrasound - guided needle biopsy , and the s4 liver mass was pathologically diagnosed as a poorly differentiated hepatocellular carcinoma after partial hepatectomy . case summarythis case describes a young non - pregnant cat that presented with uterine prolapse in association with an unusual diffuse , polypoid , fibrosing perimetritis and parametritis . following ovariohysterectomy the cat recovered fully . no intra - abdominal complications were seen on ultrasound examination 3 months postsurgery . at the time of writing , the cat remains healthy.relevance and novel informationuterine prolapse in the cat is relatively rare and usually associated with the periparturient period . inflammatory polypoid perimetritis and parametritis have not previously been documented in cats , and in dogs have only been reported in association with the administration of oestrogenic compounds . the polypoid inflammation affecting the uterus and parametrium may have contributed to increased laxity of the uterine ligaments and predisposed to the development of uterine prolapse .

some gram - negative bacteria could express extensive multidrug resistance and significantly increase the rate of nosocomial infections in the hospital ( 1 ) . among these bacteria previous studies showed that a. baumannii was already an emerging pathogen in the respiratory tract , wounds , blood and urine ( 3 ) . since the strains of a. baumannii were usually resistant to most available drugs , except colistin and tigecycline , they would be very sticky when detected on a patient . therefore , the urgent need to study the epidemiology of resistance , surveillance and control measures for a. baumannii in hospital infections was warned ( 4 ) . acinetobacter baumannii could be found in diverse sources such as food , water and soils . acinetobacter baumannii strains are able to survive for a long time even on a dry surface and some of them could toughly attach to human epithelial cells through fimbriae or side chains ( 5 ) . these characteristics indicate that a. baumannii could easily spread via surface , air or any other possible pathways . moreover , many studies showed that in a certain section of the hospital there was only one type of a. baumannii ( 1 , 6 ) . the sources of the harmful a. baumannii may be found by analyzing the molecular epidemiology of the strain in time . however , there are not enough studies on this subject . on the other hand , although physicians and surgeons all reported the threat of a. baumannii , most of the raised problems were from internal medicine or icu . the differences may be caused by the selective pressure from indiscriminate use of broad - spectrum antibiotics in different clinical departments ( 7 ) . analysis may lead to a new explanation for the formation and spread of carbapenem - resistant acinetobacter baumannii ( crab ) or carbapenem - susceptible acinetobacter baumannii ( csab ) ( 8) . however , by now few researches compared the differences of the distribution in different departments . the current study aimed to analyze the distribution of certain types of a. baumannii in clinical departments . authors tried to figure out the roles of the antibiotic - resistant genes and the therapies for the diseases in different departments with spread of crab and csab . eighty - one patients from three wards as icu , department of respiration medicine , department of burn and plastic surgery were studied . the current study was approved by the medical ethics committee of zhejiang university , shaoxing , china . all strains were identified by the vitek32 automatic bacteria identification system ( m - d corporation , us ) . the bacteria were cultured on mueller - hinton ( mh ) agar medium ( oxoid , uk ) and lb broth medium ( shenggong , china ) ( 2 ) . cultured isolations were collected and solved in boiling double distilled water for 10 minutes and then centrifuged at 12000 rpm . the supernatant was collected and used as the template for further mlst reaction . the housekeeping genes of ab ; glta , gyrb , gdhb , gpi , and rpod were selected as the standard template ( 9 , 10 ) . the sequences of the primers were designed according to those of the previous studies ( 3 , 11 ) ( table 2 ) . all primers were designed by jinsi rui corporation , china , and induced by rt - pcr kits ( drr063a , takara , japan ) . polymerase chain reaction ( pcr ) was performed as follows : initial denaturation , 95c for 5 minutes ; denaturation , 95c for 1 minute ; annealing , 56c for 30 seconds ; extension , 72c for 45 seconds . after the pcr reaction , the products were gathered and purified by pcr purification kit ( axygen , ap - pcr-250 , silicon , us ) . the test results were used to identify the strains collected from clinical specimens by referring to the ab mlst database ( 7 - 9 ) . the evaluation of antibiotic - resistance was induced by kirby - bauer test ( k - b ) ( 10 ) . a disk was impregnated with the antibiotic - compounds and then placed on the plate . concentrations of the compound were the highest next to the disk and decreased as distance from the disk increased . once the compound was effective against the bacteria at a certain concentration , no colonies would grow at the point where the concentration in the agar was greater than the effective concentration , i. e. the inhibition zone . pseudomonas aeruginosa atcc27853 and escherichia coli atcc25922 were used as the quality - control isolates ( 5 ) . the clinical and laboratory standards institute ( clsi ) standard was selected as the standard evaluation . the drug resistance ability was evaluated by studying the minimum inhibitory concentration ( mic ) by agar dilution method . the antibiotics used in the study were as follows : imipenem ( imp ) , aztreonam ( atm ) , cefepime ( fep ) , cefoperazone ( cfp ) , cefotaxime ( ctx ) , piperacillin ( pip ) , amikacin ( amk ) , and ciprofloxacin ( cip ) ( 7 ) . the target genes included methicillinase ( oxa-23 , oxa-24 , oxa-51 ) , carbapenemase ( sim , imp , spm ) , type i integrase intl 1 , and sulfonamides - resistance gene ( qace1-sul1 ) . abbreviations : f , forward primer ; r , reverse primer data were analyzed by spss software version17.0 . chi - square test was used to compare enumeration data between different groups while student s t - test for measurement data . one - way analysis of variance was used to analyze dependency of drug resistance and gene expression . eighty - one patients from three wards as icu , department of respiration medicine , department of burn and plastic surgery were studied . the current study was approved by the medical ethics committee of zhejiang university , shaoxing , china . all strains were identified by the vitek32 automatic bacteria identification system ( m - d corporation , us ) . the bacteria were cultured on mueller - hinton ( mh ) agar medium ( oxoid , uk ) and lb broth medium ( shenggong , china ) ( 2 ) . cultured isolations were collected and solved in boiling double distilled water for 10 minutes and then centrifuged at 12000 rpm . the supernatant was collected and used as the template for further mlst reaction . the housekeeping genes of ab ; glta , gyrb , gdhb , gpi , and rpod were selected as the standard template ( 9 , 10 ) . the sequences of the primers were designed according to those of the previous studies ( 3 , 11 ) ( table 2 ) . all primers were designed by jinsi rui corporation , china , and induced by rt - pcr kits ( drr063a , takara , japan ) . polymerase chain reaction ( pcr ) was performed as follows : initial denaturation , 95c for 5 minutes ; denaturation , 95c for 1 minute ; annealing , 56c for 30 seconds ; extension , 72c for 45 seconds . after the pcr reaction , the products were gathered and purified by pcr purification kit ( axygen , ap - pcr-250 , silicon , us ) . the test results were used to identify the strains collected from clinical specimens by referring to the ab mlst database ( 7 - 9 ) . the evaluation of antibiotic - resistance was induced by kirby - bauer test ( k - b ) ( 10 ) . a disk was impregnated with the antibiotic - compounds and then placed on the plate . concentrations of the compound were the highest next to the disk and decreased as distance from the disk increased . once the compound was effective against the bacteria at a certain concentration , no colonies would grow at the point where the concentration in the agar was greater than the effective concentration , i. e. the inhibition zone . pseudomonas aeruginosa atcc27853 and escherichia coli atcc25922 were used as the quality - control isolates ( 5 ) . the clinical and laboratory standards institute ( clsi ) standard was selected as the standard evaluation . the drug resistance ability was evaluated by studying the minimum inhibitory concentration ( mic ) by agar dilution method . the antibiotics used in the study were as follows : imipenem ( imp ) , aztreonam ( atm ) , cefepime ( fep ) , cefoperazone ( cfp ) , cefotaxime ( ctx ) , piperacillin ( pip ) , amikacin ( amk ) , and ciprofloxacin ( cip ) ( 7 ) . the target genes included methicillinase ( oxa-23 , oxa-24 , oxa-51 ) , carbapenemase ( sim , imp , spm ) , type i integrase intl 1 , and sulfonamides - resistance gene ( qace1-sul1 ) . data were analyzed by spss software version17.0 . chi - square test was used to compare enumeration data between different groups while student s t - test for measurement data . one - way analysis of variance was used to analyze dependency of drug resistance and gene expression . results of level of antibiotic - resistance of the isolates are shown in table 4 . all the isolates collected from the hospital under study had a certain level of resistance to all kinds of antibiotics . however , atm and imp were still relatively the most effective antibiotics . in order to further evaluate the properties of the isolates , group i , isolates resistant to more than four types of antibiotics ; group ii , isolates resistant to three or four types of antibiotics ; group iii isolates resistant to less than three types of antibiotics . it was identified that among the isolates , the ones isolated from blood had the highest level of drug - resistance . besides , among the three departments , species isolated from the icu had the highest level of drug - resistance . table 5 present distribution of the isolates in different clinical departments and clinical specimens . abbreviations : amk , amikacin ; atm , aztreonam ; cfp , cefoperazone ; cip , ciprofloxacin ; ctx , cefotaxime ; fep , cefepime ; mic , minimum inhibitory concentration ; pip , piperacillin ; mic50 , minimum inhibitory concentration required to inhibit the growth of 50% of organisms ; and mic90 , minimum inhibitory concentration required to inhibit the growth of 90% of organisms . abbreviations : bur and pla ; department of burn and plastic surgery ; icu , intensive care unit ; and res , department of respiratory medicine . percentage refers to the rate in total number ; for example , 39 ( 81.25 ) means 39 cases out of 48 , 39/48 = 81.25% . groups ; the groups i , ii , iii refer to the group classified by the study group . according to mlst analysis , two types of ab strains were sorted and identified from the clinical samples ; st208 and st218 . the detailed distribution of the two strains in clinical departments and specimens are listed in table 6 . the st208 isolates were distributed mainly in icu and respiration medicine ; while st218 in the burn and plastic surgery department . no significant correlation was found between the types and the sources of the clinical samples ( p value > 0.05 ) . but the departments where the strains came from seemed to be significantly related ( p value < 0.05 ) . according to the results of k - p drug sensitive test , the strains were divided into two groups , which were carbapenem resistant : the carbapenem resistant ab ( crab ) and the carbapenem sensitive ab ( csab ) ; 70.21% of st208 and 69.21% of st218 included crab strains , while 15.22% of st208 and 84.78% of st218 were csab . st208 strains isolated from icu and respiration medicine department were mainly crab ; while st218 strains isolated from burn and plastic surgery departments were mainly csab . abbreviations : bur and pla , department of burn and plastic surgery , icu , department of intensive care unit ; and res , department of respiration medicine . number refers to the number of the species isolated from a certain department or sample . percentage refers to the rate in total number ; for example , 40 ( 83.3 ) means 40 cases out of 48 , 40/48 = 83.3% . number in this group has a significant difference compared with those in other groups ( p value < 0.05 ) . pcr tests revealed that the prevalence of resistance inducing genes were as follows : oxa-23 81.3% , oxa-51 61.3% , sim 18.8% , oxa-24 5.3% , imp 12.3% , intl1 57.9% and qace1-sul1 61.2% . results of the current study showed that st208/crab / oxa-23 , 51 is the main molecular phenotype of the crab strains in the hospital . drug - resistance property of the isolates was related to the expression of oxa-23 and 51 ( p value < 0.05 ) . meanwhile , in st208 , there were more chances of expressions of oxa-23 , 51 compared with those in st218 ( p value < 0.05 ) . abbreviations : crab , carbapenem - resistant ab ; and csab , carbapenem - susceptible ab . there are significant differences in this group crab compared with those in csab , p value < 0.05 . data are presented as number of the isolates ( which express the gene ) per number of the stains . results of level of antibiotic - resistance of the isolates are shown in table 4 . all the isolates collected from the hospital under study had a certain level of resistance to all kinds of antibiotics . however , atm and imp were still relatively the most effective antibiotics . in order to further evaluate the properties of the isolates , group i , isolates resistant to more than four types of antibiotics ; group ii , isolates resistant to three or four types of antibiotics ; group iii isolates resistant to less than three types of antibiotics . it was identified that among the isolates , the ones isolated from blood had the highest level of drug - resistance . besides , among the three departments , species isolated from the icu had the highest level of drug - resistance . table 5 present distribution of the isolates in different clinical departments and clinical specimens . abbreviations : amk , amikacin ; atm , aztreonam ; cfp , cefoperazone ; cip , ciprofloxacin ; ctx , cefotaxime ; fep , cefepime ; mic , minimum inhibitory concentration ; pip , piperacillin ; mic50 , minimum inhibitory concentration required to inhibit the growth of 50% of organisms ; and mic90 , minimum inhibitory concentration required to inhibit the growth of 90% of organisms . abbreviations : bur and pla ; department of burn and plastic surgery ; icu , intensive care unit ; and res , department of respiratory medicine . percentage refers to the rate in total number ; for example , 39 ( 81.25 ) means 39 cases out of 48 , 39/48 = 81.25% . groups ; the groups i , ii , iii refer to the group classified by the study group . according to mlst analysis , two types of ab strains were sorted and identified from the clinical samples ; st208 and st218 . the detailed distribution of the two strains in clinical departments and specimens are listed in table 6 . the st208 isolates were distributed mainly in icu and respiration medicine ; while st218 in the burn and plastic surgery department . no significant correlation was found between the types and the sources of the clinical samples ( p value > 0.05 ) . but the departments where the strains came from seemed to be significantly related ( p value < 0.05 ) . according to the results of k - p drug sensitive test , the strains were divided into two groups , which were carbapenem resistant : the carbapenem resistant ab ( crab ) and the carbapenem sensitive ab ( csab ) ; 70.21% of st208 and 69.21% of st218 included crab strains , while 15.22% of st208 and 84.78% of st218 were csab . st208 strains isolated from icu and respiration medicine department were mainly crab ; while st218 strains isolated from burn and plastic surgery departments were mainly csab . abbreviations : bur and pla , department of burn and plastic surgery , icu , department of intensive care unit ; and res , department of respiration medicine . number refers to the number of the species isolated from a certain department or sample . percentage refers to the rate in total number ; for example , 40 ( 83.3 ) means 40 cases out of 48 , 40/48 = 83.3% . number in this group has a significant difference compared with those in other groups ( p value < 0.05 ) . pcr tests revealed that the prevalence of resistance inducing genes were as follows : oxa-23 81.3% , oxa-51 61.3% , sim 18.8% , oxa-24 5.3% , imp 12.3% , intl1 57.9% and qace1-sul1 61.2% . results of the current study showed that st208/crab / oxa-23 , 51 is the main molecular phenotype of the crab strains in the hospital . drug - resistance property of the isolates was related to the expression of oxa-23 and 51 ( p value < 0.05 ) . meanwhile , in st208 , there were more chances of expressions of oxa-23 , 51 compared with those in st218 ( p value < 0.05 ) . abbreviations : crab , carbapenem - resistant ab ; and csab , carbapenem - susceptible ab . there are significant differences in this group crab compared with those in csab , p value < 0.05 . data are presented as number of the isolates ( which express the gene ) per number of the stains . acinetobacter baumannii is widely distributed in the environment , human body and surfaces ( 12 ) . studies show that growing numbers of carbapenem - resistant pathogenic bacteria , such as crab are now appearing . the prevention and treatment of crab is an urgent issue to control nosocomial infections ( 13 - 15 ) . in the current study , all strains of a. baumannii were resistant to antibiotics in different degrees ( table 4 ) . in fact , it is a common phenomenon in hospitals in china ( 9 ) . moreover , it was found that distribution of the crab isolates were related to the wards where they came from ( table 5 ) . isolates from icu and department of respiratory medicine had higher levels of antibiotic - resistance than the strains from the surgery department . it may be due to the long - term use of antibiotics in the former wards ( 16 - 18 ) . this finding showed that the distribution of crab may be affected by the therapeutic methods . besides , authors further studied the types of the isolates in the air and hands of the doctors and nurses in the wards . it was found that in icu only st208 existed ; while in department of burn and plastic surgery only st218 was detected . considering the fact that these two departments were physically separated , it was speculated that the two different isolates may have different origins and they were separated by the spatial distance and were only epidemic in each section . currently , techniques for molecular cloning and detection are widely used to study the types of certain isolates ( 3 , 10 - 13 ) . in the current study a new technique , mlst , was used . mlst classified the strains according to the results of accurate detection of the sequences of nucleic acid from the isolates ( 19 ) . two isolates , st208 and st218 were found in the hospital , and each one had its own territory this finding showed that a certain kind of bacteria would be dominant in a certain section . the existence of anti - carbapenemase enzymes was the key factor to the prevalence of multidrug - resistant bacteria ( 20 - 22 ) . previous reports showed that five types of anti - carbapenemase enzymes , oxa-23 , 51 , sim , imp and spm could be detected in crab ( 23 ) . the current study found that oxa-23 and 51 were the most prevalent genes in the hospital . since these antibiotic resistance inducing genes were all expressed in plasmid , their distribution was determined by gene mutation and combined effect of the antibiotics used in a certain place . thus , the final results of the distribution are the natural selection in the certain therapeutic milieu in the hospital . identification of the types and genes of the isolates in a certain ward would be important to restrict the prevalence . moreover , although many microbiologists believe that the nuclear - dna is not related to the plasmid - dna in bacteria , the current study findings indicated that in st208 there were more chances for the existence of oxa-23 , 51 ( table 8) . the fact revealed that there might be certain connections between the nuclear - gene types and the appearance of plasmid genes . , there may be different isolates of ab in different wards since these departments were physically separated . the levels of antibiotic - resistance were also quite different in the isolates from different locations of the human body . species isolates from blood culture had the highest level of antibiotic - resistance . st208 and the st218 were the epidemic isolates of ab , their abilities of drug - resistance were due to the existence of oxa-23 , oxa-51 , intl 1 and qace1-sul 1 . hospital managers should take measures to supervise the epidemiology and types of the crab isolates . the study of the molecular epidemiology of these isolates from different wards and specimens were significantly important . besides , further studies on the connections between the plasmid gene expression and types of the isolates are required . high phosphorus ( p ) intake is known as one of the risk factors for impaired bone health . several researchers have investigated the adverse effects of a high p diet on bone metabolism . in human adults , a diet containing p additives increases urinary hydroxyproline excretion , a bone resorption marker . in growing male rats , elevation in parathyroid hormone ( pth ) secretion is considered one of the mechanisms by which a high p diet impairs bone metabolism . pth stimulates osteoblasts , which produce mediators of osteoclastic bone resorption such as macrophage colony - stimulating factor ( m - csf ) , interleukin-6 ( il-6 ) , or receptor activator of nf-b ligand ( rankl ) [ 46 ] . we previously reported that a high p diet increased rankl mrna expression and the osteoclast number in rats . it could therefore be deduced that high p diet - induced elevated pth secretion leads to an increase in rankl expression , which enhances osteoclastic bone resorption . overton and basu suggested that bone loss occurs with increasing age at a rate of approximately 1% per year averaged over the age range of 2976 y . in a previous mouse study by ferguson et al . , bone mass and mechanical properties approached mature levels by 12 weeks of age while age - related osteopenia was observed after 42 weeks of age . we previously reported the effects of a high p diet on mechanical properties of the femur in 4- , 12- , 24- , and 80-week - old mice . the results showed that a high p diet decreased the breaking force of the femur in 80-week - old mice and the stiffness of the femur in 24- and 80-week - old mice . we also found that a high p diet increased serum pth concentration in 12- , 24- , and 80-week - old mice , and 80-week - old mice had a higher serum pth concentration than mice at other ages . therefore , it was thought that a high p diet strongly influences aged mice in terms of pth response . the purpose of this study was to clarify the mechanism by which a high p diet affects bone metabolism in older mice . we assessed the changes in bone metabolism in young and aged mice fed a high p diet by measuring the mrna expression of bone metabolism mediators using real - time polymerase chain reaction ( pcr ) . the mice were maintained in accordance with the university guidelines for the care and use of laboratory animals . the experimental diets were based on the ain-93 g diet ( table 1 ) . a control diet containing 0.3% p and a high p diet containing 1.2% twenty - four 10-week - old ddy male mice were purchased from slc ( shizuoka , japan ) and housed individually in stainless cages in a room maintained at 22c with a 12-hour light / dark cycle . half of the mice were fed a commercial diet ( ce-2 , clea japan , tokyo , japan ) until 78 weeks of age . after the acclimatization period , 12 young ( 12-week - old ) and 12 aged ( 80-week - old ) mice were randomly divided into two experimental groups and fed the control diet or the high p diet for 4 weeks . they were given free access to the diets and distilled water . their urine samples were collected during the 5 days prior to euthanasia for the further analyses . at the end of the experimental period , all mice were euthanized under anesthesia and blood , bone , and duodenum samples were collected for analyses . the serum ca level was analyzed by atomic absorption spectrophotometry ( hitachi a-2000 ; hitachi , tokyo , japan ) according to the method of gimblet et al . . the serum p level was analyzed by colorimetry using phospha c - test wako ( wako pure chemical industries , osaka , japan ) . serum pth concentration was measured using the mouse intact pth enzyme - linked immunosorbent assay ( elisa ) kit ( alpco , nh , usa ) . serum intact osteocalcin ( oc ) concentration was measured using the mouse osteocalcin eia kit ( biomedical technologies , ma , usa ) . urinary c - terminal telopeptide of type i collagen ( ctx ) level was measured using ratlaps eia kit ( immunodiagnostic systems , boldon , uk ) . urinary creatinine level was measured using the jaffe reaction , as described by lustgarten and wenk . total rna was isolated from the homogenized femurs or duodenum by using trizol reagent ( life technologies , ca , usa ) according to the manufacturer 's specifications . the amount and purity of the rna were assessed using a nanodrop 2000c ( thermo fisher scientific , ma , usa ) . complementary dna ( cdna ) was synthesized using the high - capacity rna - to - cdna kit ( applied biosystems , ca , usa ) . for real - time pcr , the reaction mixture was prepared using the taqman gene expression master mix ( applied biosystems ) with taqman gene expression assays ( applied biosystems ) for mouse pth receptor ( assay i d : mm00441046_m1 ) , mouse rankl ( assay i d : mm00441906_m1 ) , mouse osteoprotegerin ( opg ) ( assay i d : mm01205928_m1 ) , mouse tartrate resistant acid phosphatase ( trap ) ( assay i d : mm00475698_m1 ) , mouse runt related transcription factor 2 ( runx2 ) ( assay i d : mm00501580_m1 ) , mouse osterix ( assay i d : mm04209856_m1 ) , mouse alkaline phosphatase ( alp ) ( assay i d : mm00475834_m1 ) , mouse osteopontin ( opn ) ( assay i d : mm00436767_m1 ) , mouse oc ( assay i d : mm03413826_mh ) , mouse type i collagen ( col1a1 ) ( assay i d : mm00801666_g1 ) , mouse transient receptor potential vanilloid type 6 ( trpv6 ) ( assay i d : mm00499069_m1 ) , mouse calbindin - d9k ( assay i d : mm00486654_m1 ) , mouse plasma membrane ca - atpase 1b ( pmca1b ) ( assay i d : mm01245805_m1 ) , and mouse glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) ( assay i d : mm99999915_g1 ) . real - time pcr was performed using a stepone real - time pcr system ( applied biosystems ) . the value of the young mice fed the control diet was considered to be 1.00 . after two - way analysis of variance ( anova ) , fisher 's protected least significant difference ( plsd ) test was used to determine significant differences among the groups . in both young and aged mice , there was no significant difference in the initial body weight between mice fed the control and high p diets ( table 2 ) . the initial body weight of aged mice was significantly higher than that of young mice . there were no significant differences in serum ca and p concentrations among the groups ( table 2 ) . in both young and aged mice , although there was no significant difference in serum pth concentration between young and aged mice fed the control diet , serum pth concentration was significantly higher in aged mice than in young mice fed the high p diet . in both young and aged mice , the high p diet significantly increased serum oc concentration ( table 2 ) . in mice fed both the control and high p diets , serum oc concentration was significantly lower in aged mice than in young mice . in both young and aged mice , although there was no significant difference in urinary excretion of ctx between young and aged mice fed the control diet , urinary excretion of ctx was significantly lower in aged mice than in young mice fed the high p diet . in both young and aged mice , the high p diet significantly increased mrna expression of pth receptor , rankl , trap , runx2 , osterix , alp , opn , oc , and col1a1 ( figure 1 ) compared to the control diet . in mice fed the control and high p diets , mrna expression of pth receptor , rankl , trap , runx2 , osterix , alp , opn , oc , and col1a1 was significantly lower in aged mice than in young mice . the high p diet significantly increased rankl / opg ratio in aged mice but did not in young mice . in both young and aged mice , high p diet significantly increased mrna expression of trpv6 , cabp9k , and pmca1b ( figure 2 ) compared to the control diet . in mice fed the control and high p diets , mrna expression of trpv6 and cabp9k was significantly lower in aged mice than in young mice . in humans , bone loss occurs with increasing age at a rate of approximately 1% per year averaged over the ages of 2976 years . therefore , aging is one of the risk factors for bone loss . in a previous mouse study , bone mass and mechanical properties were shown to approach mature levels by 12 weeks of age , and age - related osteopenia was observed after 42 weeks . in this study , we investigated bone metabolism by measuring markers of bone formation and resorption , serum oc concentration , and urinary ctx excretion . serum oc concentration was significantly lower in aged mice than in young mice fed the control diet , whereas there is no difference in urinary excretion of ctx between young and aged mice fed the control diet . these results showed that aged mice present a decrease in bone formation , and it appears that the balance between bone formation and resorption may be disrupted in aged mice . bone formation is mediated by osteoblasts . using gene - deficient mouse models , runx2 and osterix were shown to be essential transcription factors for osteoblast differentiation and bone formation [ 15 , 16 ] . in this study , runx2 and osterix mrna expression were significantly lower in aged mice than in young mice . these results suggest that aging leads to a reduction in osteoblast differentiation and that runx2 and osterix mrna expression changes are associated with a decrease in bone formation in aged mice . consequently , decreases in mrna expression of alp and bone matrix proteins such as opn , oc , and col1a1 also occurred in aged mice . ikeda et al . showed that the mrna expression of opn , oc , and col1a1 decreased in both cortical and trabecular bones in aged rats compared to young animals . in addition , cao et al . reported that alp and col1a1 expression declined in aged mice compared to young mice . thus , aging results in a decrease in bone formation with declined osteoblast - related gene expression . with regard to bone resorption , this study showed that rankl and trap mrna expression were decreased in aged mice compared to young mice , despite unchanging serum pth concentration . since pth stimulates rankl , the result of rankl mrna expression seems to contradict that of serum pth concentration . however , pth receptor mrna expression was decreased in aged mice compared to young mice in this study . this result suggested that pth action was suppressed , which decreased rankl mrna expression in aged mice . though urinary excretion of ctx was unchanged , expression of bone resorption - rerated genes was decreased in aged mice compared to young mice . however , it is generally known that age - related increase in serum pth contributes to the increase in bone resorption . therefore , results of serum pth concentration and bone resorption between young and aged mice in this study are contradictory . further studies with the increase in number of mice per group are needed to address these discrepancies . a decline in intestinal ca absorption is also one of the causes of age - related bone loss . the transcellular ca pathway , which is affected by 1,25-dihydroxyvitamin d ( 1,25(oh)2d ) , has been proposed to involve ca entry via trpv6 , intracellular diffusion of ca by calbindin - d9k , and basolateral extrusion of ca by pmca1b . in this study , trpv6 and calbindin - d9k mrna expression in the duodenum were decreased in aged mice compared to young mice . demonstrated that plasma 1,25(oh)2d , duodenal calbindin d protein , and ca absorption decreased with age in rats . from this study therefore , a decrease in serum 1,25(oh)2d might reflect our results on trpv6 and calbindin - d9k mrna expression . many studies have reported that high p intake induces an increase in serum pth concentration in humans [ 1 , 23 ] and animals [ 2 , 3 , 24 ] . in this study , the high p diet increased serum pth concentration in both young and aged mice and was greater in aged mice than in young mice fed the high p diet . similar to our previous study , these results suggest that the response to a high p diet in terms of pth secretion might be different and greater with aging . kidney function decreases with age , and declining kidney function causes an increase in pth secretion . . therefore , the combination of aging and high p diet might be one of the reasons that higher serum pth concentration was observed in aged mice fed the high p diet . we previously reported that a high p diet increased rankl mrna expression and bone resorption in growing rats . therefore , it was suggested that elevated pth secretion induced by the high p diet led to an increase in rankl expression , which increased bone resorption . in this study , the high p diet significantly increased urinary excretion of ctx in both young and aged mice . regarding mrna expression of bone resorption - related molecules in the femora , the high p diet significantly increased rankl and trap mrna expressions in both young and aged mice . rankl actions are inhibited by opg , which acts as a decoy receptor by blocking rankl binding to its receptor . in this study , the high p diet significantly increased the rankl / opg ratio in aged mice , whereas the ratio was unchanged in young mice . many reports have supported the assertion that the increase in rankl / opg ratio promotes osteoclastogenesis , accelerates bone resorption , and induces bone loss . our previous study showed that a high p diet decreased the breaking force and stiffness of the femur in aged mice compared to young mice . our findings show that the high p diet in the aged mice leads to increased pth secretion and consequent increases in the rankl / opg ratio accelerating osteoclastogenesis . previous studies showed that pth regulates runx2 and osterix mrna expression [ 30 , 31 ] . in this study , the high p diet significantly increased serum oc concentration and mrna expression of runx2 , osterix , alp , opn , oc , and col1a1 in both young and aged mice . from the results of bone resorption and formation markers , high bone turnover with resorption exceeding formation since high bone turnover is a risk factor for bone fracture and osteoporosis , a high p diet might be a risk factor for bone loss not only in young mice but also in aged mice . pth secretion might reflect a decrease in ca absorption in the high p diet group . although the mechanism underlying the high p diet - induced decreased ca absorption remains unknown , it is thought that the formation of insoluble ca and p salts in the intestinal lumen is an important factor . our previous study also showed that high p diet decreased ca absorption in female rats . while we did not evaluate ca absorption in this study , it is possible to estimate the decrease in ca absorption by high p diet in young and aged mice . however , this study showed that the high p diet significantly increased mrna expression of trpv6 , cabp9k , and pmca1b in both young and aged mice . previous study demonstrated that ca restriction during lactation stimulated ca - binding protein and active ca transport in jejunum and ileum . furthermore , ca deficient diet resulted in an increase in duodenal pmca mrna in chickens . these studies suggested that 1,25(oh)2d - regulated ca transporters might be stimulated by low ca status in the intestinal lumen . thus , a decrease in soluble ca induced by high p diet might lead to trpv6 , cabp9k , and pmca1b mrna expression , and these increases in 1,25(oh)2d - regulated gene expression seem to compensate for a decrease in ca absorption by the high p diet . in brief , our results suggest that high p diet accelerates the transcellular ca pathway , though absorbed amount of ca was insufficient to maintain serum pth concentration . it is known that fibroblast growth factor 23 ( fgf23 ) and 1,25(oh)2d as well as pth are key factors for ca and p metabolism . therefore , measuring serum fgf23 and 1,25(oh)2d is important to fully elucidate the mechanisms by which high p diet changes ca and p metabolism , and further studies are needed to clarify the details . in conclusion , we demonstrated that the high p diet increased bone metabolism - related gene expression in both young and aged mice . furthermore , the high p diet affected pth secretion differently in young and aged mice , leading to an increase in the rankl / opg mrna ratio in aged mice .

background : acinetobacter baumannii is the most prevalent strain in hospitals and different clinical departments.objectives:the current study aimed to investigate the genetic characteristics and resistance mechanisms of a. baumannii isolated from clinical samples in shaoxing people s hospital affiliated to zhejiang university , shaoxing , china.patients and methods : acinetobacter baumannii strains were isolated from blood , phlegm and skin of the patients hospitalized in different departments as respiratory medicine , plastic surgery and intensive care unit ( icu ) . multilocus sequence typing ( mlst ) was used to characterize the isolates . kirby - bauer test was used to evaluate antibiotic resistance of the bacteria . the expression of resistance inducing genes was detected by reverse transcription polymerase chain reaction ( rt - pcr ) . the results were analyzed and compared.results:two bacterial types , st208 , and st218 , were identified in all 140 samples . the st208 mainly came from icu and department of respiratory medicine , while st218 from department of plastic surgery ; 70.21% of st208 and 84.78% of st218 were carbapenem - resistant acinetobacter baumannii ( crab ) and carbapenem - susceptible acinetobacter baumannii ( csab ) , respectively . multidrug - resistance genes in crab isolated from the hospital mainly included , oxa-23 , oxa-5 , intl 1 and qace1-sul 1 . besides , the highest and lowest antibiotic resistance was observed in the strains isolated from blood samples and wounds , respectively.conclusions:the distribution of ab varies in different clinical departments and samples . in the hospital under study , the main types of ab were st208 and st218 . the genes which affect the ability of antibiotic - resistance were oxa-23 , oxa-51 , intl 1 and qace1-sul 1 . in this study , the effects of high phosphorus ( p ) diet on bone metabolism - related gene expression were investigated in young and aged mice . twelve- and 80-week - old ddy male mice were divided into two groups , respectively , and fed a control diet containing 0.3% p or a high p diet containing 1.2% p. after 4 weeks of treatment , serum parathyroid hormone ( pth ) concentration was significantly higher in the high p groups than in the control groups in both young and aged mice and was significantly higher in aged mice than in young mice fed the high p diet . high p diet significantly increased receptor activator of nf-b ligand ( rankl ) mrna in the femur of both young and aged mice and significantly increased the rankl / osteoprotegerin ( opg ) mrna ratio only in aged mice . high p diet significantly increased mrna expression of transient receptor potential vanilloid type 6 , calbindin - d9k , and plasma membrane ca2 + -atpase 1b in the duodenum of both young and aged mice . these results suggest that high p diet increased rankl mrna expression in the femur and calcium absorption - related gene expression in the duodenum regardless of age . furthermore , the high p diet - induced increase in pth secretion might increase the rankl / opg mrna ratio in aged mice .

despite advancements in modern medical science and the health management industry , the incidence of cerebral palsy ( cp ) continues to rise1 . the most common features of cp are decreased muscle strength and abnormal muscle tone2 . cp sufferers lack theability to generate enough force to maintain antigravity postural control , which result in abnormal postures3 . the development of movement and posture may be altered by non - progressive damage to the brain and subsequent neurological impairments ( spasticity , muscle weakness , co - contractions and visual impairment)5 . studies indicate that children and adults with both mild and severe forms of cp have postural impairments6,7,8 . the emergence of sitting postural control in early infancy changes the way infants interact with the world . from the sitting position , looking , reaching , and interacting become functional and allow exploration that supports learning and further development of motor skills . therefore , independent sitting , defined as not needing support from a caregiver or pillow while sitting , is one of the first developmental goals for every child . individual differences are present between children , and characteristic signs of developmental disorders during infancy are relatively unspecific . therefore , why a specific child is not able to achieve sitting postural control is not always clear . one method of examining postural control in adults and children is to measure the center of pressure ( cop ) at the base of support using a force platform during the task of remaining upright . cop has frequently been used to investigate postural control during standing by young children who are healthy or have cp9 , 10 . the purpose of this study was to investigate the differences of the pressure distributions of the sitting postures of typical developmental ( td ) children and children with cp . twelve cp childrens were recruited from an outpatient rehabilitation clinic . they were hemiparetic , had an mmse - k score above 2411 , could maintain an independent sitting posture without support . two of the cp children were subsequently excluded because they refused to participate in this experiment . all parents of the enrolled participants provided their written informed consent to their children s participation prior to this experiment , in accordance with the ethical principles established in the declaration of helsinki . as a result of the exclusion , this study used two school chairs mounted on a force platform to assess the quiet - sitting pressure distribution of the subjects . fsa seating assessment ( canada ) the acquisition frequency was set at 5 hz . the stated working range of the device is 0200 mmhg , with a resolution of 1 mmhg . the system was also calibrated to assign absolute pressure values to the digital output from an a / d converter connected to the sensing pad . this was done by applying a pressure distribution as similar to actual conditions as possible . then thus subjects sat on one of two school chairs according to their height . the chairs were those generally used in school . for research purposes , this study used two basic school chair because students spend a long time of day - to - day sitting on them . one chair had a 40 cm floor to seat height , a 35 cm seat depth , and a 32 cm seat width and is designed for 122.4133.5 cm height of subjects . the another had a 35 cm floor to seat height , a 38 cm seat depth , and a 35 cm seat width , and is designed for 133.6152.7 cm height of subjects . it has been used for the posture symmetry in other study13 statistical analyses were performed using pasw 18.0 . descriptive statistics were calculated ( frequency , mean , standard deviation , range ) . the mann - whitney u - tests and wilcoxon s signed rank tests were used to analyze differences between the groups and differences in lesion side , respectively . the si of the age matched td group was employed as the normal criteria . table 1table 1.general characteristics of the subjectscerebral palsygroup ( n=10)typicaldevelopmentalgroup ( n=10)age ( years)8.040.827.840.94gender ( male / female)5/54/6lesion side ( right / left)4/6symmetry index ( meansd)5.041.342.302.76 shows the general characteristics of the participants . the si of the td group was significantly closer to zero than that of the cp group of children . cp and right hemiparetic cp were respectively 4.96 ( 2.24 ) and 5.12 ( 0.83 ) with no significant difference between the hemiparetic sides . the purpose of the present study was to investigate the differences in sitting posture of children with cp while they sat on school chairs . it is known that dynamic postural control during sitting can be reliably assessed using cop data of infants who are developing typically or infants with or at risk of cp14 . the principal finding of this study is that cp children s sitting posture is asymmetrical , leaning to the less - paretic side . we thought that infants who are developing typically develop the ability to sit by exhibiting an optimal range of movement variability , whereas cp may present either too much or too little variability leading to a very rigid and narrow or unpredictable set of movement solutions to achieve independent sitting . the finding of this study is agreement with previous studies that have the dissimilarities of the cop patterns of infants with cp and td have been obviously demonstrated14 . the results can not be generalized to all cp children because the sample was limited to ten children and they were at the one stage of the developmental process . future studies should assess the relationship between symmetry of sitting posture and functional activities . bioethanol is one of the most employed liquid biofuels due to the easy adaptability of this fuel to existing engines and because this is a cleaner fuel with higher octane rating than gasoline . ethanol market grew from less than a billion liters in 1975 to more than 39 billion liters in 2006 and is expected to reach 100 billion liters in 2015 . among the widely used substrates for ethanol production are the molasses , the wastes byproduct of sugar industries from sugarcane and sugar beet . this is because they are cheap raw materials , readily available , and ready for conversion with limited pretreatments as compared with starchy or cellulosic materials , as all sugars are present in a readily fermentable form . ongoing research and development seeking to improve methods by minimizing the numbers of experiments provide information about the direct additive effects of the study variables and interaction effects using design of experiment methods . recently , this statistical technique has been successfully applied in many fields [ 58 ] . response surface methodology ( rsm ) is a combination of mathematical and statistical techniques and is used for the modeling and analysis of problems in which a response of interest is influenced by several variables , and the objective is to optimize this response . the most popular rsm design is the central composite design ( ccd ) for analysis of experimental data . the ccd is efficient and flexible , providing sufficient information about the effects of variables and overall experimental error with a minimum number of experiments . center points in ccd design are usually repeated 46 times to get a good estimate of experimental error ( pure error ) . five center points are created by default for each factor : alpha , with negative , zero and positive values ( 1 , 0 , and 1 ) . in this study , alpha value was taken as one resulting in 3 levels , lowest ( 1 ) , middle ( 0 ) , and highest ( + 1 ) which is more specifically known as central composite face centered design ( ccfd ) , in an attempt to optimize the variables : incubation period , initial ph , incubation temperature , and molasses concentration , which affect bioethanol production . the main goal of the present work was to maximize bioethanol production from sugarcane molasses in batch fermentation process using previously isolated saccharomyces cerevisiae y-39 . the application of ccfd and rsm assisted in designing , modeling , and optimizing the fermentation process by performing a series of controlled laboratory experiments . sugarcane molasses was purchased from sugars and integrated industries egyptian distillation plants in hawamdeia city , giza , egypt and stored at 4c until use . wickerham wh medium prepared according to wickerham was used for maintenance and inoculum preparation . medium for fermentation experiments was prepared as follows : 2 g kh2po4 , 10 g ( nh4)2so4 , 1 g mgso4 7h2o , and 2 g yeast extract were dissolved in 1 l distilled water , molasses concentration and ph were then adjusted according to the experimental conditions before sterilization , at 121c for 20 min to avoid contamination . a yeast strain saccharomyces cerevisiae y-39 , previously isolated for its ability to produce bioethanol from different saccharides , was used in this study . active cultures for fermentation experiments were prepared by growing y-39 in wh medium for 48 h at 30c in shaking incubator 150 rpm . harvested cells were washed twice with sterile saline ( 8.5 g nacl per 1 l distilled water ) and then resuspended in sterile saline to be used as a fresh and pure stock for inoculation . the types of sugars in molasses were determined by high performance liquid chromatography ( hplc ) , according to the method reported by madian et al . . ashes were quantified by gravimetric analysis after burning samples at 550c for 3 h , and minerals concentrations were determined by atomic absorption spectrophotometer in central analytical lab . in egyptian petroleum research institute . all other chemical characterizations of molasses were done in agricultural research center , giza , egypt . ethanol yield was measured by gas chromatography ( model 6890 , agilent ) , equipped with flame ionization detector and nominal capillary column ( 60 m 530 m 5.00 m ) . all experiments were carried out in triplicates , and the listed results are the average . batch fermentations were done in 100 ml erlenmeyer flasks fitted with rubber stoppers , containing 50 ml of culture media , inoculated with 5 ml of fresh yeast inoculum stock ( 10 mg fresh yeast / ml ) . incubation was performed in shaking incubator 100 rpm , set at temperatures according to the required experimental conditions . samples for analyses were taken at the beginning and end of fermentation at the prescribed incubation periods . response surface methodology ( rsm ) was used to optimize bioethanol production process from sugarcane molasses and investigate the influence of different fermentation process variables on the bioethanol yield . the central composite face the experimental runs were carried out according to a 2 full factorial design for the four identified design independent variables , namely , incubation period h ( x1 ) , initial ph ( x2 ) , incubation temperature , c ( x3 ) , and molasses concentration wt.% ( x4 ) , with low ( 1 ) and high ( + 1 ) levels . the total number of experiments ( runs ) was given by the simple formula [ 30 = 2 + 2k + 6 ] , where k is the number of independent variables ( k = 4 ) , this includes 16 factorial points from 24 full factorial ccfd were augmented with 6 replicates at the center point to assess the pure error . the design factors ( variables ) with low 1 and high + 1 levels , are , namely , x1 [ 24 and 72 h ] , x2 [ 5 and 7 ] , x3 [ 20 and 40c ] , and x4 [ 15 and 25 wt.% ] . the central values ; zero level chosen for experimental design were 48 h , 6 , 30c , and 20% for x1 , x2 , x3 , and x4 , respectively ( table 1 ) . once the experiments were preformed , the next step was to perform a response surface experiment to produce a prediction model to determine curvature , detect interactions among the design factors ( independent variables ) , and optimize the process , that is , determine the local optimum independent variables with maximum yield of bioethanol . the model used in this study to estimate the response surface is the quadratic polynomial represented by the following equation : ( 1)y=o+i=14ixi+i=13j = i+14ijxixj+i=14iixi2 , where y is the bioethanol yield ( g / l ) , o is the value of the fixed response at the center point of the the design , i , ij , and ii are the linear , interactive , and quadratic coefficients , respectively . xi and xj are the independent variables ( factors ) under study . the statistical software design expert 6.0.7 ( stat - ease inc . , minneapolis , usa ) was used for design of experiments , regression and graphical analyses of the data obtained , and statistical analysis of the model to evaluate the analysis of variance ( anova ) . sugarcane molasses is a dark viscous fluid with ph value of 5 and very rich in nutrients required by most microorganisms . carbon , nitrogen , phosphorus , sodium , and potassium contents are 64 , 6 , 0.3 , 0.33 , and 5.5 ( wt.% ) , respectively . non - nitrogenous compounds ( e.g. , citric acid , oxalic acid ) represent 28% ( wt.% ) . molasses is found to be rich in calcium 0.7% and contains significant quantities of trace minerals copper ( 2.2 ppm ) , zinc ( 3.91 ppm ) , manganese ( 4.74 ppm ) , iron ( 78.37 ppm ) , and magnesium ( 1370 ppm ) . sugarcane molasses is rich in fermentable sugars 55% ( wt% ) and non - fermentable sugars recorded 5% ( wt% ) . most of the chemical parameters determined in this study were in close agreement to those reported by chen and chou , who found molasses containing 4555% total sugars , 2025% reducing sugars , 2535% sucrose , 1016% ash , 0.40.8% calcium , 0.10.4% sodium , 1.55% potassium and ph 55.5 . soil and climate , the variety and maturity of the cane , and the processing conditions in the factory all influence molasses composition . consequently , considerable variation may be found in nutrient content , flavor , color , and viscosity of molasses . based on ccfd and experimental results , rsm was used to optimize fermentation process design factors ( independent variables ) . the statistical combinations of variables in coded and actual values along with the predicted and experimental responses are presented in table 2 . the regression equation characterizing the influence of different considered variables on process yield was obtained : ( 2)y=177.4 + 6.34x10.41x23.33x32.39x4 33.32x1x2 + 28.51x1x322.11x1x4 27.60x2x3 + 30.38x2x416.39x3x4 0.56x1292.58x22 + 44.08x3266.47x42 . positive sign in front of the terms indicate synergetic effect , whereas negative sign indicates antagonistic effect . pareto chart figure 1 , was used in this work to make it much easier to visualize the main and interaction effects of all factors to the response variable , that is , bioethanol yield . the model identified that within the studied range of experiments , the quadratic effect of incubation temperature and the interactive effect of initial ph and molasses concentration and that of incubation period and temperature have highly significant positive influence on the bioethanol yield . that is , with increment of both incubation period and temperature , the bioethanol yield increases , and the same occurred with increment of both ph and molasses concentration . but incubation period has a relatively low significant positive effect on bioethanol yield , while the quadratic effect of initial ph and molasses concentration , and the interactive effect of incubation period and initial ph as well as interactive effect of incubation temperature and initial ph have highly significant inverse effect on the bioethanol yield . the interactive effect of incubation period and molasses concentration and that of incubation temperature and molasses concentration have a significant negative impact on the bioethanol yield . initial ph , incubation temperature , molasses concentration , and the quadratic effect of incubation period seem to have negative impact on bioethanol yield . thus , bioethanol yield decreases with increase of the initial ph and incubation period , incubation temperature and initial ph , incubation period and molasses concentration or incubation temperature and molasses concentration . the validity of the fitted model was evaluated , and its statistical significance was controlled by f - test . the analysis of variance ( anova ) for the response surface full quadratic model is given in table 3 . it can be indicated that the model is highly statistically significant at 95% confidence level , with f - value of 82.14 , and very low probability p value of < 0.0001 . the values of the determination coefficients , r and radj which measure the model fitting reliability for model ( 2 ) , were calculated to be 0.9871 and 0.9751 , respectively . this suggests that approximately 98.71% of the variance is attributed to the variables and indicated a high significance of the model . thus , only 0.0129 of the total variations can not be explained by the model which ensures the good adjustment of the above model to experimental data . confirmation of the adequacy of the regression model was reflected also by the good agreement between experimental and predicted values of response variables as shown in table 2 . the lack of fit test is performed by comparing the viability of the current model residuals to the variability between observations at replicate settings of the factors . the lack of fit was statistically significant with f - value of 53.24 and p value of 0.0002 . a significant lack of fit suggests that there may be some systematic variation unaccounted for in the hypothesized model . this may be due to the exact replicate values of the independent variable in the model that provide an estimate of pure error . the relationship between predicted and experimental values of bioethanol yield is shown in figure 2 . it can be seen that there is a high correlation ( r = 0.9853 ) between the predicted and experimental values indicating that the predicted and experimental values were in reasonable agreement . it means that the data fit well with the model and give a convincingly good estimate of response for the system in the experimental range studied . figure 3 shows the normal probability plots of the standardized residuals for bioethanol production efficiency . a normal probability plot indicates that if the residuals follow a normal distribution , in which case the points will follow a straight line . since some scattering is expected even with the normal data , as shown in figure 3 , it can be assumed that the data is normally distributed . thus , the obtained normal probability plot indicates a good validity for the approximation of the quadratic regression model . , points of observed runs were scattered randomly within the constant range of residuals across the graph . that is , the model is adequate , and there is no reason to suspect any violation of the independence or constant variance assumption in all runs . the standardized residuals versus run plot represented in figure 4(b ) shows randomly scattered points ranged between 2.8 ; the errors were normally distributed and insignificant . the perturbation plot figure 5 shows the comparative effects of all independent variables on bioethanol yield . the sharp curvature of the three factors : initial ph , incubation temperature , and molasses concentration shows that the response , bioethanol yield , was very sensitive to these three variables . the comparatively semiflat incubation period curve shows less sensitivity of bioethanol yield towards the incubation period . thus , the incubation period with the studied range of experiments has no major function in the fermentation process compared to the other three factors . the empirical predicted quadratic model for response ( bioethanol yield ) in terms of process variables ( incubation period , initial ph , incubation temperature , and molasses concentration ) are plotted in three - dimensional diagrams ( figure 6 ) , to investigate the interaction among the variables and to determine the optimum condition of each factor for maximum bioethanol yield . figure 6(a ) represents the effects of varying , incubation period and initial ph on bioethanol yield at constant incubation temperature 38c and molasses concentration 18 wt% . however , increase in bioethanol yield occurred with an increase in incubation period at ph range from 5.5 to 6.5 . further increase in initial ph would decrease the bioethanol yield . according to this interaction effects , a significant positive impact is detectable ; that is , bioethanol yield increased with the increase of incubation temperature and time . figure 6(c ) shows the interactive effect of incubation period h and molasses concentration wt.% on bioethanol production at constant initial ph 5.6 and incubation temperature 38c . it is obvious that with the increase of molasses concentration above 20 wt% , bioethanol yield decreases . g / l obtained at higher incubation period 72 h and 20 wt.% molasses concentration . figure 6(d ) shows the cooperative effect of incubation temperature and initial ph on bioethanol yield at constant incubation period 72 h and molasses concentration 18 wt% . as shown in figure 6(d ) , at low and high ph values , the bioethanol yield decreases . the maximum bioethanol yield 257 g / l was obtained at high incubation temperature 40c and initial ph of 6 . figure 6(e ) illustrates the interactive effect of initial ph and molasses concentration on bioethanol yield at constant incubation period 72 h and temperature 40c . where , bioethanol yield is low at both low and high molasses concentrations and initial ph . but bioethanol yield increases within ph range 5.56.5 and molasses concentration 18%22% with maximum yield of 257 g / l at ph and molasses concentration of 6 and 20% , respectively . figure 6(f ) represents the interactive effect of incubation temperature and molasses concentration on bioethanol yield at constant incubation period 72 h and initial ph 5.6 . it is obvious that with the increase in temperature , the bioethanol yield increases reaching its maximum at 40c , while at low and high molasses concentrations the yield decreases and increased within 18%22% . g / l is obtained at molasses concentration and incubation temperature of 20% and 40c . the optimization process was carried out to determine the optimum value of bioethanol production efficiency , using the design expert 6.0.7 software . according to the software optimization step , the desired goal for each operational condition ( x1 incubation period , x2 initial ph , x3 incubation temperature , and x4 molasses concentration ) was chosen within the studied range . the response ( bioethanol production ) was defined as maximum to achieve the highest performance . the program combines the individual desirability into a single number and then searches to optimize this function based on the response goal . accordingly , the optimum working conditions and respective bioethanol production were established , and the results are presented in table 4 . an additional experiment was then performed to confirm the optimum results . as shown , the maximum bioethanol production was 255 g / l at incubation period of 71 h , initial ph 5.6 , incubation temperature 38c , and molasses concentration of 18% . recording average of 1.08 and 0.87% , respectively , indicating that process optimization by ccfd was capable and reliable to optimize bioethanol production from sugarcane molasses using saccharomyces cerevisiae y-39 . rsm and ccfd proved to be reliable and powerful tool for modeling , optimizing and studying the interactive effects of four process variables ( incubation period , initial ph , incubation temperature , and molasses concentration ) of bioethanol production from batch fermentation of sugarcane molasses using locally isolated saccharomyces cerevisiae y-39 . a highly significant ( r = 0.9871 , p < 0.0001 ) regression quadratic model equation was obtained by analyzing the data of a 2 factorial design . the maximum predicted and actual bioethanol yields are 253 and 255

[ purpose ] the purpose of this study was to investigate the differences in symmetry of sitting posture between typical developmental ( td ) children and hemi - cerebral palsy ( cp ) children . [ subjects and methods ] a school chair mounted on a force platform was used to assess the quiet - sitting pressure distribution of 10 td and 10 cp children . [ results ] the symmetry index of the td children was significantly closer to zero than that of the cp children irrespective of the latter group s hemiparetic side . [ conclusions ] sitting posture on school chairs of cp children was more asymmetrical than that of td children . a statistical model was developed in this study to describe bioethanol production through a batch fermentation process of sugarcane molasses by locally isolated saccharomyces cerevisiae y-39 . response surface methodology rsm based on central composite face centered design ccfd was employed to statistically evaluate and optimize the conditions for maximum bioethanol production and study the significance and interaction of incubation period , initial ph , incubation temperature , and molasses concentration on bioethanol yield . with the use of the developed quadratic model equation , a maximum ethanol production of 255 g / l was obtained in a batch fermentation process at optimum operating conditions of approximately 71 h , ph 5.6 , 38c , molasses concentration 18% wt.% , and 100 rpm .

in 1989 , caffe et al . published a method for organ culture in which the neuroretina was placed with the photoreceptor layer facing downward on rafts made of nitrocellulose filters and polyamide gauze grids . since then , mammalian retinal organ cultures have been commonly used to research retinal physiology and pathobiology . retinal cell dynamics have shown that organotypic models can be comparable with in vivo conditions , especially those of the outer retina . thus , retinal cultures are still used in electrophysiological studies to monitor drug effects on retinal cell functionality , to evaluate neurotrophic factors or physical tension on retinal cells , and to study the molecular basis of potential therapies for photoreceptor death . our group has previously used neuroretina explant cultures to evaluate modifications induced by exogenous cells such as blood - derived macrophages or by cytokines such as tumor necrosis factor alpha ( tnf- ) that are implicated in the pathobiology of proliferative vitreoretinopathy . we have also established a mixed coculture model composed of three cellular layers , the neuroretina , the rpe , and adipose tissue - derived stem cells , to evaluate the neuroprotective effects of stem cells . there are some obvious limitations of these culture systems , such as the axotomy of ganglion cells as part of the dissection procedure and the absence of a blood supply . thus , degenerative changes in retinal cells , especially at the inner retina , could differ from in vivo conditions . the rpe is a monolayer of pigmented , cuboidal epithelial cells that are closely associated with the photoreceptor outer segments . the most important functions of the rpe are the synthesis and maintenance of the interphotoreceptor matrix , photoreceptor membrane turnover , and retinoid metabolism . rpe association is supported by the fact that neuroretinas adjoined to rpe monolayer cultures have better - preserved tissue architecture in culture studies . the rpe is also considered a key element in the development of some retinal diseases and , importantly , in the physiopathology of retinal detachment and central serous chorioretinopathy . in these pathologies , the neuroretina and the rpe furthermore , the rpe secretes across the apical membrane neuroprotective factors that helps to maintain retinal homeostasis . thus , an adequate ex vivo tool for studying retinal degeneration and the importance of molecular signaling across the subretinal space after physical separation of the neuroretina and the rpe would be inherent to overcome the complexities that are inherent in vivo studies . in this study , we developed and characterized a novel organotypic coculture system in which the central cone - dominated porcine neuroretina was cocultured with rpe cells that were maintained physically separated from the neuroretina by the culture medium and a porous membrane . this arrangement prevented any physical contact between the cocultured cells but allowed signal molecules to pass between them . this model could be useful for studying in vitro the interactions between the neuroretina and the rpe when they lose their natural contact , as happens in several retinal diseases such as retinal detachment and central serous chorioretinopathy . further , this ex vivo model will allow study of the role of rpe - secreted factors and the evaluation of potential therapies for reducing the progression of neuroretina degeneration . fifteen porcine eyes from animals aged 68 months were obtained from the local slaughterhouse and processed within 2 h of death . immediately after enucleation , the eyes were immersed in ice - cold transport medium composed of dulbecco s modified eagle medium ( dmem ) supplemented with a 10% antibiotic - antimycotic mixture containing penicillin , streptomycin , and amphotericin b ( gibco , invitrogen , paisley , uk ) and transported on ice to the laboratory . under aseptic conditions in a laminar airflow hood , the eyes were dissected free from all periocular tissue . after full immersion of the eyeballs in 70% ethanol during 2 min , the eyes were washed in clean dmem supplemented with 10% antibiotic - antimycotic at room temperature . briefly , the eyes were dissected at the ora serrata to exclude the iris and lens . the vitreous was then removed from the posterior eyecup with cotton swabs , and then the neuroretina was detached and discarded . the remaining eye cup was covered with 0.05% trypsin - tetrasodium ethylene diamine tetra - acetate ( trypsin - edta , gibco ) for 30 min at 37 c . rpe cells were removed by filling the eye cup with dmem supplemented with 10% fetal bovine serum ( fbs ; gibco ) plus 1% antibiotic - antimycotic mixture ( complete dmem ) and swabbing gently . after resuspension in complete dmem , the cells were plated in 25 cm flasks ( nunc , roskilde , denmark ) . the rpe cells were maintained in complete dmem under standard culture conditions of 37 c in an atmosphere of 5% co2 with 95% humidity . rpe cell morphology was evaluated with a nikon eclipse ts100 inverted - phase contrast microscope ( nikon instruments inc . , louis , mo ) was used to determine viability and cell numbers . after reaching > 90% confluence , the cells were trypsinized with 0.05% trypsin - edta , washed , and resuspended in pbs ( gibco ) . passage 2 rpe cells were seeded ( 30,000 cells / cm ) on the bottom of transwell culture plates ( corning inc . , corning , ny ) and grown for 24 h in complete dmem to allow cellular adhesion before coculturing with neuroretina explants . the porcine cone - enriched visual streak was identified , as described by hendrickson and hicks , and two 55 mm adjacent explants from each eye were obtained with castroviejo corneal scissors ( john weiss & son ltd . , milton keynes , uk ) from the temporal area 1 mm superior to the optic disc ( figure 1 ) . the neuroretina explants were laid over the transwell membranes ( 24-mm diameter with 0.4-m pore polycarbonate membrane insert ; product # 3412 , corning inc . ) with the photoreceptor layer facing the membrane . nine neuroretina explants were cocultured with rpe cells but physically separated from them by the culture medium overlying the rpe cells and the transwell membrane ( figure 2 ) . cultures were maintained in 1:1 neurobasal - a / dmem supplemented with 10% fetal bovine serum , 2% b-27 ( gibco ) , 1% l - glutamine ( sigma - aldrich , st . louis , mo ) , and 1% antibiotic - antimycotic mixture and maintained in standard culture conditions . the culture medium level ( 1.5 ml as suggested by the manufacturer ) was maintained in contact with the support membrane beneath the explants and changed with freshly prepared , warmed medium every day . the explants were harvested for analysis after 9 days of culture , as previously described by our group , and were cut into two halves for subsequent processing . two fresh central neuroretina specimens were used as culture day 0 samples and processed in parallel . two neuroretina explants ( 55 mm ) were obtained from each eye at the porcine cone - enriched visual streak superotemporal to the optic disc . . schematic view of the coculture model of the porcine neuroretina explant and isolated rpe cells . the neuroretina explant was placed over a cell culture membrane , and the rpe cells were placed on the bottom of the cell culture insert and cocultured together in the same well . in this configuration , the cell culture membrane physically separated them , but signal molecules could pass through the porous membrane . rpe cells that were cocultured with neuroretina explants for 9 days were fixed in the transwell culture plates with ice - cold methanol ( panreac quimica s.a.u . , barcelona , spain ) for 15 min at 4 c . primary antibodies ( table 1 ) for zonula occludens protein 1 ( zo-1 ) , rpe - specific 65 kda protein ( rpe65 ) , and cellular retinaldehyde binding protein ( cralbp ) were diluted in pbs containing 0.5% triton x-100 , 10% goat serum , and 1% bovine serum albumin ( bsa ; all sigma - aldrich ) . the antibodies were applied directly to the cultures and incubated overnight at 4 c . after washing in pbs , the corresponding species - specific secondary antibodies to immunoglobulin gamma conjugated to alexa fluor 488 or 568 ( green and red ; molecular probes , eugene , or ) were applied at a 1:200 dilution for 1 h. nuclei were stained with 10 g / ml 4,6-diamino-2-phenylindole dihydrochloride ( dapi , molecular probes ) . finally , the cells were washed in pbs , mounted in fluorescent mounting medium ( dakocytomation inc . , carpinteria , ca ) , and coverslipped . rpe : retinal pigment epithelium cells ; nr : neuroretina explant neuroretina samples were fixed overnight in 1% paraformaldehyde and 1% glutaraldehyde ( both panreac quimica s.a.u . ) in phosphate buffer ( pb , gibco ) . after gradual dehydration in an ethanol series , the pieces were embedded in low - viscosity epoxy resin ( spurr , taab , aldermaston , uk ) . semithin sections ( 1 m ) were obtained with an ultramicrotome ( lkb bromma 8800 ultratome iii , freiburg , germany ) and stained with 1% toluidine blue in 3% sodium tetraborate ( both panreac quimica s.a.u . ) . for immunohistochemistry , the other half of the samples were fixed in 4% paraformaldehyde in pb , ph 7.4 , for 2 h and then subjected to sucrose ( panreac quimica s.a.u . ) sections ( 5 m ) were cut on a cryostat ( leica instruments , nussloch , germany ) and mounted on glass slides ( superfrost plus ; menzel - glser , braunschweig , germany ) . the neuroretinas were immunostained for the phenotype - specific markers ( table 1 ) rhodopsin ( rho ) , calbindin d-28k ( cb ) , glial fibrillary acidic protein ( gfap ) , and cralbp . specific combinations of antibodies were diluted in pbs containing 0.5% triton x-100 and incubated overnight at 4 c . thereafter , the corresponding species - specific secondary antibodies conjugated to alexa fluor 488 and/or 568 were applied at a 1:200 dilution for 1 h. nuclei were stained with 10 g / ml dapi . finally , the sections were washed in pbs , mounted in fluorescent mounting medium , and coverslipped . the primary antibodies used in this work have been used in previous studies and have been well characterized by our group and other authors regarding specific cell - type immunostaining in whole fixed porcine eyes and in porcine neuroretina samples [ 6 - 8,16 ] . furthermore , control samples in which primary antibodies were omitted were processed in parallel , and no immunoreactivity was found in any case . samples were analyzed with a leica dm4000b light microscope ( leica microsystems , wetzlar , germany ) equipped for epifluorescence , leica hcx pl fluotar ph2 20x/0.50 and 40x/0.75 and leica n plan 63x/0.8 objectives ( leica microsystems ) were used , and images were obtained with a leica dfc490 digital camera ( leica microsystems ) . brightness and contrast were minimally adjusted and final figures composed with pixelmator 3.4 twist ( apple , cupertino , ca ) . the thickness of the neuroretina and the thickness of the outer nuclear layer ( onl ) and the inner nuclear layer ( inl ) were measured on toluidine blue histological images with image j 1.47v ( nih image , national institute of health , bethesda , md ) . neuroretina thickness was determined between the outer and the inner limiting membranes . in each case , six measurements were performed in 20x images from each of the neuroretina explants ( n=20 ) . statistical analysis was performed using r statistical software version 3.1.0 ( foundation for statistical computing , vienna , austria ) . a kruskal one - way anova ( anova ) was used to compare the mean thicknesses of freshly isolated neuroretinas and those cultured for 9 days alone or for 9 days in coculture with rpe cells . the homogeneity of variance assumption was checked with the robust brown - forsythe levene - type test using the group medians as implemented in r lawstat package . when the homogeneity of variance was not validated , the welch test was used for comparison of the mean values . pair - wise comparisons were performed with the student t test with bonferroni correction for multiple testing . rpe cells from the porcine eyes ( n=5 ) were obtained as previously described . briefly , the eyes were dissected at the ora serrata to exclude the iris and lens . the vitreous was then removed from the posterior eyecup with cotton swabs , and then the neuroretina was detached and discarded . the remaining eye cup was covered with 0.05% trypsin - tetrasodium ethylene diamine tetra - acetate ( trypsin - edta , gibco ) for 30 min at 37 c . rpe cells were removed by filling the eye cup with dmem supplemented with 10% fetal bovine serum ( fbs ; gibco ) plus 1% antibiotic - antimycotic mixture ( complete dmem ) and swabbing gently . after resuspension in complete dmem , the cells were plated in 25 cm flasks ( nunc , roskilde , denmark ) . the rpe cells were maintained in complete dmem under standard culture conditions of 37 c in an atmosphere of 5% co2 with 95% humidity . rpe cell morphology was evaluated with a nikon eclipse ts100 inverted - phase contrast microscope ( nikon instruments inc . , louis , mo ) was used to determine viability and cell numbers . after reaching > 90% confluence , the cells were trypsinized with 0.05% trypsin - edta , washed , and resuspended in pbs ( gibco ) . passage 2 rpe cells were seeded ( 30,000 cells / cm ) on the bottom of transwell culture plates ( corning inc . , corning , ny ) and grown for 24 h in complete dmem to allow cellular adhesion before coculturing with neuroretina explants . the porcine cone - enriched visual streak was identified , as described by hendrickson and hicks , and two 55 mm adjacent explants from each eye were obtained with castroviejo corneal scissors ( john weiss & son ltd . , milton keynes , uk ) from the temporal area 1 mm superior to the optic disc ( figure 1 ) . the neuroretina explants were laid over the transwell membranes ( 24-mm diameter with 0.4-m pore polycarbonate membrane insert ; product # 3412 , corning inc . ) with the photoreceptor layer facing the membrane . nine neuroretina explants were cocultured with rpe cells but physically separated from them by the culture medium overlying the rpe cells and the transwell membrane ( figure 2 ) . cultures were maintained in 1:1 neurobasal - a / dmem supplemented with 10% fetal bovine serum , 2% b-27 ( gibco ) , 1% l - glutamine ( sigma - aldrich , st . louis , mo ) , and 1% antibiotic - antimycotic mixture and maintained in standard culture conditions . the culture medium level ( 1.5 ml as suggested by the manufacturer ) was maintained in contact with the support membrane beneath the explants and changed with freshly prepared , warmed medium every day . the explants were harvested for analysis after 9 days of culture , as previously described by our group , and were cut into two halves for subsequent processing . two fresh central neuroretina specimens were used as culture day 0 samples and processed in parallel . were obtained from each eye at the porcine cone - enriched visual streak superotemporal to the optic disc . . schematic view of the coculture model of the porcine neuroretina explant and isolated rpe cells . the neuroretina explant was placed over a cell culture membrane , and the rpe cells were placed on the bottom of the cell culture insert and cocultured together in the same well . in this configuration , the cell culture membrane physically separated them , but signal molecules could pass through the porous membrane . rpe cells that were cocultured with neuroretina explants for 9 days were fixed in the transwell culture plates with ice - cold methanol ( panreac quimica s.a.u . , primary antibodies ( table 1 ) for zonula occludens protein 1 ( zo-1 ) , rpe - specific 65 kda protein ( rpe65 ) , and cellular retinaldehyde binding protein ( cralbp ) were diluted in pbs containing 0.5% triton x-100 , 10% goat serum , and 1% bovine serum albumin ( bsa ; all sigma - aldrich ) . the antibodies were applied directly to the cultures and incubated overnight at 4 c . after washing in pbs , the corresponding species - specific secondary antibodies to immunoglobulin gamma conjugated to alexa fluor 488 or 568 ( green and red ; molecular probes , eugene , or ) were applied at a 1:200 dilution for 1 h. nuclei were stained with 10 g / ml 4,6-diamino-2-phenylindole dihydrochloride ( dapi , molecular probes ) . finally , the cells were washed in pbs , mounted in fluorescent mounting medium ( dakocytomation inc . , carpinteria , ca ) , and coverslipped . rpe : retinal pigment epithelium cells ; nr : neuroretina explant neuroretina samples were fixed overnight in 1% paraformaldehyde and 1% glutaraldehyde ( both panreac quimica s.a.u . ) in phosphate buffer ( pb , gibco ) . after gradual dehydration in an ethanol series , the pieces were embedded in low - viscosity epoxy resin ( spurr , taab , aldermaston , uk ) . semithin sections ( 1 m ) were obtained with an ultramicrotome ( lkb bromma 8800 ultratome iii , freiburg , germany ) and stained with 1% toluidine blue in 3% sodium tetraborate ( both panreac quimica s.a.u . ) . for immunohistochemistry , the other half of the samples were fixed in 4% paraformaldehyde in pb , ph 7.4 , for 2 h and then subjected to sucrose ( panreac quimica s.a.u . ) sections ( 5 m ) were cut on a cryostat ( leica instruments , nussloch , germany ) and mounted on glass slides ( superfrost plus ; menzel - glser , braunschweig , germany ) . the neuroretinas were immunostained for the phenotype - specific markers ( table 1 ) rhodopsin ( rho ) , calbindin d-28k ( cb ) , glial fibrillary acidic protein ( gfap ) , and cralbp . specific combinations of antibodies were diluted in pbs containing 0.5% triton x-100 and incubated overnight at 4 c . thereafter , the corresponding species - specific secondary antibodies conjugated to alexa fluor 488 and/or 568 were applied at a 1:200 dilution for 1 h. nuclei were stained with 10 g / ml dapi . finally , the sections were washed in pbs , mounted in fluorescent mounting medium , and coverslipped . the primary antibodies used in this work have been used in previous studies and have been well characterized by our group and other authors regarding specific cell - type immunostaining in whole fixed porcine eyes and in porcine neuroretina samples [ 6 - 8,16 ] . furthermore , control samples in which primary antibodies were omitted were processed in parallel , and no immunoreactivity was found in any case . samples were analyzed with a leica dm4000b light microscope ( leica microsystems , wetzlar , germany ) equipped for epifluorescence , leica hcx pl fluotar ph2 20x/0.50 and 40x/0.75 and leica n plan 63x/0.8 objectives ( leica microsystems ) were used , and images were obtained with a leica dfc490 digital camera ( leica microsystems ) . brightness and contrast were minimally adjusted and final figures composed with pixelmator 3.4 twist ( apple , cupertino , ca ) . the thickness of the neuroretina and the thickness of the outer nuclear layer ( onl ) and the inner nuclear layer ( inl ) were measured on toluidine blue histological images with image j 1.47v ( nih image , national institute of health , bethesda , md ) . neuroretina thickness was determined between the outer and the inner limiting membranes . in each case , six measurements were performed in 20x images from each of the neuroretina explants ( n=20 ) . statistical analysis was performed using r statistical software version 3.1.0 ( foundation for statistical computing , vienna , austria ) . a kruskal one - way anova ( anova ) was used to compare the mean thicknesses of freshly isolated neuroretinas and those cultured for 9 days alone or for 9 days in coculture with rpe cells . the homogeneity of variance assumption was checked with the robust brown - forsythe levene - type test using the group medians as implemented in r lawstat package . when the homogeneity of variance was not validated , the welch test was used for comparison of the mean values . pair - wise comparisons were performed with the student t test with bonferroni correction for multiple testing . isolated porcine rpe cells started to adhere on culture day 1 and to form clusters of cells at day 4 in flasks ( figure 3a ) . these cells were polygonal and contained pigment . at day 7 of culture in flasks , the rpe cells were almost confluent in a monostratified epithelium , had a polygonal shape , and had pigmented cytoplasmic granules ( figure 3b ) . however , some cells had dedifferentiated morphology , with increased size and reduced pigment content ( figure 3b , arrowheads ) . cellular characteristics were maintained until passage 2 , when the rpe cells were cocultured with neuroretina explants . at coculture day 9 ( figure 3c ) , the rpe cells formed a confluent monolayer in the bottom of the cell culture inserts , retained the polygonal shape , and still had some pigment content ( figure 3c , insert ) ; some cells looked dedifferentiated ( figure 3c , arrowheads ) . cellular morphology and immunochemistry of rpe cells during the culture and coculture period . at day 4 of culture in the flasks ( a ) , the rpe cells formed clusters of pigmented , polygonal cells . at day 7 in the flasks ( b ) , the rpe cells reached confluence and maintained morphological characteristics . dedifferentiated cells were present ( b , arrowheads ) . at 9 days of coculture with the neuroretina explants ( c ) , the rpe cells in the cell culture inserts were confluent , monolayered , pigmented , and polygonal - shaped ( c , insert ) . dedifferentiated cells were still present ( c , arrowheads ) . at 9 days of coculture , the rpe cells in the cell culture inserts showed variable zo-1 immunoexpression at the cell periphery ( d , green ; nuclei , blue ) and maintained cytoplasmic rpe65 ( e , red ; nuclei , blue ) . cralbp was not detectable ( f ) scale bars=100 m ( a c ) and 20 m ( d f ) . to assess preservation of the rpe morphology and phenotype during coculture with the neuroretina explants , the rpe cells were examined with antibodies against zo-1 , which is a peripheral adaptor protein of tight junction structure between rpe cells . the expression of the rpe65 protein , which is located in the cytoplasm and involved in the production of 11-cis - retinal and in visual pigment regeneration , and cralbp , a retinoid - binding protein implicated in vitamin a metabolism and found in the rpe apical microvilli , was also detected with immunohistochemistry . nine days after coculture with the neuroretina explants , the rpe cells showed variable zo-1 immunoexpression at the polygonal cell periphery ( figure 3d ) and maintained the expression of cytoplasmic rpe65 ( figure 3e ) . neuroretina explants stained with toluidine blue showed the general overview of all layers of the porcine retina ( figure 4 ) . in the freshly isolated neuroretina explants ( figure 4a , d ) , the characteristic neuroretina architecture was apparent , with the notable presence of cone photoreceptors corresponding to the porcine visual streak . the delicate structures of the photoreceptor outer and inner segments ( os and is ) were well preserved before culturing . in the absence of cocultured rpe cells , the retina structure at 9 days of culture ( figure 4b , e , f ) was disorganized , and cellular vacuolization was present throughout the retina layers . photoreceptor os were lost while the remaining is appeared shorter and swollen compared to the freshly isolated neuroretina explants . in the onl , photoreceptor cell bodies were oriented to form degenerative rosette - like structures ( figure 4b , e , asterisks ) . the mller cells were hypertrophied , the nuclei were translocated to the onl , and cytoplasmic pigmented granules were present ( figure 4f , open arrow and open arrowheads ) . furthermore , cellular extensions of mller cells covered the photoreceptor ( figure 4b , f , arrows ) . at 9 days of coculture with rpe cells ( figure 4c , g ) , the cellular architecture of the neuroretina explants was better preserved , and the retina layers were more easily discerned than those in the neuroretina explants cultured alone . photoreceptor is were condensed and swollen ( figure 4 g , arrowheads ) ; no rosette - like formation was evident . cellular extensions over the photoreceptors were present ( figure 4c , g , arrows ) . neuroretina morphology during the culture and coculture period . in the freshly isolated retina explants ( a and d ) , the porcine central retina showed the characteristic highly organized layered structure . at 9 days of culture alone ( b , e , and f ) , photoreceptor os were lost , and the cell bodies formed degenerative rosette - like structures ( b and e , asterisks ) . mller cells were hypertrophied , with nuclei translocated to the outer nuclear layer ( f , open arrow ) and cell cytoplasm pigmented granules ( f , open arrowheads ) . cellular extensions over photoreceptors were also present ( b and f , arrows ) . at 9 days of coculture with rpe cells ( c and g ) , the layered retinal morphology was preserved with condensed photoreceptor is still present ( g , arrowheads ) . os = outer segments ; is = inner segments ; olm = outer limiting membrane ; onl = outer nuclear layer ; opl = outer plexiform layer ; inl = inner nuclear layer ; ipl = inner plexiform layer ; gcl = ganglion cell layer . , the neuroretina explants were examined with antibodies against rho , an opsin protein present in rod os , and cb , a calcium - binding protein present in cone photoreceptors . additionally , to assess the degree of glial cell activation , the neuroretina explants were immunostained with antibodies against gfap , an intermediate filament protein present in glial cells , and against cralbp , found in mller cells . in the freshly fixed neuroretina explants , the rods maintained normal long and straight os morphology expressing rho ( figure 5a ) . at 9 days of culture without the rpe , partial displacement of rho immunoexpression was detected at the swollen rod is ( figure 5b ) . additionally , photoreceptor nuclei were displaced to the is region ( figure 5b asterisks ) . after 9 days of coculture with rpe , rho expression ( figure 5c ) was evident at the onl ( figure 5d arrows ) and was displaced to shorter and swollen rod is . neuroretina immunohistochemistry during the culture and coculture period . in the freshly isolated retina explants ( a ) , rho immunoreactivity ( green ) showed the normal appearance of rod outer segments ( os ) . at 9 days of neuroretina explants culture alone ( b ) , rho was scarcely detected , and the inner segments ( is ) were not discernable . photoreceptor nuclei appeared at the is region ( b , arrowhead ) . at 9 days in coculture with rpe cells ( c ) , rho was displaced to the short and swollen rod is and to the outer nuclear layer ( arrows ) . in the freshly isolated retina explants ( d ) , cb ( red ) showed cone photoreceptors with normal morphology . at 9 days of neuroretina culture alone ( e ) , cone morphology was markedly altered as revealed with cb immunostaining . at 9 days in coculture with the rpe ( f ) , cone morphology underwent degeneration . in the freshly isolated retina explants ( g ) , cralbp ( green ) showed mller cells with normal morphology . reduced immunostaining for gfap ( red ) was observed . at 9 days of neuroretina culture alone ( h ) , gfap was upregulated at the cytoplasm of glial cells , and gfap - positive extensions formed a layered - like structure outside the retinal tissue ( arrows ) . at 9 days in coculture with rpe ( i ) , cralbp was scarcely detected , and gfap was reduced compared to explants cultured alone . os = outer segments ; is = inner segments ; onl = outer nuclear layer ; inl = inner nuclear layer ; gcl = ganglion cell layer . freshly fixed neuroretina explants , cones were immunoreactive for cb and had a normal morphology ( figure 5d ) . at 9 days of culture without the rpe , the cb immunostained cones showed markedly altered morphology ( figure 5e ) . at 9 days of coculture with the rpe , the cones underwent morphological degenerative changes ( figure 5f ) . in the freshly fixed neuroretina explants , cralbp immunoexpression revealed mller cells with normal morphology ( figure 5 g ) . gfap immunostaining was limited to the innermost layers of the neuroretinal tissue . at 9 days of culture without rpe , gfap was clearly upregulated in the cytoplasm of the glial cells ( figure 5h ) . gfap cellular processes extended outside the retinal tissue and formed layered structures ( figure 5h , arrows ) . at 9 days of coculture with rpe , cralbp was scarcely present , and glial cell immunoexpression of gfap was reduced compared with cultures without rpe ( figure 5i ) . gfap processes were also present over the outer limiting membrane ; the thickness of the cellular extensions was reduced ( figure 5i , arrows ) . the thickness of the neuroretina , the inl , and the onl changed over time during the culture period ( figure 6 ) . the thickness of freshly isolated neuroretinas ( n=2 ) was 135.2917.02 m . after 9 days of culture alone , the thickness of the neuroretinas ( n=9 ) was 92.2814.94 m , while that of the rpe - cocultured neuroretinas ( n=9 ) was 109.354.25 m ( p<0.0001 ) . the onl thickness of the freshly isolated samples was 34.641.43 m . after 9 days of culture alone , the onl thickness was 21.73.57 m , while that of the rpe - cocultured neuroretinas was 30.823.16 m ( p<0.0001 ) . the inl thickness of the freshly isolated samples was 35.541.81 m . after 9 days of culture alone , the inl thickness was 21.892.68 m , while that of the rpe - cocultured neuroretinas was 27.882.35 m ( p<0.0001 ) . porcine neuroretina thickness during the culture and coculture period . neuroretina tissue thickness ( a ) on day 9 of culture and on day 9 of coculture with rpe was reduced compared to the freshly isolated tissue . the thickness of the neuroretina cultures alone on day 9 was significantly less than that of the neuroretinas that had been cocultured with rpe . onl ( b ) and inl ( c ) thickness on day 9 of culture and on day 9 of coculture with rpe was reduced compared to the freshly isolated tissue . the onl ( b ) and inl ( c ) thickness of the neuroretina cultures alone on day 9 was significantly less than that of the neuroretinas that had been cocultured with rpe . isolated porcine rpe cells started to adhere on culture day 1 and to form clusters of cells at day 4 in flasks ( figure 3a ) . these cells were polygonal and contained pigment . at day 7 of culture in flasks , the rpe cells were almost confluent in a monostratified epithelium , had a polygonal shape , and had pigmented cytoplasmic granules ( figure 3b ) . however , some cells had dedifferentiated morphology , with increased size and reduced pigment content ( figure 3b , arrowheads ) . cellular characteristics were maintained until passage 2 , when the rpe cells were cocultured with neuroretina explants . at coculture day 9 ( figure 3c ) , the rpe cells formed a confluent monolayer in the bottom of the cell culture inserts , retained the polygonal shape , and still had some pigment content ( figure 3c , insert ) ; some cells looked dedifferentiated ( figure 3c , arrowheads ) . cellular morphology and immunochemistry of rpe cells during the culture and coculture period . at day 4 of culture in the flasks ( a ) , the rpe cells formed clusters of pigmented , polygonal cells . at day 7 in the flasks ( b ) , the rpe cells reached confluence and maintained morphological characteristics . dedifferentiated cells were present ( b , arrowheads ) . at 9 days of coculture with the neuroretina explants ( c ) , the rpe cells in the cell culture inserts were confluent , monolayered , pigmented , and polygonal - shaped ( c , insert ) . dedifferentiated cells were still present ( c , arrowheads ) . at 9 days of coculture , the rpe cells in the cell culture inserts showed variable zo-1 immunoexpression at the cell periphery ( d , green ; nuclei , blue ) and maintained cytoplasmic rpe65 ( e , red ; nuclei , blue ) . cralbp was not detectable ( f ) scale bars=100 m ( a c ) and 20 m ( d f ) . to assess preservation of the rpe morphology and phenotype during coculture with the neuroretina explants , the rpe cells were examined with antibodies against zo-1 , which is a peripheral adaptor protein of tight junction structure between rpe cells . the expression of the rpe65 protein , which is located in the cytoplasm and involved in the production of 11-cis - retinal and in visual pigment regeneration , and cralbp , a retinoid - binding protein implicated in vitamin a metabolism and found in the rpe apical microvilli , was also detected with immunohistochemistry . nine days after coculture with the neuroretina explants , the rpe cells showed variable zo-1 immunoexpression at the polygonal cell periphery ( figure 3d ) and maintained the expression of cytoplasmic rpe65 ( figure 3e ) . neuroretina explants stained with toluidine blue showed the general overview of all layers of the porcine retina ( figure 4 ) . in the freshly isolated neuroretina explants ( figure 4a , d ) , the characteristic neuroretina architecture was apparent , with the notable presence of cone photoreceptors corresponding to the porcine visual streak . the delicate structures of the photoreceptor outer and inner segments ( os and is ) were well preserved before culturing . in the absence of cocultured rpe cells , the retina structure at 9 days of culture ( figure 4b , e , f ) was disorganized , and cellular vacuolization was present throughout the retina layers . photoreceptor os were lost while the remaining is appeared shorter and swollen compared to the freshly isolated neuroretina explants . in the onl , photoreceptor cell bodies were oriented to form degenerative rosette - like structures ( figure 4b , e , asterisks ) . the mller cells were hypertrophied , the nuclei were translocated to the onl , and cytoplasmic pigmented granules were present ( figure 4f , open arrow and open arrowheads ) . furthermore , cellular extensions of mller cells covered the photoreceptor ( figure 4b , f , arrows ) . at 9 days of coculture with rpe cells ( figure 4c , g ) , the cellular architecture of the neuroretina explants was better preserved , and the retina layers were more easily discerned than those in the neuroretina explants cultured alone . photoreceptor is were condensed and swollen ( figure 4 g , arrowheads ) ; no rosette - like formation was evident . cellular extensions over the photoreceptors were present ( figure 4c , g , arrows ) . ( a and d ) , the porcine central retina showed the characteristic highly organized layered structure . at 9 days of culture alone ( b , photoreceptor os were lost , and the cell bodies formed degenerative rosette - like structures ( b and e , asterisks ) . mller cells were hypertrophied , with nuclei translocated to the outer nuclear layer ( f , open arrow ) and cell cytoplasm pigmented granules ( f , open arrowheads ) . cellular extensions over photoreceptors were also present ( b and f , arrows ) . at 9 days of coculture with rpe cells ( c and g ) , the layered retinal morphology was preserved with condensed photoreceptor is still present ( g , arrowheads ) . os = outer segments ; is = inner segments ; olm = outer limiting membrane ; onl = outer nuclear layer ; opl = outer plexiform layer ; inl = inner nuclear layer ; ipl = inner plexiform layer ; gcl = ganglion cell layer . , the neuroretina explants were examined with antibodies against rho , an opsin protein present in rod os , and cb , a calcium - binding protein present in cone photoreceptors . additionally , to assess the degree of glial cell activation , the neuroretina explants were immunostained with antibodies against gfap , an intermediate filament protein present in glial cells , and against cralbp , found in mller cells . in the freshly fixed neuroretina explants , the rods maintained normal long and straight os morphology expressing rho ( figure 5a ) . at 9 days of culture without the rpe , partial displacement of rho immunoexpression was detected at the swollen rod is ( figure 5b ) . additionally , photoreceptor nuclei were displaced to the is region ( figure 5b asterisks ) . after 9 days of coculture with rpe , rho expression ( figure 5c ) was evident at the onl ( figure 5d arrows ) and was displaced to shorter and swollen rod is . neuroretina immunohistochemistry during the culture and coculture period . in the freshly isolated retina explants ( a ) , rho immunoreactivity ( green ) showed the normal appearance of rod outer segments ( os ) . at 9 days of neuroretina explants culture alone ( b ) , rho was scarcely detected , and the inner segments ( is ) were not discernable . photoreceptor nuclei appeared at the is region ( b , arrowhead ) . at 9 days in coculture with rpe cells ( c ) , rho was displaced to the short and swollen rod is and to the outer nuclear layer ( arrows ) . in the freshly isolated retina explants ( d ) , cb ( red ) showed cone photoreceptors with normal morphology . at 9 days of neuroretina culture alone ( e ) , cone morphology was markedly altered as revealed with cb immunostaining . at 9 days in coculture with the rpe ( f ) , reduced immunostaining for gfap ( red ) was observed . at 9 days of neuroretina culture alone ( h ) , gfap was upregulated at the cytoplasm of glial cells , and gfap - positive extensions formed a layered - like structure outside the retinal tissue ( arrows ) . at 9 days in coculture with rpe ( i ) , cralbp was scarcely detected , and gfap was reduced compared to explants cultured alone . os = outer segments ; is = inner segments ; onl = outer nuclear layer ; inl = inner nuclear layer ; gcl = ganglion cell layer . cones were immunoreactive for cb and had a normal morphology ( figure 5d ) . at 9 days of culture without the rpe , the cb immunostained cones showed markedly altered morphology ( figure 5e ) . at 9 days of coculture with the rpe , the cones underwent morphological degenerative changes ( figure 5f ) . in the freshly fixed neuroretina explants , gfap immunostaining was limited to the innermost layers of the neuroretinal tissue . at 9 days of culture without rpe , gfap was clearly upregulated in the cytoplasm of the glial cells ( figure 5h ) . gfap cellular processes extended outside the retinal tissue and formed layered structures ( figure 5h , arrows ) . at 9 days of coculture with rpe , cralbp was scarcely present , and glial cell immunoexpression of gfap was reduced compared with cultures without rpe ( figure 5i ) . gfap processes were also present over the outer limiting membrane ; the thickness of the cellular extensions was reduced ( figure 5i , arrows ) . the thickness of the neuroretina , the inl , and the onl changed over time during the culture period ( figure 6 ) . the thickness of freshly isolated neuroretinas ( n=2 ) was 135.2917.02 m . after 9 days of culture alone , the thickness of the neuroretinas ( n=9 ) was 92.2814.94 m , while that of the rpe - cocultured neuroretinas ( n=9 ) was 109.354.25 m ( p<0.0001 ) . the onl thickness of the freshly isolated samples was 34.641.43 m . after 9 days of culture alone , the onl thickness was 21.73.57 m , while that of the rpe - cocultured neuroretinas was 30.823.16 m ( p<0.0001 ) . the inl thickness of the freshly isolated samples was 35.541.81 m . after 9 days of culture alone , the inl thickness was 21.892.68 m , while that of the rpe - cocultured neuroretinas was 27.882.35 m ( p<0.0001 ) . neuroretina tissue thickness ( a ) on day 9 of culture and on day 9 of coculture with rpe was reduced compared to the freshly isolated tissue . the thickness of the neuroretina cultures alone on day 9 was significantly less than that of the neuroretinas that had been cocultured with rpe . onl ( b ) and inl ( c ) thickness on day 9 of culture and on day 9 of coculture with rpe was reduced compared to the freshly isolated tissue . the onl ( b ) and inl ( c ) thickness of the neuroretina cultures alone on day 9 was significantly less than that of the neuroretinas that had been cocultured with rpe . * p<0.02 ( n=20 ) and * * p<0.0001 ( n=20 ) . onl = outer nuclear layer ; inl = inner nuclear layer . the present study described the development and characterization of a novel coculture model of cone - dominated porcine central retina and rpe cells to closely simulate ex vivo the subretinal space microenvironment . retinal degeneration is a common finding of many retinal diseases , and in some cases , such as retinal detachment and central serous chorioretinopathy , it is in part the consequence of physical separation between the neuroretina and the rpe . this separation starts a cascade of events that result in cellular changes throughout the retina . these events are in part responsible for the poor functional results that can occur even after successful reattachment surgery . currently , research in retinal degeneration is largely limited due to the difficulty in obtaining specimens from freshly detached human retinas and the limitations of experimental animal models . in this sense , importantly , they closely simulate in vivo retinal cellular and molecular dynamics and have been the source of improved knowledge of retinal physiopathology in recent decades . nevertheless , these previously reported culture models lack the influence of the rpe cells that are necessary for maintaining the viability and functionality of the outermost retinal layers . furthermore , rpe cells secrete various growth and trophic factors that act on the neuroretina and the choroidal endothelium . the rpe also plays an important role in the retinal degeneration process and is considered a key element in retinal detachment physiopathology . there are some obvious limitations of these culture systems , such as the absence of choroidal and retinal blood flow , the lack of vitreous , and the axotomy of ganglion cells , that may considerably limit the study of the inner retina modifications . however , neuroretina organotypic cultures are still considered an adequate tool for improving knowledge of retinal physiopathology . other authors have previously described models in which rpe monolayers were cocultured in contact with the neuroretina . those models are complex to develop and do not adequately mimic a retinal pathology related to the separation between the neuroretina and the rpe . our purpose was to study and characterize the differences between neuroretinas cultured alone and those cocultured with rpe cells while physically separated from each other . this novel model could be the basis for studying the interaction between the neuroretina and rpe cells when they are separated in vivo because no such studies currently exist . in this model , neuroretina explants were cultured over cell culture membranes that physically separated the rpe cells cultured on the bottom of the cell culture inserts . thus , the porous cell culture membranes inhibit rpe cell migration into the retinal tissue and direct contact with neuroretina cells ; while molecular exchange among the different cell types can occur . in this scenario , rpe - secreted factors can diffuse through the cell culture membrane pores and influence neuroretina cell dynamics , thus recreating the subretinal space milieu . the porcine retina possesses some characteristics that make this species particularly useful in retinal research , such as retinal extent , ultrastructure , and the lack of a tapetum . the retinal parenchyma is quite similar to that of humans , with a double circulation system and a central zone , the visual streak . this zone is a broad horizontal retinal streak above the optic disc , with high cone density and without direct vascularization . unlike the human fovea , the porcine retina has a rod density that is similar to that of humans , and the cellular dynamics after neuroretina rpe separation are comparable in both species , especially those of key cells such as mller and rpe cells . therefore , in the coculture model that we have developed , we used explants from the porcine cone - dominated area centralis because of the similarity with the human central retina . as shown with phase contrast microscopy , rpe cells largely maintained their morphological characteristics throughout the coculture period . furthermore , these cells maintained the expression of zo-1 outlining the polygonal shape of the rpe cells within the monolayer , and rpe65 supporting the preservation of the rpe phenotype , without dedifferentiation to form other cell types or undergoing an epithelial mesenchymal transition . as previously described in primary rpe cell cultures , a few clusters of cells became partially dedifferentiated . cralbp immunoexpression was not observed in rpe cells cocultured with neuroretinas , probably due to the loss of normal cell polarization , especially seen as the disappearance of the apical microvilli . the ability of rpe in culture to secrete neurotrophic factors has been described by other authors . in the cultures in the present study , the neuroretina explants without the rpe showed degenerative alterations in the tissue and retina cells as previously described . however , the presence of degenerative rosette - like structures reported here were not observed by our group in previous work in which the central retina was not used . our immunohistochemical studies revealed retinal cell degenerative modifications as we and others previously described in organotypic porcine retina cultures . although rho and cralbp immunoexpression was better preserved and lower levels of gfap were detected in the neuroretinas cocultured with rpe cells , we did not quantify the gfap or cralbp labeling , and this will be part of future experiments . however , it is not clear if that is related to cellular swelling or to better cellular viability . in this sense , it will be useful to explore cell death and survival in future studies . in any case , the neuroretina explants cocultured with rpe cells maintained better preserved tissue and cellular characteristics and significantly better conserved tissue and nuclear layer thicknesses . these findings were consistent through the complete 5-mm neuroretina explants and were observed in all the experiments performed . based on the morphological and immunohistochemical results found in this study , neuroretina preservation in cocultures with rpe cells may be partially linked to neuroprotective factors secreted by rpe cells . the secretion of neurotrophic / neuroprotective factors by rpe cells is stimulated during retinal damage . our group has developed and standardized a novel coculture model of central cone dominated porcine neuroretina that was supplemented with rpe cells maintained separately within the same culture wells . this coculture system mimics ex vivo the subretinal space that develops during retinal detachment ( rd ) and other disease conditions of the retina . compared to the neuroretina explant cultures alone , the presence of cocultured rpe provided improved neuroretina architecture and thickness , better preservation of rho and cralbp immunoexpression , and lower levels of gfap . these data suggest that the cocultured rpe , while not in direct contact with the neuroretina explant , had a neuroprotective role . this effect may be linked to the beneficial effects of neurotrophic factors secreted or induced by rpe cells during coculture . furthermore , the proposed model will be useful to better study interactions between the rpe and the neuroretina and to test neuroprotective and/or anti - inflammatory drugs for retinal degenerative diseases . our group has developed and standardized a novel coculture model of central cone dominated porcine neuroretina that was supplemented with rpe cells maintained separately within the same culture wells . this coculture system mimics ex vivo the subretinal space that develops during retinal detachment ( rd ) and other disease conditions of the retina . compared to the neuroretina explant cultures alone , the presence of cocultured rpe provided improved neuroretina architecture and thickness , better preservation of rho and cralbp immunoexpression , and lower levels of gfap . these data suggest that the cocultured rpe , while not in direct contact with the neuroretina explant , had a neuroprotective role . this effect may be linked to the beneficial effects of neurotrophic factors secreted or induced by rpe cells during coculture . furthermore , the proposed model will be useful to better study interactions between the rpe and the neuroretina and to test neuroprotective and/or anti - inflammatory drugs for retinal degenerative diseases . ectopic kidney is a relatively rare renal anomaly however , an endourologist does encounter stone disease in an ectopic kidney occasionally . factors such as anomalous blood vessels and tortuous ureter with high insertion can lead to poor drainage and predisposition to the formation of renal calculi in these patients . the common management option for such stones is laparoscopy or ultrasound guided percutaneous nephrolithotomy ( pcnl ) . we report our experience with the use the recently described micro - pcnl or microperc for two such cases . two male patients , aged 57 and 60 years respectively , presented to us with calculi in ectopic pelvic kidneys . the first patient had undergone a flexible ureterorenoscopy elsewhere during which the stone could not be reached due to difficult angulation and inflamed tissue leading to poor vision . computed tomography urogram revealed an ectopic malrotated left kidney lying over the sacrum . there was a 13 mm 11 mm sized calculus in the renal pelvis [ figure 1a and b ] . in the second patient , computed tomography urogram revealed a normal right kidney and an ectopic malrotated left kidney lying over the sacrum . there was an 18 mm 17 mm sized calculus ( 1232 hu ) in the pelvis and a 5 mm 4 mm sized calculus ( 423 hu ) in the lower calyx of the ectopic kidney [ figure 1c and d ] . ( a ) x - ray kub showing renal calculus in pelvis of pelvic ectopic kidney with double j stent in situ , ( b ) computed tomography urogram showing the pelvicalyceal system anatomy , ( c ) x - ray kub showing renal calculus ( yellow arrow ) in ectopic kidney , ( d ) computed tomography urogram showing the pelvicalyceal system anatomy ( location of pelvic stone - yellow arrow ; location of lower calyceal stone - red arrow ) both procedures were performed under general anesthesia . in lithotomy position , ureteric catheterization was carried out under cystoscopic guidance using 7 fr ureteric catheters over a 0.035 inch glidewire ( terumo , tokyo , japan ) . the position was changed to supine - oblique with a sandbag under the ipsilateral hemipelvis to move the overlying bowel away from the kidney . an ultrasound probe was pressed against the anterior abdominal wall to displace the bowel away from the line of access . further , colour doppler was used to rule out any significant blood vessel in the path of needle puncture . three - way connector was attached to the needle , allowing saline irrigation , passage of a 0.9 mm flexible microperc telescope , and a 272 m holmium : yttrium aluminum garnet ( ho : yag ) laser fiber [ figure 2 ] . the stones were completely fragmented to dust with the laser . an x - ray and ultrasound was obtained on the first post - operative day to document stone clearance and to rule out any fluid collection . the urethral catheter was removed on the first post - operative day and the patient was discharged . ( a ) surface view of patient position and ultrasound guided percutaneous renal access , ( b ) intraoperative ultrasonography picture showing entry of puncture needle ( yellow arrows ) into lower calyx containing calculus ( red arrow ) , ( c ) confirmation of access into lower calyx by antegrade contrast study to delineate pelvicalyceal system , ( d ) intraoperative surface view showing microperc instruments , ( e ) puncture site at the end of the procedure ( yellow arrow ) , ( f ) post - operative x - ray kub showing complete clearance operating times were 30 and 35 min respectively . in the first patient , ureteric catheter was left in situ for 1 day . in the second patient , ureteric catheter was replaced by a double - j stent at the end of the procedure . visual analog pain scores ( 1 - 10 scale ) on first post - operative day were two and three respectively . first post - operative day and 1 month follow - up x - ray and ultrasound kub revealed complete stone clearance and no evidence of fluid collection in the abdomen . options for managing small renal calculi in pelvic ectopic kidney are shock wave lithotripsy ( swl ) , retrograde intrarenal surgery ( rirs ) , ultrasound or laparoscopy guided pcnl . since these kidneys are surrounded by bowel and bone , moreover , the clearance of fragmented stones is also impaired due to high insertion of ureter and impaired pyeloureteral motility due to surrounding fibrous bands . rirs can be technically demanding due to abnormal and tortuous course of the ureter of a pelvic kidney . this was exemplified by our first patient in whom the stone could not be reached with flexible ureteroscope and the procedure had to be abandoned . the potential hazards in percutaneous access in an ectopic kidney are : ( 1 ) risk of injury to surrounding bowel , particularly if track dilatation is carried out through the bowel , ( 2 ) abnormal vasculature resulting in bleeding from tract dilatation and ( 3 ) spillage of fluid into peritoneal cavity . microperc is a minimally invasive form of pcnl in which percutaneous renal access and stone fragmentation are achieved in a single - step using a 16 g needle . since dilatation is not performed , potential hazards associated with it are avoided . fluid collection is less likely during puncture and at the end of the procedure since the needle puncture site closes quickly . both our patients had rapid post - operative recovery , probably due to lack of fluid spillage and hence no paralytic ileus . though laparoscopic guidance has been advocated for percutaneous access of an ectopic kidney , in experienced hands , ultrasound guidance can provide a safe entry into the appropriate calyx . appropriate patient positioning to move bowel away from the kidney , placing a sand bag to push the kidney towards the anterior abdominal wall and compression with the ultrasound transducer allow safe access into the kidney . further , the use of colour doppler rules out any significant blood vessel along the path of needle puncture . the hemoglobin drop in both of our patients was minimal , supporting the safety of this procedure . in laparoscopy guided pcnl , an abdominal drain may need to be left in situ for a prolonged period because of persistent urinary leakage . the average hospital stay in a series of 15 patients who underwent laparoscopy guided pcnl was 4.8 days ( 4 - 11 days ) . the patients in that series had sequential removal of nephrostomy tube , urethral catheter and abdominal drain , which lead to the prolonged post - operative stay . thus , ultrasound guided microperc is a safe and efficient technique in the management of small renal calculi in ectopic kidneys and it is a minimally invasive procedure with short hospital stay .

purposeto develop and standardize a novel organ culture model using porcine central neuroretina explants and rpe cells separated by a cell culture membrane.methodsrpe cells were isolated from porcine eyes , expanded , and seeded on the bottom of cell culture inserts . neuroretina explants were obtained from the area centralis and cultured alone ( controls ) on cell culture membranes or supplemented with rpe cells in the same wells but physically separated by the culture membrane . finally , cellular and tissue specimens were processed for phase contrast , cyto-/histological , and immunochemical evaluation . neuroretina thickness was also determined.resultscompared to the neuroretinas cultured alone , the neuroretinas cocultured with rpe cells maintained better tissue structure and cellular organization , displayed better preservation of photoreceptors containing rhodopsin , lower levels of glial fibrillary acidic protein immunoexpression , and preservation of cellular retinaldehyde binding protein both markers of reactive gliosis . neuroretina thickness was significantly greater in the cocultures.conclusionsa coculture model of central porcine neuroretina and rpe cells was successfully developed and standardized . this model mimics a subretinal space and will be useful in studying interactions between the rpe and the neuroretina and to preclinically test potential therapies . management of stone disease in an ectopic kidney is challenging . laparoscopy or ultrasound guided percutaneous nephrolithotomy and retrograde intra - renal surgery are the preferred techniques for these stones . we performed ultrasound guided microperc using a 16 g needle for the management of renal calculi in pelvic ectopic kidneys in two patients . there was no intraoperative or post - operative complication . both patients had complete stone clearance and were discharged on the first post - operative day . ultrasound guided microperc is a safe and effective option for the management of small renal calculi in pelvic ectopic kidneys .

intra - radicular posts are commonly used to restore endodontically treated teeth if their remaining coronal tissue could no longer provide adequate support and retention for the restoration . although the use of prefabricated posts has gained popularity , for several years custom cast dowel and core has been used to retain restorations with clinical evidence of success . one important advantage of this post system is that the dowel will fit a flared or irregularly shaped canal more closely than prefabricated post systems do . however , although endodontic therapy has shown a high success rate , adverse situations that require endodontic retreatment are not rare . when signs , symptoms and radiographic images suggest the failure of endodontic treatment , an atraumatic and efficient post removal is essential for optimal non - surgical endodontic management . drills and extractors exert high force on the root and may result in root fractures . ultrasonic energy is transmitted to the post , causing cracks in the cement thus facilitating its removal . recent evidence suggests that ultrasonic vibration is a safe and fast method for post removal . however , several in vitro studies have evaluated post removal with ultrasonic devices by using roots of extract teeth included in resin cylinders . this procedure is used in order to facilitate handling and test application . however clinically , the root is enveloped by periodontal ligament ( pdl ) , which presents higher resiliency than resins used for root inclusion . this difference in resiliencies enables pdl to dissipate the ultrasonic energy more easily than when the root is included in a resin cylinder . thus , the facility found in some in vitro studies for post removal with ultrasound activation may not be consistent with the clinical reality . the simulation of the pdl in these tests would be important to bring the laboratory findings to clinical application . the aim of this in vitro study was to evaluate the effect of simulated periodontal ligament ( spdl ) on custom cast dowel and core removal using an ultrasonic device . the null hypothesis was that the spdl does not influence the action of the ultrasonic device used to remove the cast dowel and core from the root canal . thirty - two human maxillary canines with mature apices , un - pronounced flattening , roots with no curves and single canal were selected for this study . crowns were removed in order to obtain a 15-mm - long root remainder . for the endodontic treatment , the root canals were prepared according to a crown - down technique , using stainless - steel k - files and # 2 to # 4 gates - glidden drills ( dentsply maillefer ; ballaigues , switzerland ) . all enlargement procedures were followed by irrigation with a 2.5% sodium hypochlorite solution . instrumented root canals were obturated with gutta - percha cones and sealer-26 resin sealer ( dentsply , petrpolis , rj , brazil ) using the lateral condensation technique . the filled roots were stored in relative humidity for at least 72 h to allow the resin sealer to set . the specimens were randomly allocated according to presence of spdl and application of ultrasonic vibration . half of the roots were included directly in self - cured acrylic resin cylinders ( jet clssico , so paulo , sp , brazil ) without spdl . the external surfaces of the root remainders were dipped into melted wax ( epoxiglass , diadema , sp , brazil ) , resulting in a 0.2 to 0.3-mm - thick wax layer . after resin polymerization , the roots were removed from the cylinder , the wax removed from the root surface creating a space in the resin cylinder . the polyether impression material ( impregum f , 3m / espe , seefeld , germany ) was mixed and placed in the space created in the resin cylinder . the tooth was re - inserted into the cylinder and the excess material removed with a scalpel blade . in order to allow tensile testing without the root becoming dislodged , the roots included in resin cylinder with spdl were fixed to cylinders with two stainless steel wires about 1 mm in diameter ( figure 2 ) . note the presence of two wires in order to prevent the root dislodgement during testing post - holes were prepared by standardizing the length at 8 mm and preparation was performed with a size 6 largo drill ( dentsply maillefer ) . this drill was used with a low - speed handpiece attached to a parallelometer . the root canal impressions were made with self - cured resin acrylic ( duralay , reliance dental , worth , il , usa ) . the dowel and cores were cast in a nickel - chromium alloy ( wironia , bego , bremen , germany ) . all custom cast dowel and cores were luted with dual - cured resin cement ( panavia f ; kuraray , osaka , japan ) , in accordance with the manufacturer 's instructions . after the storage period , half of specimens of each inclusion type ( with or without spdl ) were submitted to ultrasonic vibration . this was applied by the same calibrated operator , using a piezoelectric ultrasonic device ( enac , osada electric co ltd . , tokyo , japan ) , and an st 09 tip ( osada electric co. ltd . ) , at maximum power under water cooling . the vibration was applied for 1 min to the buccal , mesial , lingual , distal , and incisal surfaces , successively , with total application time of 4 min for each sample ( figure 3 ) . samples were positioned in a universal testing machine ( model 4411 , instron corp . , canton , ma , usa ) and the ring of the core was attached to the load cell ( 500 n ) . tensile load was applied at a crosshead speed of 0.5 mm / min until the cast dowel and core was dislodged ( figure 4 ) . the factors evaluated were " presence of spdl " and " ultrasonic vibration application " . post - hoc tests were calculated using tukey`s multiple - comparison test ( =0.05 ) . application of ultrasonic vibration application of tensile load at samples until dislodgment of the custom cast dowel and core . a and b - without simulated periodontal ligament ; c and d - with simulated periodontal ligament there was a statistically significant effect for the factors " presence of spdl " ( p<0.01 ) , " ultrasonic vibration application " ( p<0.01 ) and for interaction between factors ( p<0.05 ) . the means tensile strength values in kgf ( sd ) necessary to dislodge the cast post - and - cores and the results of the tukey 's test are shown in table 1 . when ultrasonic vibration was not applied , samples with spdl presented the lowest tensile bond strength values . no significant differences were observed in the presence or absence of spdl in samples submitted to ultrasonic vibration . ultrasonic vibration application led to the lowest values when the pdl was not simulated , but had no effect on samples with spdl . tensile strength means values in kgf ( sd ) necessary to dislodge the cast post - and - cores the influence of pdl is often omitted in in vitro tests that evaluate post removal using ultrasonic devices , as opposed to other studies that evaluate the in vitro fracture resistance of restored teeth . the pdl is an important structure for distributing the stress generated by load application on teeth . based on this , elastomeric materials have been used to reproduce the pdl in several studies . polyether impression material is adequate for such purpose because of its ease of use , consistency and deformation limit . one difficulty of using spdl in tensile tests is the possibility of it being dislodged during load application . in the present study , roots were fixed in a resin cylinder with two stainless steel wires to prevent dislodgment of root and allow testing . this methodology enabled tensile load application on samples included with spdl , but it had an influence on the tensile bond strength values . samples included in the cylinder with the presence of spdl presented lower bond strength values than those directly included in resin , when the ultrasonic vibrations were not applied . one possible explanation for this may be related to the forces resulting during tensile load application . during the test , the deformation of wires used to fix the root allowed a slight dislodgement of the root . thus , the tensile load is more concentrated at the interface between resin cement and dowel for the samples included with spdl . this results in the need for lower loads to remove these cast dowel and cores . despite this influence on the load distribution , the simulation of pdl also intervened in the effectiveness of the ultrasonic vibration to reduce the dowel retention . the tensile bond strength of the samples included in resin cylinders with spdl was not altered by use of the ultrasound device , as opposed to samples included without spdl . the reduction in cast dowel and core retention with the application of ultrasonic energy has been demonstrated by several studies . the ultrasonic device used in this study has a piezoelectric transducer that transforms electricity into ultrasonic vibrations . quartz crystals within the transducer are vibrated by the electricity flowing through them . by applying an alternating electrical field across the crystal , these ultrasonic vibrations are transmitted through the dowel and core , fracturing the cement interposed between the dowel and the root canal walls and facilitate their removal . several studies has reported that the type of luting agent can influence the post retention and removal procedure . posts cemented with resin cements usually require greater force for their removal when compared to those cemented with zinc phosphate or glass ionomer cements . in this study , the cast dowel and cores were luted with the resin cement panavia f. this cement contains the resin monomer 10-mdp ( 10-methacryloyloxydecyl di - hydrogen phosphate ) in its composition , which bonds to metal oxides . considering that it is essential for the vibrations to reach the resin cement in order to facilitate dowel and core removal , the root inclusion method may affect the efficacy of the ultrasonic device . the high rigidity of acrylic resin used for sample inclusion does not allow for root movement during the ultrasound application . thus , approximately all the energy dispensed by the tip of the ultrasonic device is transmitted through the cast dowel and core to reach the resin cement . on the other hand , the root mobility permitted by impression material may to reduce the energy that reaches the resin cement . thus , the resin cement was submitted to lower strain , which was not sufficient to reduce the retention of cast dowel and core . the latter situation is closer to clinical reality than directly including the root in resin cylinders . therefore , within the limitations of this study , it was demonstrated that the pdl simulation had a significant effect on custom cast dowel and core removal with ultrasonic vibrations . this means that several of the in vitro studies that evaluated ultrasonic devices for dowel and core removal may have overestimated their efficacy . the ultrasonic vibration had no effect on cast dowel - and - core retention when the pdl was simulated . the present outcomes demonstrate the importance of this simulation during in vitro evaluation to avoid overestimating the efficacy of ultrasonic vibration used for cast dowel - and - core removal . human metapneumovirus ( hmpv ) is an rna virus in the pneumovirinae subfamily of the paramyxoviridae family that was first isolated in the netherlands in 2001 and has subsequently been identified worldwide . it has been implicated as a significant cause of hospitalization for young children , second only to respiratory syncytial virus ( rsv ) in infants hospitalized with acute respiratory infections ( aris ) . hmpv has been detected in 1.543.0% of patients with aris . while it circulates predominantly in the winter , seroprevalence studies have shown that almost all children over five years of age have evidence of past infection . clinical syndromes associated with hmpv infection are similar to those of rsv infection , ranging from mild upper respiratory tract infections to wheezing and severe lower respiratory tract infections requiring mechanical ventilation . rare cases of fatalities have been associated with hmpv and it has been implicated in a handful of cases of encephalitis . although hmpv infections have been diagnosed in adults , their greatest impact occurs in children . a significant association with hmpv and wheezing is seen in young children , and hmpv has been linked to apparent life - threatening events in infants . hmpv has been associated with aris with super - infections as a result of staphylococcus aureus and streptococcus pneumoniae . to begin to understand the impact of hmpv on our institution , we analyzed children admitted to our tertiary care center in southeast michigan with respiratory symptoms during the respiratory season of 20062007 through an observational , retrospective study . the primary purposes of our study were to establish the utility of testing for hmpv in children who were admitted to our hospital during the respiratory virus season and to compare the impact of hmpv and rsv on the healthcare system . we identified a convenience sample of 256 nasopharyngeal ( np ) specimens from children younger than 18 years of age admitted with respiratory symptoms between november 1 , 2006 and may 31 , 2007 . the specimens were obtained by a np wash or swab based on the admitting physician s discretion . after routine testing by direct fluorescent antibody ( dfa ) and/or culture for rsv , parainfluenza viruses 13 , influenza viruses a and b , adenovirus , and rhinovirus , the np specimens were frozen at 70c and later subjected to nucleic acid extraction using the easymag system ( biomrieux , durham , nc , usa ) and following the manufacturer s protocol with slight modifications . two hundred microliters of each specimen were pre - treated with 20 units of dnase ( new england biolabs , uk ) at 37c for 45 min before extraction . extracts were used as the template for detection of hmpv using the nuclisense real - time analyte specific reagent ( asr ) assay performed on the easyq instrument ( biomrieux ) . a proprietary internal control containing the same primer binding sites as the hmpv target with unique internal sequences targeted by a separate molecular beacon probe were spiked into each specimen before extraction to monitor amplification integrity . laboratory personnel were blinded to the clinical data and the clinical investigator was blinded to the laboratory results . only the first specimen from which a virus was identified per admission was considered . the -test was used for analysis of categorical variables , comparing hmpv - positive patients to rsv - positive patients as well as patients who were negative for both hmpv and rsv . the student s t - test was used for an analysis that compared continu ous variables . of the 256 specimens , rsv was identified in 52 ( 20.3% ) , hmpv in 18 ( 7.0% ) , influenza in 9 ( 3.5% ) , rhinovirus in 5 ( 2.0% ) , parainfluenza in 8 ( 3.1% ) , and adenovirus in 4 ( 1.6% ) . three specimens had co - infections : hmpv and rsv in two and rsv and influenza a in one . hmpv was detected primarily in specimens collected between january and march ( 83.4% ) , while rsv was uniformly detected in those from november through february and then decreased into may . no hmpv was detected in specimens obtained in april and may ( figure 1 ) . because the primary goal of our study was to compare patients with hmpv to those with rsv , and because the number of patients with isolated viruses other than hmpv or rsv was low , cases with viruses other than hmpv or rsv and those with no specific virus isolated were combined as a separate group for the additional analyses detailed in the following section . while several studies have shown that hmpv occurs more in older children than does rsv , other reports showed no difference in age predilection , gender predominance , or presence of underlying medical disorders . in our study , the majority of hmpv - positive patients were aged 1324 months ( n=7 , 43.8% ) , whereas most rsv - positive patients ( n=35 ; 71.4% ) were younger than 12 months of age ( p<0.01 ) . underlying medical disorders considered were prematurity , chronic lung disease , bronchopulmonary dysplasia , asthma , congenital heart disease , congestive heart failure , immunosuppression , immunodeficiencies , hematological and solid organ malignancies , diabetes , and renal failure . we also found that children attending daycare or school were not at increased risk of being positive for hmpv or rsv or for having a respiratory illness not associated with either of these viruses ( table 1 ) . table 1demographic data.hmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and rsv ( % ) n=188age 012 months4 ( 25.0)*35 ( 71.4)77 ( 41.0 ) * 1324 months7 ( 43.8)9 ( 18.4)38 ( 20.2 ) 25 months5 ( 31.2)5 ( 10.2)73 ( 38.8)male7 ( 43.8)26 ( 53.1)98 ( 52.1)daily activities home11 ( 68.8)37 ( 75.5)118 ( 62.8 ) school / daycare5 ( 31.2)12 ( 24.5)70 ( 37.2)presence of underlying medical condition11 ( 68.8)32 ( 65.3)140 ( 74.5)p values not listed here were > 0.05 and thus deemed not statistically significant;*p<0.01 . p values not listed here were > 0.05 and thus deemed not statistically significant ; hmpv has previously been reported to be a rare cause of community - acquired pneumonia . we found that hmpv - positive patients were more likely to be diagnosed with pneumonia ( 37.5% ) than were the other two groups ( 14% , p=0.04 for rsv - positive , p=0.02 for negative for both hmpv and rsv ) . in addition , hmpv - positive patients were equally likely to be diagnosed with bronchiolitis as were rsv - positive children ( approximately 30% ) but less likely than children with respiratory symptoms who were negative for hmpv and rsv ( 11% , p=0.02 ) . in our study , the rate of abnormal chest radiographs was comparable in all three groups , but peribronchial cuffing was more likely to be present in hmpv - positive patients than in the other two groups ( table 2 ) . table 2clinical features.featurehmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and rsv ( % ) n=188pneumonia6 ( 37.5)*,**7 ( 14.3)*27 ( 14.4)**bronchiolitis5 ( 31.2)14 ( 29.2)21 ( 11.2)abnormal chest radiograph14 ( 87.5)35 ( 77.8)123 ( 71.9)peribronchial cuffing6 ( 37.5),5 ( 11.1)18 ( 10.4)oxygen supplementation12 ( 75.0)38 ( 77.6)104 ( 55.3)antibiotic use13 ( 81.2)25 ( 51.0)136 ( 72.3)antiviral use0 ( 0)1 ( 2.0)4 ( 2.1)steroid use8 ( 50.0)15 ( 30.6)66 ( 35.1)mechanical ventilation2 ( 12.5)2 ( 4.1)26 ( 13.8)icu admission6 ( 37.5)6 ( 12.2)50 ( 26.6)pvalues not listed here were > 0.05 and thus deemed not statistically significant;*p=0.04;**p=0.02;p=0.02;p<0.01;p=0.03;p=0.02 . pvalues not listed here were > 0.05 and thus deemed not statistically significant ; in order to compare severity of illness across the three patient populations , we examined their hospital course . mean duration of hospital stay was 6 days ( range 137 d ) for hmpv - positive patients , 6 days ( range 1112 d ) for rsv - positive patients , and 12 days ( range 1117 d ) for patients negative for hmpv and rsv ( p=0.83 for hmpv vs. rsv ; p=0.06 for hmpv vs. negative for hmpv and rsv ) . hmpv - positive patients were more likely to be treated with antibiotics than were rsv - positive patients ( 81.2% vs. 51.0% , p=0.03 ) and were more likely to be admitted to the intensive care unit ( icu ) ( 37.5% vs. 12.2% , p=0.02 ) . hmpv - positive patients were as likely to require oxygen supplementation , mechanical ventilation , and steroid use as were rsv - positive patients ( table 2 ) . infections with hmpv and rsv have been reported to be clinically indistinguishable , but subtle differences were identified in our study . the most common presenting symptom for hmpv - positive patients was fever , which occurred more often in hmpv - positive patients than in the other two patient populations . hmpv - positive patients were also more likely than patients negative for hmpv and rsv to have decreased urine output ( table 3 ; 43.8% vs. 14.4% , p<0.01 ) . on physical examination , hmpv - positive patients were more likely than rsv - positive patients to exhibit focal decreased breath sounds ( 18.8% vs. 2.0% , p=0.01 ) and were more likely than patients negative for hmpv and rsv to exhibit signs of otitis media ( 18.8% vs. 4.8% , p=0.02 ) . otherwise , there were no differences in the signs and symptoms noted in hmpv and rsv - positive patients ( table 4 ) . table 3clinical symptoms.hmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and rsv ( % ) n=188fatigue6 ( 37.5)9 ( 18.4)153 ( 81.4)fever15 ( 93.8)*,**30 ( 61.2)*102 ( 54.3)**rash1 ( 6.2)1 ( 2.0)10 ( 5.3)vomiting5 ( 31.2)19 ( 38.8)67 ( 35.6)diarrhea4 ( 25.0)11 ( 22.4)31 ( 16.5)poor feeding9 ( 56.2)30 ( 61.2)80 ( 42.6)decreased urine output7 ( 43.8)14 ( 28.6)27 ( 14.4)watery eyes1 ( 6.2)1 ( 2.0)7 ( 3.7)red eyes0 ( 0)1 ( 2.0)4 ( 2.1)rhinorrhea6 ( 37.5)30 ( 61.2)74 ( 39.4)congestion8 ( 50.0)25 ( 51.0)61 ( 32.4)ear pain1 ( 6.2)2 ( 4.1)7 ( 3.7)sore throat1 ( 6.2)1 ( 2.0)15 ( 8.0)cough13 ( 81.2)44 ( 89.8)128 ( 68.1)rapid breathing4 ( 25.0)12 ( 24.5)30 ( 16.0)difficulty breathing9 ( 56.2)29 ( 59.2)80 ( 42.6)apnea2 ( 12.5)1 ( 2.0)12 ( 6.4)pvalues not listed here were > 0.05 and thus deemed not statistically significant;*p=0.01;**p=<0.01;p<0.01 . pvalues not listed here were > 0.05 and thus deemed not statistically significant ; table 4clinical signs.hmpv+(% ) n=16rsv+(% ) n=49negative for hmpv and rsv ( % ) n=188respiratory distress6 ( 37.5)24 ( 49.0)63 ( 33.5)respiratory failure1 ( 6.2)0 ( 0)15 ( 8.0)tachypnea5 ( 31.2)16 ( 32.7)40 ( 21.3)retractions4 ( 25.0)*30 ( 61.2)*48 ( 25.5)crackles4 ( 25.0)10 ( 20.4)28 ( 14.9)rhonchi2 ( 12.5)6 ( 12.2)17 ( 9.0)wheezing6 ( 37.5)23 ( 46.9)48 ( 25.5)rales0 ( 0)2 ( 4.1)7 ( 3.7)focal decreased breath sounds3 ( 18.8)1 ( 2.0)13 ( 6.9)tachycardia5 ( 31.2)18 ( 36.7)46 ( 24.5)poor perfusion1 ( 6.2)4 ( 8.2)13 ( 6.9)conjunctivitis1 ( 6.2)0 ( 0)5 ( 2.7)phayrngitis2 ( 12.5)1 ( 2.0)10 ( 5.3)signs of otitis media3 ( 18.8)8 ( 16.3)9 ( 4.8)lymphadenopathy of head / neck region1 ( 6.2)0 ( 0)11 ( 5.9)pvalues not listed here were > 0.05 and thus deemed not statistically significant;*p=0.01;p=0.02;p=0.02 . hmpv was the second most commonly identified respiratory virus during the respiratory season of 20062007 in our study . our results confirm the previous findings that children with symptomatic hmpv infection are older than those infected with rsv , possibly because of differences in the upper respiratory tract or lung anatomy of older children that allow for hmpv acquisition . in our study , although a previous report found that hmpv is a rare cause of community - acquired pneumonia among hospitalized patients ( 4.9% ) , 37.5% of our hmpv - positive patients were admitted with a diagnosis of pneumonia . this difference is likely because of a requirement of three independent radiologists interpretations of the radiographs in the earlier study compared to the diagnosis of one admitting physician in our study . hmpv - positive patients were more likely than the other two groups to have peribronchial cuffing on their chest radiographs , evidence suggesting that these patients have interstitial edema , likely a result of the disruption of the respiratory epithelial structure and inflammation that hmpv has been shown to cause in animal studies . hmpv may have a stronger predilection for the respiratory epithelial cells than rsv , corroborated by reports that hmpv - positive patients frequently exhibited signs of otitis media . further research is needed on the pathogenesis of hmpv in humans , specifically regarding the ability of hmpv to infect human respiratory epithelial cells compared to rsv and other viruses . the present study demonstrates , as previously reported , that hmpv - positive patients were as likely to be severely ill as were rsv - positive patients . in fact , in our study , hmpv - positive patients were more likely than rsv - positive patients to be admitted to the icu even though they were not more likely to have an underlying medical illness . in addition , hmpv - positive patients were more likely than were rsv - positive patients to receive antibiotics . during the study time period , our institution did not routinely test specimens for hmpv . physicians may have used antibiotics more often in patients only retrospectively shown to be hmpv - positive because they were not aware of a specific virus contributing to these patients illnesses . the finding of increased antibiotic use in children with other aris , most of whom had negative viral cultures , corroborates this suggestion . it is also possible that the increased diagnosis of pneumonia in the hmpv - positive patients contributed to the increased use of antibiotics . this study , however , shows that there may be subtle differences in the clinical features of hmpv and rsv infections . as previously reported , fever was the most common presenting symptom for hmpv - positive patients and was more common in hmpv - positive children than in the other two groups . hmpv - positive patients were also more likely to experience decreased urinary output than patients negative for hmpv and rsv . more information is needed to compare the level of inflammation produced by hmpv with that of other viruses . our study was limited by extraction of the data from a convenience sample and by the fact that only one respiratory viral season was studied . in addition , the signs and symptoms associated with the studied viruses may have been exaggerated as only hospitalized patients were included . lastly , we acknowledge the li mi tations associated with the use of different methods for detecting different viruses in our study . the real - time asr assay that we used to detect hmpv was likely more sensitive than the dfa- and culture - based techniques used to detect rsv and other viruses . as such , it is possible that children infected with rsv were assigned to the group of patients not infected with hmpv or rsv . even with this potential drawback , however , our data strongly suggest that hmpv was common in the patient population included in this study . furthermore , as molecular methods gain more widespread use for the detection of many respiratory pathogens , continued studies assessing the correlation between laboratory and clinical information are warranted . our study confirms that hmpv is a significant pathogen particularly in young children and is frequently associated with respiratory symptoms resulting in hospitalization . in general , the clinical manifestations of hmpv- and rsv - associated infections in children are similar , although our data suggest subtle differences in illness presentation but not in severity . our data also suggest that underidentification of children with hmpv may lead to inappropriate use of antibiotics . therefore , considering the high prevalence of hmpv , the severity of hmpv illness , and the ease and accuracy of detection , routine diagnostic testing for hmpv should be implemented . increasingly , hmpv is being recognized as a significant cause of disease in other populations such as elderly patients and immunocompromised cases , highlighting other groups that would likely benefit from routine testing for hmpv . future studies with expanded patient populations will help to determine how identifying cases with hmpv - associated disease will allow clinicians to anticipate the patient s clinical course , identify cohort patients appropriately , and decrease the use of unnecessary antibiotics .

objectivethe aim of this study was to evaluate the effect of simulated periodontal ligament ( spdl ) on custom cast dowel and core removal by ultrasonic vibration.material and methodsthirty - two human maxillary canines were included in resin cylinders with or without spdl made from polyether impression material . in order to allow tensile testing , the roots included in resin cylinders with spdl were fixed to cylinders with two stainless steel wires . post - holes were prepared by standardizing the length at 8 mm and root canal impressions were made with self - cured resin acrylic . cast dowel and core sets were fabricated and luted with panavia f resin cement . half of the samples were submitted to ultrasonic vibration before the tensile test . data were analyzed statistically by two - way anova and tukey 's post - hoc tests ( p<0.05).resultsthe ultrasonic vibration reduced the tensile strength of the samples directly included in resin cylinders . there was no difference between the values , whether or not ultrasonic vibration was used , when the pdl was simulated . however , the presence of spdl affected the tensile strength values even when no ultrasonic vibration was applied.conclusionsimulation of pdl has an effect on both ultrasonic vibration and tensile testing . human metapneumovirus ( hmpv ) is a recently discovered virus that causes respiratory illness in children that can lead to hospitalization . our study was undertaken to further understand hmpv - associated illness , compare clinical characteristics of hmpv and respiratory syncytial virus ( rsv ) , and establish the utility of routine screening for hmpv . we retrospectively identified hmpv - associated illnesses described among children with respiratory symptoms admitted to a tertiary care center in southeast michigan during the 20062007 respiratory viral season . a convenience sample of 256 nasopharyngeal specimens was subjected to nucleic acid extraction and amplification to identify those specimens positive for hmpv . a medical record review was undertaken to retrieve demographic and clinical data of patients with hmpv , comparing them to rsv - positive patients and patients evaluated for respiratory symptoms who were negative for hmpv and rsv . we found that hmpv was the second most commonly identified virus after rsv . hmpv - positive patients were older than rsv - positive patients . among hmpv - positive patients , pneumonia was diagnosed in 37.5% and bronchiolitis in 31.2% , peribronchial cuffing was present on chest radiographs of 37.5% , antibiotic treatment was used in 81.2% , and admission to the icu was seen in 37.5% . finally , hmpv - positive patients were more likely to have fever than rsv - positive patients or patients negative for hmpv and rsv . we concluded that hmpv is a major pathogen associated with hospitalization of children and with the same severity of illness as rsv but in a slightly older population . because of the apparent prevalence and severity of illness , routine screening should be implemented .

recent whole transcriptome studies have revealed that about three quarters of the human genome is capable of being transcribed , while protein - coding regions account for just 2% of the genome ( 13 ) . therefore , the vast majority of transcribed sequences do not encode proteins , and are called non - coding rna ( ncrna ) . accumulating evidence shows that non - coding rnas play key roles in various biological processes , such as imprinting control , the circuitry controlling pluripotency and differentiation , immune responses , and chromosome dynamics ( 4 ) . ncrnas are as important as protein - coding genes to cellular functions ( 5,6 ) . notably , a growing number of long ncrnas ( lncrnas ) , which are considered to be > 200 nt in length and are often multiexonic ( 7 ) , have been implicated in disease etiology ( 810 ) . it is therefore of great importance to collect lncrna information and store this information in a one - stop knowledge gateway for lncrnas , the noncode database . the development of high - throughput sequencing methodologies has reduced the cost of rna sequencing , and as a result there has been an explosive rise in the number of newly identified lncrnas . established a consensus set of 384 066 predicted transcripts from 7256 rna - seq libraries , which were designated as the mitranscriptome assembly ( 11 ) . since then the revolutionary advancement of sequencing methods , such as single - molecule long - read techniques , leads us closer to the real lncrna transcriptome . given sufficient material , amplification - free sequencing of full - length cdna molecules noncode has collected data from literature published since the last update and includes the latest versions of several public databases ( ensembl ( 13 ) , refseq(14 ) , lncrnadb ( 15 ) and gencode ( 16 ) ) . after the removal of false and redundant lncrnas , noncode contains a total of 527,336 transcripts . in addition to the identification of new lncrnas , data on the genetics and biochemical properties of lncrnas has accumulated rapidly . of the papers retrieved from pubmed for lncrnas , we found that the vast majority studied lncrna function , especially the relationship between lncrnas and disease ( 810 ) . in large - scale searches for single - base differences between diseased and healthy individuals , about 40% of the disease - related differences show up in genomic regions outside of protein - coding genes . this implicates non - coding regions as vital for genetic risk factors of disease ( 2 ) . in order to enable a systematic compilation and integration of this information the sources for these annotations were derived from literature mining , differential lncrna analysis utilizing public rna - seq data and microarray data and mutation analysis from public genome - wide association study ( gwas ) data . along with the ever increasing number of lncrnas and the amount and functional study data , genome - wide conservation information is required for biologists to study the mechanisms of lncrna actions . in order to explore the conservation information of lncrnas , noncode collected six new mammalian species ( chimpanzee , gorilla , orangutan , rhesus macaque , opossum and platypus ) ( 17 ) . users can browse the conserved counterparts of any human lncrna gene in other species through a phylogenetic tree layout . similar to the former iterations of noncode ( 1821 ) , the source of noncode 2016 includes the previous versions of noncode , the collated literature and other public databases . noncoding , non - coding , no code , non - code , lncrna and lincrna , and found 6532 new articles since 1 june 2013 ( the last collection date for noncode ) . we retrieved the newly identified lncrnas and their annotations from the supplementary material or web site of these articles . together with the newest data from ensembl , refseq , lncrnadb , gencode and the old versions of noncode data , literature data were processed through a standard pipeline for each species . all of the input data were processed into bed or gtf formats based on one assembly version , for example , hg38 for human and mm10 for mouse.combination . all of the normalized data files were combined together using the cuffcompare program in the cufinks suite ( 22 ) . after eliminating redundancy , every new transcript i d and the accompanying resources were extracted.filtering protein - coding rna . firstly , the rna was compared with the coding rna in refseq and ensembl , and the = and c transcripts were excluded . secondly , the rna was filtered through the coding - non - coding index ( cnci ) ( 23 ) program and only the rnas considered non - coding by cnci were kept.information retrieval . we assigned each transcript a name according to the criterion of noncode v4 and extracted basic information such as location ( 24 ) , exons , length , assembly sequence , source , etc.advanced annotation . advanced annotations included expression profiles , predicted functions , conservation , disease information , etc . human expression profiles were collected from 16 tissues of the human bodymap 2.0 data ( ena archive : erp000546 ) and eight cell lines ( geo accession no . gse30554 ) , while mouse data was collected from six different tissues ( ena archive : erp000591 ) . functions for the lncrna genes were predicted by lnc - gfp ( 25 ) , a coding non - coding co - expression network ( 26,27 ) based global function predictor.web presence . more annotation information has been added and a more user - friendly interface has been introduced . all of the input data were processed into bed or gtf formats based on one assembly version , for example , hg38 for human and mm10 for mouse . all of the normalized data files were combined together using the cuffcompare program in the cufinks suite ( 22 ) . after eliminating redundancy , firstly , the rna was compared with the coding rna in refseq and ensembl , and the = and c transcripts were excluded . secondly , the rna was filtered through the coding - non - coding index ( cnci ) ( 23 ) program and only the rnas considered non - coding by cnci were kept . we assigned each transcript a name according to the criterion of noncode v4 and extracted basic information such as location ( 24 ) , exons , length , assembly sequence , source , etc . advanced annotations included expression profiles , predicted functions , conservation , disease information , etc . human expression profiles were collected from 16 tissues of the human bodymap 2.0 data ( ena archive : erp000546 ) and eight cell lines ( geo accession no . gse30554 ) , while mouse data was collected from six different tissues ( ena archive : erp000591 ) . functions for the lncrna genes were predicted by lnc - gfp ( 25 ) , a coding non - coding co - expression network ( 26,27 ) based global function predictor . more annotation information has been added and a more user - friendly interface has been introduced . noncode contains 527,336 lncrna transcripts from 16 species ( human , mouse , cow , rat , chimpanzee , gorilla , orangutan , rhesus macaque , opossum , platypus , chicken , zebrafish , fruitfly , caenorhabditiselegans , yeast and arabidopsis , ) . according to the definition of lncrna genes ( 18 ) , a total of 101,700 and 86,935 genes were generated from 167,150 and 130,558 lncrnas from human and mouse ( shown in table 1 ) , respectively . following the nomenclature of noncode v4 ( 18 ) , both lncrna transcripts and genes were designated systematically : non+ three characters ( representing a species ) + t ( transcript ) or g ( gene ) + six sequential numbers . noncode has annotated expression profiles from all the human and mouse transcripts and genes , and a large number of these genes were annotated with predicted functions . definitive evidence has proven that transcription of the non - coding genome has produced functional rnas ( 1 ) . in particular , lncrnas have been implicated in biological , developmental , and pathological processes , and acted through mechanisms such as chromatin reprogramming , cis regulation at enhancers , and post - transcriptional regulation of mrna processing ( 28 ) . lncrnas are therefore considered to be important regulators of tissue physiology and disease processes including cancer ( 11 ) . although we have collected functional interactions between ncrnas and biomolecules in npinter ( 2931 ) , we think it is also necessary to include disease information into noncode . recent published papers have been explored , and the proven associations between noncode transcripts and diseases has been integrated into the latest version . the noncode assembly also assessed the overlaps of transcripts with the unique disease - associated single - nucleotide polymorphisms ( snps ) from a catalog of gwass ( 32 ) and the snp database ( dbsnp ) ( 33 ) . there were also a lot of relational data between lncrnas and diseases which were analyzed from rna - seq and microarray data . after collecting the basic data , we compared it with the lncrnas in noncode and retained data that overlapped with noncode lncrnas . noncode 2016 contains 1110 lncrnas which were related to 284 diseases . among these associations , disease related data acquisition pipeline in the lncrna gene description pages , users can retrieve the related diseases of the entry , and also get the source of the information , such as the pmid(s ) of the reference paper(s ) . there is also mutational information retrieved from the literature , gwass and the dbsnp database . compared to protein - coding genes and small rnas ( e.g. mirnas and snornas ) , several reports have suggested that lncrnas are modestly conserved ( 11 ) . most lncrnas are less conserved in sequence ( 34 ) , but there are still many lncrnas that are conserved in their genomic loci , exonic sequences and promoter regions ( 35 ) . these are preserved across multiple species , attesting to their important functional potentials ( 36 ) . benefiting from next - generation sequencing technologies , ncrnas are now more easily identied via transcript sequencing . noncode has added six new species , mainly from multi - species rna - seq data ( 37,38 ) . an evolutionary tree from 12 commonly studied species ( human , mouse , cow , rat , chicken , zebrafish , chimpanzee , gorilla , orangutan , rhesus macaque , opossum and platypus ) was constructed using methods introduced in phylononcode ( 39 ) . each human lncrna gene counterpart from the other listed species can be retrieved through browsing the evolutionary tree ( shown in figure 2 ) . the counterpart of each lncrna was computed using the ucsc liftover tool ( 40 ) . in brief , liftover utilized blastz ( 41 ) , an independent implementation of the gapped blast algorithm specifically designed for aligning two long genomic sequences , as a core algorithm to detect homologous regions in other genomes . after mapping to the second species , however , a proper method of in silico transcript reconstruction is an ongoing challenge . according to an assessment by the paul bertone group , < 40% of known transcripts the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current - generation rna - seq data ( 42 ) . furthermore , multiple amplification steps during library preparation complicate the quantification of expression levels . to some extent given sufficient material , amplification - free and fragmentation - free sequencing of full - length cdna molecules provides a more direct view of rna molecules ( 12 ) . noncode contacted the authors of the third - generation single - molecule long - read survey of the human transcriptome paper ( 12 ) . after our analysis , the single - molecule lncrna transcripts were included into noncode . to meet the quality demands of researchers users can choose the subset which is considered high quality . the quality controls include the source of the data , literature support , other database support and long - read sequencing method support . the controls also include selection of exon numbers , the lengths of the transcripts and prediction tools support . the web interface will return the subset according to the conditions users chose and allow users to download the data . noncode 2016 contains 527,336 lncrnas from 16 different species , this compares favorably with other lncrna databases . for example , lncipedia ( human only ) contains 111 685 transcripts ( 43 ) , lncrnator ( human , mouse , fly , zebrafish , worm and yeast ) contains 34 605 transcripts ( 44 ) , while the lncrnawiki ( human only ) contains 105 255 transcripts ( 45 ) . as mentioned above , technical limitations imposed by short - read sequencing lead to a number of computational challenges in transcript reconstruction and quantification . for many transcripts , automated methods failed to identify all of the constituent exons , and in cases in which all exons were reported , the protocols tested often failed to assemble the exons into complete isoforms ( 42 ) . for example , although we have obtained all the data from mitranscriptome ( 11 ) , which contains 384 066 human lncrnas from 7256 rna - seq libraries , the detection of precise refseq splicing patterns from mitranscriptome was only 31% , and the fraction of annotated genes within the entire mitranscriptome was only 46% . although it is reasonable to assume that unannotated transcription is unique to specific lineages , the low refseq detection rate was unusual . we therefore made a decision that noncode would not include mitranscriptome data in the current version . in the future , we will attempt to make clear the real reason(s ) . perhaps a more comprehensive construction tool is required to answer this question . national high technology research and development program ( 863 program ) of china [ 2014aa021103 , 2014aa021502 ] ; training program of the major research plan of the national natural science foundation of china ; national natural science foundation of china [ 31371320 , 31401119 ] ; chinese academy of science strategic project of leading science and technology [ xda01020402 ] . funding for open access charge : national high technology research and development program ( 863 program ) of china [ 2014aa021103 ] . the chemical modification of proteins is an established tool for studying the structure , function , and regulation of this class of biopolymer . moreover , in recent years , a great deal of effort has been directed toward the modification of proteins for therapeutic applications . traditionally , protein conjugation chemistries have exploited the reactivity of surface - exposed nucleophilic amino acids , such as cysteine or lysine , however , these methods typically result in heterogeneous mixtures of products , which can complicate biological studies or efficacious medicinal applications . to address this concern , several strategies have been developed for the site - specific modification of proteins , ranging from total chemical synthesis ( usually via native chemical ligation , ncl ) to the genetic incorporation of unnatural amino acids or bio - orthogonal functional groups . in between these two extremes the most widely used protein semisynthesis technique is an extension of ncl termed expressed protein ligation ( epl ) in which a recombinant protein -thioester building block is ligated to a synthetic molecule equipped with a 1,2-aminothiol moiety ( most commonly an n - terminal cys - containing peptide ) through the formation of a native peptide bond ( scheme 1 ) . since its inception , epl has been applied to a wide variety of proteins , including enzymes , ion channels , transcription factors , transmembrane receptors , and antibodies ( for reviews see refs ( 15 and 16 ) ) . one of the basic requirements of epl is a thioester group at the c - terminus of a recombinant protein . this reactive handle is introduced by exploiting a process known as protein splicing ( scheme 1 ) , which is mediated by an autoprocessing domain called an intein . protein splicing typically takes place through the formation of one or more protein thioester intermediates , which ultimately resolve to form a native peptide bond between the sequences flanking the intein ( referred to as n- and c - exteins ) . by using appropriate intein mutants , it is possible to intercept these intermediates with exogenous thiols , resulting in an n - extein of choice being cleaved from the mutant intein as a reactive -thioester derivative suitable for chemical ligation . int and int represent n- and c - intein fragments , respectively . despite the many successes of epl , the approach often suffers from low overall efficiency due to complications associated with the generation of protein -thioesters . in particular , fusions to inteins are , to varying extents , susceptible to premature extein cleavage , both in vivo and during initial purification from cell lysates , which reduces the isolated yield of the intein fusion needed for the subsequent thiolysis step . importantly , the cleaved extein side - product is unreactive toward epl , and its separation from the desired -thioester or the ligation product is often difficult for large proteins , such as antibodies . to compound matters , the thiolysis reaction itself can be slow and inefficient , further strengthening the need to develop customized purification regimes , involving multiple chromatographic steps , to isolate the desired product from complex mixtures . collectively , these technical issues mean that a considerable investment in time and resources is usually required before a semisynthetic protein is obtained in useful quantities . ( a ) schematic of the procedure for the isolation of an -thioester derivative of a protein of interest ( poi ) using engineered split intein fragments ( int and int ) . epl can be performed in a one - pot fashion during thiolysis from the split intein or immediately after elution , without need of any further purification . ( b ) sequences of npu ( wt ) and the npuaa mutant used on the split intein column . catalytic residues mutated in npuaa are shown in bold , and the linker sequence added for immobilization onto the solid support is underlined . sequences are numbered according to the intein sequence alignment shown in figure s2 . to overcome the various drawbacks associated with the intein thiolysis process central to epl , we envisioned an alternative strategy based on naturally occurring split inteins . unlike inteins used in standard epl , which are contiguous polypeptides that catalyze protein splicing in cis , split inteins consist of two discrete polypeptides , herein termed int and int , which , upon association , catalyze protein splicing in trans ( protein trans - splicing , pts , scheme 1 ) . split inteins have two important properties that make them attractive for an improved epl strategy . first , cognate int and int pairs often bind tightly to one another ; dissociation constants in the low nanomolar range , reflecting extremely fast on - rates , have been reported for members of the naturally split dnae inteins from cyanobacteria . this ability of split inteins to self - associate has recently been exploited by lu et al . as part of a traceless protein purification system , in this case using an artificially split intein pair . the potential utility of split inteins in epl is further enhanced by the remarkable splicing efficiency of some members of the family . for instance , many of the split dnae inteins have half - lives for the splicing reaction of less than a minute , as compared to several hours in the case of the cis - splicing inteins commonly used in epl . moreover , recent mechanistic investigations indicate that these ultrafast dnae inteins have a highly activated n - terminal splice junction , making them superior reagents for protein -thioester generation . given the unique properties of split inteins , in particular , the ultrafast split dnae inteins , we conceived the integrated protein modification system shown in figure 1 in which the split intein association is employed both to purify the desired protein from complex biological mixtures and to trigger the generation of a desired protein -thioester for epl . in principle , this complementation system should address the major issues attendant to the standard epl protocol , including premature cleavage of the intein , which can not occur in the case of a split intein fragment absent its cognate partner . to implement our system , we designed a mutant of the ultrafast nostoc punctiforme ( npu ) split dnae intein suitable for efficient -thioester generation . specifically , we mutated the catalytic c - terminal residue in the int fragment ( asn137 ) and the first residue in the c - extein ( cys+1 ) to ala , to allow for efficient build up of the desired splicing intermediates upon exposure to an n - extein - npu fusion ( figure 1 ) . preliminary studies showed that mixing n - extein - npu fusions ( where n - extein corresponded to various model proteins ) with the mutant npu ( npuaa ) led to highly efficient n - extein -thioester formation in a thiol - dependent manner ( figures s3 and s4 ) . importantly , only very low levels of intein cleavage ( i.e. , unwanted hydrolysis ) were observed in the absence of thiols , thereby fulfilling a requirement of our integrated strategy . encouraged by these results , we adapted the system for the one - pot purification and generation of c - terminally modified proteins by taking full advantage of the strong and specific interaction between the split intein fragments . accordingly , the npuaa peptide was immobilized on a solid support through a unique cys residue engineered within its c - extein region ( figure s5 ) . the resulting npuaa column ( hereafter referred to as int column ) was then evaluated as an affinity - modification resin . three test proteins maltose binding protein ( mbp ) , ubiquitin ( ub ) , and protein histidine phosphatase type 1 ( phpt1 ) were genetically fused to npu and expressed in escherichia coli . in each case , cells were lysed , and the soluble fraction was loaded onto the int column to allow binding of the npu tagged protein to the immobilized npuaa . after a brief incubation ( 5 min at rt ) the column was extensively washed to remove contaminants , and thiolysis was triggered by addition of a buffer containing the thiol , mercaptoethansulfonate ( mes ) . in all three cases , the desired -thioester protein eluted from the int column with high recovery ( 7595% ) and high purity ( 95% as determined by rp - hplc and mass spectrometry ) ( figure 2 ) . total isolated yields of purified protein -thioesters varied from one protein to another and ranged from 2.5 mg ( per l of bacterial culture ) for ub - mes to 40 mg for mbp - mes . the calculated loading capacity of the int column used in these experiments was 36 mg of protein per ml of beads ( 0.12 mols / ml ) , but higher or lower loadings could easily be achieved by modifying the amount of npu - aa immobilized on the solid support . furthermore , we showed that the int column could be regenerated and reused at least 5 times with only minimal loss of capacity ( figures s11 and s12 ) . the utility of the -thioester derivatives of ub , mbp , and phpt1 obtained from the column was demonstrated by ligating each of them to an n - terminal cys - containing fluorescent peptide ( cgk(fl ) ) to give the corresponding semisynthetic products in excellent yield ( figure s10 ) . importantly , one - pot thiolysis / ligation reactions could be carried out , which allowed us to obtain a site - specifically modified protein directly from cell lysates without isolating the intermediate thioester ( figure 3 ) . ( a ) mbp , ( b ) phpt1 , and ( c ) ub mercaptoethansulfonate ( mes ) -thioesters were purified in one step from e. coli cell lysates using the int column . the purifications were monitored by coomassie stained sds - page analysis ( top ) ( inp : input , ft : column flow - through , w1 - 3 : washes , e1 - 4 : elutions , and bds : resin beads ) . rp - hplc ( detection at 214 nm ) and esi - tof ms analysis of the eluted fractions ( bottom left and right , respectively ) confirmed the identity of all protein -thioesters and indicated high purity . one - pot purification / ligation of ubiquitin to the h - cgk(fluorescein)-nh2 peptide ( cgk(fl ) ) . ub - npu from e. coli cell lysates was bound to the int column , as shown in figure 2 , and after removal of contaminants through extensive washes , intein cleavage and ligation were triggered by addition of 200 mm mes and 1 mm cgk(fl ) peptide . coomassie stained sds - page analysis and in gel fluorescence of the purification / ligation ( left ) . rp - hplc ( detection at 214 and 440 nm ) and esi - tof ms ( right ) of the eluted fractions confirm the desired ligated protein was obtained in one step directly from cell lysates with a ligation yield close to 95% ( quantified by rp - hplc ) . an attractive feature of epl is that it can allow for the preparation of site - specifically modified proteins in a virtually traceless manner . this is contingent on the ability to efficiently generate recombinant protein -thioesters when any of the 20 proteinogenic amino acids are present at the c - terminus of the protein . the activity of split inteins is known to be sensitive to the identity of the amino acids immediately flanking the splice junction . thus , we were eager to test the generality of our strategy and asked whether we could generate -thioesters of all 20 amino acids , using ubiquitin as the n - extein template . twenty ub - npu fusion proteins were individually expressed in e. coli and purified over the int column as before . thiol - induced cleavage yields from the solid support were determined by sds - page analysis of the eluted and resin beads fractions , and levels of competing side reactions ( mainly hydrolysis ) were measured by rp - hplc and esi - tof ms . the results clearly show that for most amino acids > 60% of the bound proteins were recovered after mes treatment . furthermore , 8095% of the eluted material was the desired -thioester product . the only exceptions were pro and glu , for which recovery were 49 and 50% , respectively ( figure 4 ) . the asn mutant displayed high levels of cleavage from the split intein , but almost no -thioester could be isolated due to side - chain cyclization to form a c - terminal succinimide . a second problematic residue was asp , for which we observed some premature cleavage during initial binding to the int resin . moreover , rp - hplc analysis of the eluted fractions upon thiolysis revealed two species with the molecular weight of the desired -thioester . these results were not wholly unexpected , as asp is known to cleave prematurely from contiguous inteins through side - chain cyclization , and its -thioesters have been reported to migrate to the side - chain carboxylate yielding mixtures of - and -isomers . these minor constraints aside , it is clear from these studies that our streamlined epl system is compatible when the majority of amino acids are present as the last residue in the protein of interest . the 20 mutants of the protein ub - x - npu were expressed in e. coli varying the identity of the c - terminal amino acid of ub ( x ) from the wt gly to all other proteinogenic amino acids . thiol - induced cleavage yields from the int column were calculated from the sds - page analysis of the eluted fractions and left over resin beads . ratios of -thioester vs side products were determined from rp - hplc and esi - tof ms analysis of the eluted fractions . the major competing reaction for all amino acids was hydrolysis with the exception of asn for which its succinimide form was isolated instead . * protein semisynthesis frequently requires the preparation of protein fragments , which are often poorly behaved and need to be purified in the presence of strong chaotropic agents . we first confirmed that the model protein ub - npu could bind the int column in the presence of 2 and 4 m urea and that the corresponding ub -thioester could be generated with similar yields as obtained under native conditions ( figure s14 ) . this is consistent with a previous study , which demonstrated npu dnae could splice in the presence of high concentration of denaturants . we then turned to a more challenging target , namely a fragment of histone h2b ( residues 1116 ) , a polypeptide that is prone to aggregation and difficult to generate as an -thioester derivative using standard epl procedures . we expressed human histone h2b(1116 ) fused to npu in e. coli , extracted it from inclusion bodies in 6 m urea , and diluted it to 2 m urea prior to loading on the int - intein column . the incubation was performed for 3 h at ph 6.0 to maximize binding while avoiding premature cleavage through hydrolysis . the ph was then raised to 7.2 , and thiolysis was carried out for 36 h at rt ( note that the presence of the denaturant slows down the thiolysis rate ) . using these conditions , hh2b(1116)-mes was obtained in excellent purity ( > 90% by rp - hplc ) and isolated yield ( 20 mg per l of culture ) . this represents a significant improvement over previous protocols which afford less protein ( 4 mg per l of culture ) and require the use of multiple chromatographic purification steps including rp - hplc . importantly , the hh2b(1116)-mes thioester obtained from the int column could be directly used in epl reactions without further purification . accordingly , we successfully ligated the protein to a hh2b(117125 ) peptide containing an acetylated lys at position 120 to yield semisynthetic hh2b - k120ac ( figure 5 ) . coomassie stained sds - page analysis of hh2b(1116 ) -thioester generation in the presence of 2 m urea ( sup : cell lysate supernatant , trit : 1% triton wash of the inclusion bodies , inp : solubilized inclusion bodies used as input for the int column ) . e1e3 were collected after 18 h of incubation with mes and e4e6 after an additional 18 h. e1e6 were pooled , concentrated to 150 m , and ligated to the peptide h - cvtk(ac)ytsak - oh at 1 mm for 3 h at rt . ( b ) rp - hplc ( left ) of the ligation reaction mixture and ms ( right ) of the ligated hh2b - k120ac product . finally , we tested our streamlined epl methodology for the modification of a monoclonal antibody . the site - specific modification of antibodies has become highly desirable in the area of biopharmaceuticals and diagnostics . currently , most commercially utilized methods to conjugate cargo to antibodies are relatively nonspecific and result in polydisperse mixtures that may vary from batch - to - batch . since this heterogeneity can adversely affect both efficacy and safety of the conjugate indeed , protein semisynthesis via standard epl and pts has recently been used to generate monoclonal antibody conjugates with full activity . given this , we were eager to see whether our streamlined epl process could be used in the facile generation of antibody conjugates . as a model immunoglobulin ( igg ) for our studies , we used an antibody against the dec205 receptor , a c - type lectin found predominantly on dendritic cells . accordingly , we designed a construct in which npu was fused to the c - terminus of the heavy chain of the antibody ( dec205-npu ) . initial expression tests of dec205-npu in 293 t cells resulted in very low levels of the antibody being secreted ( figure 6a ) . we have observed previously that the identity of the int can have an effect on expression levels of its fusions . consequently , we asked whether we could obtain higher levels of secreted dec205-int by varying the identity of the intein n - fragment . several new dec205-int constructs were generated in which int corresponded to the n - fragment of a series of ultrafast split dnae inteins , namely , ava , csp , cra , cwa , mcht , oli , and ter . we also tested an npu mutant ( c - s ) where the noncatalytic cysteines , cys28 and cys59 , were mutated to ser , to determine whether these residues influence secretion and maturation of the igg tetramer ( figure 6a ) . importantly , each of the int fragments in this set can cross - react with the c - fragment of npu without significant loss of splicing efficiency . thus , the int column already in hand is compatible with all of these dec205-int fusions . the contiguous mxe gyra intein was also fused to dec205 to test whether the use of n - intein fragments negatively affected expression levels compared to a full - length intein . an expression screen of this dec205-int library was performed in 293 t cells , revealing that the ava and csp fusions reproducibly exhibited higher expression levels than the other n - inteins , with the former being the best . indeed , the expression levels of the dec - ava construct were at least as good as the dec - gyra construct . based on this , the dec205-ava fusion was chosen and purified over the int column in an analogous manner to that of the soluble proteins described above ( figure 6b ) . elutions from the column contained the thiolyzed dec205 , which was subsequently ligated to the cgk(fl ) peptide ( figure 6c ) . size exclusion chromatography ( sec ) coupled to multiple angle light scattering ( mals ) analysis confirmed that the antibody retained its tetrameric folded state after thiolysis and ligation ( figure 6d ) . we also performed ms analysis of the deglycosylated and fully reduced antibody , which confirmed the formation of a stable , nonreducible amide bond between the dec205 heavy chain and the fluorescent peptide with a 75% yield ( figure 6e ) . importantly , we demonstrated that the semisynthetic dec - cgk(fl ) retains its ability to bind the dec205 receptor to the same extent as a control dec205 , previously shown to be fully functional in vivo(46 ) ( figure 7 ) . binding of dec - cgk(fl ) to the dec205 receptor could be monitored not only by flow cytometry using an antimouse igg secondary antibody ( figure 7 ) but also through the site - specifically incorporated fluorescein ( figure s20 ) . purification of a monoclonal antibody -thioester using a split intein and its site - specific modification . ( a ) test expression of dec205 genetically fused to the contiguous mxe gyra intein and different split dnae inteins through the c - terminus of its heavy chain ( hc ) . western blot of 293 t cell supernatants of several dec205-int fusions using an antibody against mouse igg . ( c ) elution fractions containing dec205-mes were concentrated to 20 m and ligated to the cgk(fl ) fluorescent peptide at 1 mm for 48 h at rt . ( d ) sec - mals analysis of the ligated antibody showing that it retains its tetrameric structure after thiolysis and ligation ( mw = 151 kda , mw calcd = 148 kda ) . ( e ) esi - tof ms analysis of degycosylated and fully reduced hc after ligation , showing 75% of the hc is labeled . ( a ) dose dependent binding of dec205-cgk(fl ) to cho cells expressing the mouse dec205 receptor monitored by flow cytometry using a pe - labeled -mouse igg . binding to control cho / neo cells , which do not express the receptor is shown in gray . ( b ) as in ( a ) but using a control -dec205 antibody . we have shown that split dnae inteins can be engineered for the efficient generation and isolation of protein -thioesters . furthermore , the strategy can be seamlessly integrated with epl , and one pot purification / ligations can be performed without isolation of the -thioester intermediates . we note , however , that the ligation step ( i.e. , ncl ) still requires the use of high concentrations ( high m or above ) of n - terminal cysteine peptide for efficient reactions . the strong and specific interaction between the two intein fragments facilitates the purification of protein -thioesters under a variety of conditions , including strong denaturants , and the isolation of these products directly from cell lysates proceeds significantly faster than via many mainstream epl strategies , which often take multiple days and several intermediate enrichment / purification steps . additionally , while split intein fusions are usually associated with low levels of expression , we show here that certain dnae int fragments express to the same levels as commonly used contiguous inteins when fused to a monoclonal ab . moreover , the absence of premature cleavage events allowed us to generate semisynthetic proteins , such as h2b - k120ac in far superior yields than those of previously established protocols . importantly , we have shown the utility of this methodology for the modification of complex biomolecules such as an igg . thus , streamlined epl via our split dnae intein column should provide an efficient route to site - specifically modified proteins for basic biochemical research as well as translational applications . npu - aa - cys - ome peptide ( 0.5 mols per ml of resin ) was dissolved in 2 column volumes ( cv ) of coupling buffer ( 50 mm trishcl at ph 8.5 , 250 l for 125 l resin ) and treated with 25 mm tcep from a 1 m stock for 15 min . the peptide solution was then added to 1 cv of agarose sulfolink resin ( from pierce , loading : 18.4 mol iodoacetyl groups / ml of resin ) in a small fritted column and incubated for 15 min on a nutator , followed by 30 min standing at rt . the column flow - through was collected , and the column was washed twice with 2 cv of coupling buffer . unreacted iodoacetyl groups on the resin were blocked by a treatment with 50 mm h - cys - ome in coupling buffer for 15 min on a nutator , followed by 30 min standing at rt . column was washed twice with 1 cv of coupling buffer , 2 cv of 1 m nacl , and finally 2 cv of water . int columns were stored in storage buffer ( 100 mm phosphate , 150 mm nacl , 1 mm edta , 0.05% nan3 , ph 7.2 ) , at 4 c for up to 2 weeks . e. coli bl21(de3 ) cells transformed with the desired poi - npu plasmid were grown in 1 l of lb containing 100 g / ml of ampicillin at 37 c until od600 = 0.6 . after harvesting the cells by centrifugation ( 10 500 rcf , 30 min ) , the cell pellets were transferred to 50 ml conical tubes with 20 ml of high - salt binding buffer ( 100 mm phosphate , 500 mm nacl , 1 mm edta , 1 mm tcep , ph 7.2 ) with complete protease inhibitor cocktail ( roche ) and stored at 80 c . resuspended cells were lysed by sonication , and the soluble fraction recovered by centrifugation ( 17 000 rcf , 10 min ) . onto 62.5 l of int column , 300 l of the soluble fraction were loaded and incubated at rt for 5 min . after incubation , the flow - through was collected , and the column was washed with 300 l of high - salt binding buffer , 300 l of wash buffer ( 100 mm phosphate , 300 mm nacl , 1 mm edta , 1 mm tcep , ph 7.2 ) , and 300 l of binding buffer ( 100 mm phosphate , 150 mm nacl , 1 mm edta , 1 mm tcep , ph 7.2 ) . the column was capped and incubated with 150 l of elution buffer ( 100 mm phosphate , 150 mm nacl , 200 mm mes , 10 mm tcep , 1 mm edta , ph 7.2 ) for 18 h. flow - through was collected , and the column was washed three times with 150 l elution buffer . onto 62.5 l of int column , 300 l cell lysate containing ub - npu was loaded and incubated as described before . the column was washed with 300 l high - salt binding buffer , 300 l wash buffer , and 300 l binding buffer . the column was capped and incubated with 75 l epl buffer ( 100 mm phosphate , 150 mm nacl , 200 mm mes , 50 mm mpaa , 1 mm edta , 10 mm tcep , ph 7.9 ) containing 1 mm cgk(fl ) peptide for 18 h. the flow - through was collected , and the column was washed three times with 75 l elution buffer . gels were first imaged using the green fluorescence channel on a ge imagequant las 4010 imager and then coomassie stained . t cells were transiently cotransfected with antimouse - dec205-lc and antimouse - dec205-hc - ava using lipofectamine 2000 ( invitrogen ) , according to the manufacturer s instructions . after 4 days incubation at 37 c with 5% co2 , cell supernatants were harvested and spun down at 2000 rcf for 20 min at 4 c , filtered through a 0.22 m filter , and supplemented with complete protease inhbitors . for a typical purification , 50 ml of dec205-ava transfected cell supernatants were concentrated to a final volume of 5 ml and exchanged into binding buffer . the resulting ab solution ( input ) was applied to an int column of 300 l beads ( loading : 1.8 mol npuc peptide / ml ) and incubated at rt for 30 min . column flow - through was collected , and column washed three times with 3 cv of wash buffer and once with 3 cv of binding buffer . the column was capped and incubated with 3 cv of ab elution buffer ( 100 mm phosphate , 150 mm nacl , 200 mm mes , 1 mm tcep , 1 mm edta , ph 7.2 ) for 20 h. the column flow - through was collected , and the column washed three times with 3 cv of ab elution buffer . ligation was initiated by addition of 1 mm cgk(fl ) peptide and 1 mm tcep and adjusting ph to 7.58.0 . the reaction was incubated in the dark at rt for 48 h and monitored by sds - page imaged using a fluorescence scanner and coomassie staining . once the reaction was completed , the ligated ab was diluted to 200500 l and dialyzed into 100 mm phosphate , 150 mm nacl , 1 mm edta , 1 mm tcep , ph 7.2 .

noncode ( http://www.bioinfo.org/noncode/ ) is an interactive database that aims to present the most complete collection and annotation of non - coding rnas , especially long non - coding rnas ( lncrnas ) . the recently reduced cost of rna sequencing has produced an explosion of newly identified data . revolutionary third - generation sequencing methods have also contributed to more accurate annotations . accumulative experimental data also provides more comprehensive knowledge of lncrna functions . in this update , noncode has added six new species , bringing the total to 16 species altogether . the lncrnas in noncode have increased from 210 831 to 527,336 . for human and mouse , the lncrna numbers are 167,150 and 130,558 , respectively . noncode 2016 has also introduced three important new features : ( i ) conservation annotation ; ( ii ) the relationships between lncrnas and diseases ; and ( iii ) an interface to choose high - quality datasets through predicted scores , literature support and long - read sequencing method support . noncode is also accessible through http://www.noncode.org/. chemically modified proteins are invaluable tools for studying the molecular details of biological processes , and they also hold great potential as new therapeutic agents . several methods have been developed for the site - specific modification of proteins , one of the most widely used being expressed protein ligation ( epl ) in which a recombinant -thioester is ligated to an n - terminal cys - containing peptide . despite the widespread use of epl , the generation and isolation of the required recombinant protein -thioesters remain challenging . we describe here a new method for the preparation and purification of recombinant protein -thioesters using engineered versions of naturally split dnae inteins . this family of autoprocessing enzymes is closely related to the inteins currently used for protein -thioester generation , but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active . taking advantage of the strong affinity between the two split intein fragments , we devised a streamlined procedure for the purification and generation of protein -thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site - specifically modified monoclonal antibody .

ameloblastoma is a benign but locally aggressive neoplasm of the odontogenic epithelium.1 accounting for 11% of all odontogenic tumors , ameloblastoma is the most common odontogenic neoplasm affecting the jaws , yet it only accounts for 1% of all tumors of the maxilla and mandible.24 the average age of patients presenting with ameloblastoma is 36 years , with men and women being equally affected.5 ameloblastomas are classified as solid / multicystic , intraosseous , or unicystic , with peripheral subtypes.6,7 the radiographic appearance of ameloblastoma , dentigerous cysts , and odontogenic keratocysts is similar and for this reason a biopsy is recommended to obtain a precise diagnosis of ameloblastoma.8 although a benign tumor , ameloblastoma is aggressive because of the myeline nature of its growth and because its conservative treatment is associated with a high rate of recurrence ( 50%90% ) . primary resection of this benign tumor is , therefore , considered the only predictable curative form of therapy.711 unfortunately , this treatment leaves the patient with a defect in the affected region of the jaw . a multi - disciplinary approach is needed for the patient s complete rehabilitation , including bone grafting , the planned placement of implant , and the prosthetic work . sometimes the orthodontist s role in this multidisciplinary approach is to create an optimum occlusal relationship and sufficient space to allow for successful reconstruction of the affected region of the jaw . a 31-year - old caucasian female was referred to the graduate orthodontic clinic of aristotle university of thessaloniki following mandibulectomy and immediate replacement of removed bone with autologous calvarial bone graft for the purposes of evaluation and treatment of occlusal disturbances . the patient s medical / dental history referred to a tumor , which was histologically classified as follicular ameloblastoma . according to the history , the tumor was located in the posterior right mandibular region encompassing three teeth , specifically the first and second premolars and the first molar . the first author performed a partial mandibular resection and an immediate reconstruction utilizing bone graft taken from the patient s calvaria . extraoral examination revealed an orthognathic profile without facial asymmetry and with the lips competent at rest ( figure 2 ) . intraoral examination revealed amalgam restorations in all maxillary and mandibular second molars , in the left first maxillary premolar , and the left mandibular first molar . clinical examination indicated satisfactory dental hygiene and periodontal condition , with absence of bleeding on probing and no significant pocket depths . an analysis of dental casts revealed a class i malocclusion characterized by a 3 mm overjet , 3 mm of overbite , moderate crowding in the anterior region of both dental arches ( 3 mm crowding in the maxillary dental arch and 6 mm in the mandibular one , respectively ) , a labially positioned mandibular right canine , a 2 mm deviation of the lower dental arch midline to the right , moderate bolton tooth size discrepancy ( mandibular anterior relatively larger than maxillary ones ) , and an edentulous space due to the aforementioned surgical procedure ( figure 2 ) . analysis of the cephalometric radiograph indicated harmonious sagittal and vertical skeletal relationships ( sna : 83 , snb : 80 , facial angle : 90 , anb : 3 , individual anb : 3.5 ) ( figure 3 ) . the problem list for this patient included the presence of a class i malocclusion characterized by a condition of moderate crowding in the anterior region of both dental arches , a labially positioned mandibular right canine , a mild deviation of the mandibular dental arch midline to the right , a moderate tooth size discrepancy , and an edentulous space in the right posterior area of the mandible due to the extractions of the right premolars and first molar . the patient s chief complaint referred to the need for optimum prosthetic rehabilitation and a desire for improvement of dental esthetics . a comprehensive orthodontic treatment aiming to correct the above - mentioned occlusal problems was proposed and accepted by the patient . the placement of the two dental implants took place by the first author prior to the orthodontic treatment and according to the orthodontic treatment plan . a setup of the patient s dental casts was made to facilitate accurate determination of the exact positions of the two implants . following their placement , a lingual arch was banded on the lower dental arch for anchorage and space preservation . fixed appliances ( .018 x .022 , roth ) were bonded on the maxillary teeth in order to level and align the dental arch . following the placement of a bracket , the mandibular right canine was uprighted and brought into the dental arch by means of a t - loop . then , fixed appliances were bonded to the remaining mandibular teeth and reproximation took place on the lower incisors . the fixed appliances were removed after alignment and leveling of both dental arches , crowding alleviation , and correction of dental arch midlines . essix maxillary and mandibular splints were used for retention until the placement of prostheses ( figure 4 ) . new essix mandibular retainer was delivered following the end of prosthetic work . due to the patient s advanced stage of pregnancy ( 9 month ) and the relevant inconvenience , at this stage it was not possible to place a bonded lingual retainer in the lower anterior teeth , as was originally planned . superimposition of the initial and final tracings of the lateral cephalometric x - rays indicated that slight labial proclination of the upper and lower incisors occurred post - treatment ( figure 5 ) . prosthodontic rehabilitation of the partially edentulous right mandibular dental arch region was achieved through the placement of two implants and two crowns , respectively ( figure 6 ) . a 31-year - old caucasian female was referred to the graduate orthodontic clinic of aristotle university of thessaloniki following mandibulectomy and immediate replacement of removed bone with autologous calvarial bone graft for the purposes of evaluation and treatment of occlusal disturbances . the patient s medical / dental history referred to a tumor , which was histologically classified as follicular ameloblastoma . according to the history , the tumor was located in the posterior right mandibular region encompassing three teeth , specifically the first and second premolars and the first molar . the first author performed a partial mandibular resection and an immediate reconstruction utilizing bone graft taken from the patient s calvaria . extraoral examination revealed an orthognathic profile without facial asymmetry and with the lips competent at rest ( figure 2 ) . intraoral examination revealed amalgam restorations in all maxillary and mandibular second molars , in the left first maxillary premolar , and the left mandibular first molar . clinical examination indicated satisfactory dental hygiene and periodontal condition , with absence of bleeding on probing and no significant pocket depths . an analysis of dental casts revealed a class i malocclusion characterized by a 3 mm overjet , 3 mm of overbite , moderate crowding in the anterior region of both dental arches ( 3 mm crowding in the maxillary dental arch and 6 mm in the mandibular one , respectively ) , a labially positioned mandibular right canine , a 2 mm deviation of the lower dental arch midline to the right , moderate bolton tooth size discrepancy ( mandibular anterior relatively larger than maxillary ones ) , and an edentulous space due to the aforementioned surgical procedure ( figure 2 ) . analysis of the cephalometric radiograph indicated harmonious sagittal and vertical skeletal relationships ( sna : 83 , snb : 80 , facial angle : 90 , anb : 3 , individual anb : 3.5 ) ( figure 3 ) . the problem list for this patient included the presence of a class i malocclusion characterized by a condition of moderate crowding in the anterior region of both dental arches , a labially positioned mandibular right canine , a mild deviation of the mandibular dental arch midline to the right , a moderate tooth size discrepancy , and an edentulous space in the right posterior area of the mandible due to the extractions of the right premolars and first molar . the patient s chief complaint referred to the need for optimum prosthetic rehabilitation and a desire for improvement of dental esthetics . a comprehensive orthodontic treatment aiming to correct the above - mentioned occlusal problems was proposed and accepted by the patient . the placement of the two dental implants took place by the first author prior to the orthodontic treatment and according to the orthodontic treatment plan . a setup of the patient s dental casts was made to facilitate accurate determination of the exact positions of the two implants . following their placement , a lingual arch fixed appliances ( .018 x .022 , roth ) were bonded on the maxillary teeth in order to level and align the dental arch . following the placement of a bracket , the mandibular right canine was uprighted and brought into the dental arch by means of a t - loop . then , fixed appliances were bonded to the remaining mandibular teeth and reproximation took place on the lower incisors . the fixed appliances were removed after alignment and leveling of both dental arches , crowding alleviation , and correction of dental arch midlines . essix maxillary and mandibular splints were used for retention until the placement of prostheses ( figure 4 ) . new essix mandibular retainer was delivered following the end of prosthetic work . due to the patient s advanced stage of pregnancy ( 9 month ) and the relevant inconvenience , at this stage it was not possible to place a bonded lingual retainer in the lower anterior teeth , as was originally planned . superimposition of the initial and final tracings of the lateral cephalometric x - rays indicated that slight labial proclination of the upper and lower incisors occurred post - treatment ( figure 5 ) . prosthodontic rehabilitation of the partially edentulous right mandibular dental arch region was achieved through the placement of two implants and two crowns , respectively ( figure 6 ) . ameloblastoma is a benign odontogenic tumor arising from the residual epithelial components of tooth development . it is a slow growing , locally aggressive tumor capable of causing facial deformity , with a high recurrence rate due its capacity to infiltrate trabecular bone . bone grafts replace the surgically removed bone , with autologous bone grafting being the most desirable . it is typically harvested from intraoral sources ( e.g. , chin ) or extraoral sources ( e.g. , iliac crest , fibula , calvarial bone ) . the most commonly used graft material for alveolar ridge reconstruction is free autogenous iliac bone.12 in this case , however , autologous calvarial bone grafts were used to reconstruct the missing mandibular bone following the surgical resection of the tumor and the removal of three teeth in the region . the advantages of calvarial bone grafting include good integration , absence of pain from the donor site , and no visible scar . these advantages , however , are not applicable in the case of thin calvaria bone with a thickness of less than 5 mm.12 recent reports on the use of calvarial bone grafting for the reconstruction and subsequent placement of dental implants have presented good clinical outcomes , with low rates of graft resorption and high implant survival rates.1316 the results of these studies have showed that calvarial bone grafting appears to be less prone to resorption than iliac grafts are . in this case , this goal could have been achieved by the placement of two implants and a bridge , replacing all three teeth . however , this treatment plan would not have addressed the patient s chief complaint , nor would it result in optimum functionality and esthetics . accordingly , the placement of the two implants was decided in relation to the orthodontic treatment plan , aiming for an optimum result . the two implants were placed in the posterior region of the edentulous area , hence replacing only two of the missing teeth , with the extra space being used to correct crowding and to improve dental occlusion . the outcome of this interdisciplinary approach was the satisfactory restoration of both occlusal function and esthetics . a multidisciplinary approach , including oral surgery , orthodontics , and prosthodontics was able to provide a patient diagnosed with follicular ameloblastoma in the right mandibular region with a satisfactory occlusion following partial mandibular resection and removal of adjacent teeth in the affected site . the laparoscopic removal of a cervical stump following a supra cervical ( subtotal ) hysterectomy was first described by nezhat et al , and they concluded that the cervical stump could be removed laparoscopically by an experienced surgeon . the advantages of the laparoscopic approach included possible stump adhesiolysis , providing adequate postoperative vault support , and assessment of the pelvic lymph nodes . the 43-year - old , presented with a history of persistent p v discharge and occasional post - coital bleeding . she had undergone subtotal hysterectomy in 1994 , due to postpartum hemorrhage following a normal delivery . a colposcopic biopsy done in january 2009 , reported severe dysplasia of the cervix , with a human papillomavirus ( hpv ) effect and crypt extension . there was a strong family history of cancer of the cervix , as her mother had succumbed to the disease . on general examination she was in fair general condition , well - built and well - nourished , with adequate hydration . the hemoglobin was 13.3 g / dl , blood sugar was 5.3 mmols / l , urea and electrolytes were normal . an initial diagnosis of abnormal pap smear was entertained and the patient opted for a laparoscopic trachelectomy , with the option of a laparotomy , after discussing all her options . there were dense adhesions in the pouch of douglas involving the bowel and the cervical stump . the pelvic lymph nodes were clearly visualized ( after intracervical methylene blue injection ) and did not appear to be enlarged . gentle adhesiolysis was undertaken using sharp dissection , bipolar cautery , and a harmonic scalpel . the vaginal vault was subsequently opened over the ceramic cup of a clermont ferrand elevator . a cystoscopy with retrograde ureteral catheterization , to confirm the integrity of the bladder and ureters , was undertaken . the cervical stump after laparoscopic trachelectomy at one week of follow - up the patient was well . a postoperative intravenous urogram ( ivu ) confirmed that both the ureters and bladder were intact . subtotal hysterectomy was developed as a procedure in the 1990s , and is regarded as a safe option to total abdominal hysterectomy in the management of benign uterine conditions and in obstetrics , due to severe postpartum hemorrhage . okaro et al , in an assessment of the long - term outcomes of laparoscopic supracervical hysterectomy analyzed the case records of 70 consecutive women undergoing the procedure . of these , 24.3% ( 17 cases ) reported symptoms related to the cervical stump , within 14 months of the original surgery . in his series 14 of these patients underwent laparoscopic trachelectomy , one had only laparoscopic adhesiolysis and two underwent a laparotomy with trachelectomy due to dense bowel adhesions on the cervical stump . histologically the stumps showed endometriosis ( 23.5% ) and mild dysplasia in 7.6% of the patients . in this case our patient presented with persistent p v discharge and occasional post - coital bleeding . the subsequent pap smears were abnormal . in a retrospective of 41 patients undergoing laparoscopic subtotal hysterectomy , van der stege et al , noted that 98% of the patients were satisfied with their procedure , with 10% of them having monthly spotting . they concluded that although laparoscopic hysterectomy for benign diseases was a satisfactory procedure , special attention should be paid to careful management of the cervical stump . hilger et al , reviewed the indications of 310 trachelectomies performed at the mayo clinic from 1974 to 2003 . they included stump prolapse ( 4% ) , fibroid mass ( 1% ) , cervical dysplasia ( 6% ) , carcinoma in situ ( 5% ) , irregular bleeding ( 2% ) , and cervicitis ( 53% ) . the complications following vaginal trachelectomies were encountered in 80% of the procedures against 37% in the abdominal procedure . in our report the cervical stump confirmed carcinoma in situ .

this case report describes the combined surgical , orthodontic and prosthodontic rehabilitation of an adult female patient with a previous history of follicular ameloblastoma , which was treated through partial mandibulectomy and an immediate replacement of missing bone with an autologous calvarial bone graft . orthodontic treatment was undertaken in order to restore occlusal disturbances and obtain sufficient space for two dental implants and an optimum prosthodontic rehabilitation . a 43-year - old , who underwent a subtotal hysterectomy for postpartum hemorrhage following a normal delivery , 10 years ago , presented with a history of persistent vaginal discharge and post - coital bleeding . a pap smear reported moderate dysplasia , and a subsequent colposcopic biopsy reported severe dysplasia with crypt extension . the patient underwent a laparoscopic trachelectomy , and histology of the stump reported cervical squamous carcinoma in situ , with no microinvasion .

biological membranes are mainly composed of phospholipids , sphingolipids , cholesterol , and membrane - associated proteins . these molecules are nonhomogeneously distributed in membranes and can rearrange , leading to the formation of membrane domains with highly differentiated molecular compositions and supramolecular architectures , which are stabilized by lateral interactions among the membrane components . although glycosphingolipids ( gsls ) were originally thought to be structural components of plasma membranes , several experiments suggested that gsls are involved in the regulation of numerous cellular functions . the membrane lipid bilayer is a stable structure , constituting a physical boundary between intra- and extracellular environments . gsls are expressed on the surface of cellular membranes . based on their physicochemical properties , especially their many hydroxyl and acetamide groups , which can act as hydrogen bond donors and acceptors , gsls form clusters through cis interactions . there is a general consensus on the roles played by the ceramide moiety of gsls in promoting the formation and stabilization of membrane lipid domains . in addition , ceramide was also shown to be involved in gsl - mediated functions and several biological activities [ 3 , 4 ] . we recently showed that very long fatty acid chains of ceramide , such as c24:0 and c24:1 , are responsible for the direct connection between lactosylceramide ( laccer , cdw17 ) and palmitoylated signal transducer molecules . moreover , the phosphorylated product of sphingosine , sphingosine-1-phosphate ( s1p ) , was shown to be important in immunological , especially inflammatory reactions [ 4 , 6 ] . this review describes the role of the fatty acid chains of ceramide in gsl - mediated outside - in signaling in promoting gsl - enriched domain - mediated cellular functions , as well as the activities of s1p in inflammatory reactions of keratinocytes in human . the most studied gsl - enriched domains are membrane lipid microdomains , called lipid rafts , defined by their gsl- and cholesterol - rich nature , enrichment in gpi - anchored proteins and membrane - anchored signaling molecules , and cytoskeletal association [ 7 , 8 ] . as shown in artificial membrane models , gsls tend to form clusters , with this cluster formation confirmed in intact cells by immunoelectron microscopy [ 1012 ] . the gsl - enriched microdomains on plasma membranes have a diameter of 50100 nm and include signal transducer molecules , such as src family kinases [ 11 , 12 ] . gsls that contain saturated fatty acid chains with higher transition temperatures show ordered , less fluid , liquid phase . cholesterol is composed of a highly hydrophobic sterol - ring system and 3-hydroxy moiety , the only hydrophilic part of the molecule . the small cholesterol sterol - ring system and the ceramide moiety of sphingolipids are thought to interact via hydrogen bonds and hydrophobic van der waal 's interactions . in addition , hydrophilic interactions between sugar moieties of gsls promote the lateral association of gsls and cholesterol . phospholipids tend to be loosely packaged in bilayers , resulting in the formation of liquid - disordered membranes that allow rapid lateral and rotational movement of lipids . these interactions result in the separation of gsl- and cholesterol - enriched lipid microdomains from other phospholipids in the cell membrane and the formation of distinct domains . electron microscopy using labeled anti - gsl antibodies has revealed gsl clusters on the surface of glycosphingolipid / phosphatidylcholine ( pc ) liposomes , even in the absence of sphingomyelin ( sm ) and cholesterol . the anti - laccer mabs t5a7 and huly - m13 recognized laccer on human neutrophils , but only t5a7 recognized laccer on mouse neutrophils . interestingly , huly - m13 but not t5a7 can be used for immunoprecipitation , suggesting a difference in binding and/or cluster formation of huly - m13 and t5a7 to laccer - enriched microdomains . indeed , stimulated emission depletion ( sted ) superresolution microscopy showed that t5a7 and huly - m13 bind to different regions of the same laccer / dioleoylphosphatidylcholine ( dopc ) liposomes ( figure 1 ) . laccer - enriched microdomains are composed of laccer , sm , phospholipids , and cholesterol . surface plasmon resonance analysis showed that reduction of the laccer content in the dopc / cholesterol / laccer / sm lipid layer markedly decreased huly - m13 but not t5a7 binding to laccer , suggesting that the content of laccer in laccer - enriched microdomains affects the binding avidity of huly - m13 to laccer . in contrast , the molecular species of pc , including dopc , dipalmitoylphosphatidylcholine ( dppc ) , and palmitoyl - oleoyl - phosphatidylcholine ( popc ) , did not affect the binding avidity of huly - m13 to laccer - coated plastic wells . lactose inhibited the binding of huly - m13 to laccer / dopc liposome - coated and dopc / laccer mixture - coated plastic wells , suggesting that huly - m13 binds only to laccer clusters in laccer - enriched microdomains . in contrast , the binding avidity of t5a7 to laccer - coated plastic wells was much weaker than its binding avidity to dopc / laccer- , popc / laccer- , and dppc / laccer mixture - coated wells , suggesting that the binding of t5a7 to laccer is affected by pc . the ability of lactose to inhibit the binding of t5a7 to dopc / laccer liposome - coated plastic wells was similar to its ability to inhibit the binding of huly - m13 . in contrast , lactose inhibition of t5a7 binding to dopc / laccer mixture - coated plastic wells was significantly lower than its inhibition of huly - m13 binding , suggesting that t5a7 recognizes the pc - enhanced three - dimensional structure of laccer clusters . thus , huly - m13 may bind to the core region of lactose clusters in laccer - enriched domains , while t5a7 binds to dispersed laccer clusters in the phase boundary regions of these microdomains . these findings suggest that the specificities of these antibodies against the same gsls are dependent on the organizations of the gsls and molecules surrounding the gsl - enriched domains . disorders of the degradation of gsls sometimes cause human diseases [ 18 , 19 ] . for degradation , , those molecules are constitutively degraded by their suitable catabolic enzymes . when the activities of lysosomal enzymes are impaired , degradation is not able to proceed normally and undegraded molecules accumulate in the organelle and intracellular membranes , causing several metabolism diseases . for instance , genetic disorder of glucocerebrosidase ( gba ) ( ec 3.2.1.45 ; ) , gaucher disease , results in accumulation of glccer and its deacetylated form glucosylsphingosine is caused by abnormality of gba . gaucher disease is a multisystem disorder whose features include peripheral blood cytopenias , hepatosplenomegaly , bone disease , and neurological manifestations in some cases . the form of intravenous enzyme replacement therapy in the 1990s has been developed and resulted in dramatic improvements in haematological and visceral disease . recognition of complications , including multiple myeloma and parkinson disease , has challenged the traditional macrophage - centric view of the pathophysiology of this disorder . however , the pathways by which enzyme deficiency results in the clinical manifestations of this disorder remain obscure . in spinal cords of amyotrophic lateral sclerosis ( als ) patients , levels of gm1 , gm3 , laccer , glccer , galcer , and ceramide were significantly elevated . furthermore , glucocerebrosidase-1 , glucocerebrosidase-2 , hexosaminidase , galactosylceramidase , -galactosidase , and -galactosidase activities were also elevated in those patients . inhibition of glucosylceramide synthesis accelerated disease course in als model mice , whereas infusion of exogenous gm3 significantly slowed the onset of paralysis and increased survival . these observations suggest that gsls and their metabolism are likely important participants in pathogenesis of als . further studies about gsl metabolism pathways in gsl - related disease will serve to advance our understanding of other associated disorders . over the last 30 years , many studies have indicated that gsls expressed on the cell surface may act as binding sites for microorganisms . the binding avidities of microorganisms to several types of gsl [ 2427 ] suggest that gsls are involved in host - pathogen interactions . indeed , microorganisms have been shown to recognize and enter host cells via gsl - enriched membrane microdomains on the cells . among gsls , laccer has been well described to bind to several kinds of microorganisms , including viruses and fungi . for instance , candida albicans specifically bind to laccer though the binding of -1,6-long glucosyl side - chain - branched -1,3-glucan to laccer - enriched domains [ 26 , 29 ] . it is also well known that microorganisms - derived toxins , such as shiga toxin , specifically bind to gsls [ 3032 ] . furthermore , a sphingolipid metabolite , ceramide , has been demonstrated to play a crucial role in pulmonary infection and inflammation .ceramide , which is degraded product of gsls and sphingomyelin , has been reported to form ceramide - rich membrane platforms and involve uptake of several microorganisms including pseudomonas aeruginosa . abnormal amounts of enzymes involved in the synthesis of ceramide have been demonstrated in emphysematic smokers and in patients with severe sepsis . gsls have been reported to interact with membrane proteins and modulate the properties of these proteins [ 2 , 25 ] . in addition , certain proteins , including glycosylphosphatidylinositol- ( gpi- ) anchored and palmitoylated proteins , tend to enter gsl - enriched membrane microdomains . however , the mechanism by which gsls interact with proteins and mediate outside - in signaling is unclear . the ceramide moiety consists of a long chain base linked to a fatty acid chain . sphingosine containing c18 carbons [ ( 2s,3r,4e)-2-amino-1,3-dihydroxy - octadecene ] is generally the main structure in mammals , but a structure containing 20 carbons is relatively abundant in neurons . ceramide is synthesized by ceramide synthases ( cers ) 16 , each of which uses a restricted subset of fatty acyl - coas for n - acylation of the sphingoid long chain base . the expression levels of genes encoding cers are tissue specific , suggesting that the molecular varieties and expression patterns of gsls are associated with the functions of these cells . although gsl - enriched microdomains have been implicated in a number of important membrane events [ 2 , 38 , 39 ] , the molecular mechanisms responsible for gsl - mediated cell functions are still unclear . one of the main issues centers around the association of gsls with signal transducer molecules localized on the cytosolic side . laccer , along with the src family kinase lyn , forms lipid microdomains on the plasma membranes of human neutrophils and is involved in several cellular functions , including chemotaxis , phagocytosis , and superoxide generation , highly dependent on lyn [ 16 , 29 , 38 ] . hl-60 cells differentiated into neutrophilic lineage cells by dmso ( d - hl-60 ) were found to acquire superoxide generating activity , but not through laccer , despite their expression of laccer on plasma membranes . most laccer and lyn were recovered in the microdomain fractions of neutrophils and d - hl-60 cells . lipidomics analysis revealed that laccer in the neutrophil plasma membrane was mainly composed of molecular species containing c16:0 , c24:1 , and c24:0 fatty acid chains , whereas over 70% of laccer in the plasma membranes of d - hl-60 cells contained c16:0 fatty acid chains , but only about 14% were c24:1 and c24:0 . lyn was immunoprecipitated by anti - laccer antibody in neutrophils but not d - hl-60 cells . importantly , lyn was coimmunoprecipitated by anti - laccer antibody from the detergent resistant membrane ( drm ) fraction of plasma membranes from c24:0 and c24:1 , but not c16:0 or c22:0 , laccer - loaded d - hl-60 cells . anti - laccer antibody induced superoxide generation from d - hl-60 cells loaded with c24:0-laccer , but not c16:0-laccer . lyn colocalized with laccer - enriched domains of d - hl-60 cells loaded with c24:0-laccer , but not c16:0-laccer . these results suggested that the c24 fatty acid chain of laccer is indispensable for connecting lyn with laccer - enriched microdomains . knockdown of lyn molecules by human lyn - specific short interfering rna ( sirna ) in d - hl-60 cells completely abolished the effects of c24:1-laccer loading function , suggesting that lyn is crucial for c24-laccer - mediated neutrophil function . experiments using azide - photoactivatable tritium - labeled c24- and c16-laccer revealed that c24- but not c16-laccer directly associated with lyn and a heterotrimeric g protein subunit gi . these results confirm a specific direct interaction between c24-laccer and the signal transduction molecules lyn and gi , which are associated with the cytoplasmic layer via palmitic acid chains ( figure 2 ) . laccer species with very long fatty acids are indispensable for lyn - coupled laccer - enriched membrane microdomain - mediated neutrophil functions . gpi - anchored proteins are composed of glycerol phospholipids , which do not have c24 fatty acid chains , suggesting that gpi - anchored proteins are not able to form large clusters by themselves and can not directly connect with signal transduction molecules through fatty acid chains . to mediate cell functions , gpi - anchored proteins require signal transduction molecule - coupled transmembrane proteins or gsl - enriched domains , such as laccer - enriched domains . the epidermis consists of a single layer of proliferating undifferentiated keratinocytes , the stratum basale , and several superficial layers of the stratum spinosum and stratum granulosum ( sg ) , which form the stratum corneum ( sc ) . the sc acts as an air - liquid interface barrier to avoid drying of tissues in contact with air . ceramide is the main component of sc and is important for the water retention and permeability barrier functions of sc . sphingosine can be phosphorylated by sphingosine kinase to form s1p , a molecule involved in a wide range of cellular functions , including growth , differentiation , survival , chemotaxis , angiogenesis , and embryogenesis , in various types of cells [ 44 , 45 ] . s1p was shown to inhibit keratinocyte proliferation , to promote corneocyte differentiation , and to chemoattract keratinocytes . roles of s1p in skin immunological functions have been demonstrated in mouse models [ 45 , 4751 ] . mice are the good experimental tool of choice for the majority of immunologists , and the study of immune responses in mice has provided considerable insight into human immune system function . however , there are significant differences in immunological reactions between mice and human . little is known , however , about the role of ceramide metabolites in the immunological functions of differentiating keratinocytes . a neutral cdase from pseudomonas aeruginosa an17 ( pacdase ) isolated from a patient with atopic dermatitis ( ad ) was shown to require detergents to hydrolyze ceramide . staphylococcus aureus - derived lipids , which consist primarily of cardiolipin and phosphatidylglycerol , enhanced the pacdase hydrolysis of normal ceramide and of human skin - specific omega - hydroxyacyl ceramide in the absence of detergents . a three - dimensionally cultured human primary keratinocyte ( 3d keratinocyte ) culture system has been utilized to simulate epidermal differentiation at its air - liquid interface , resulting in the generation of basal , spinous , and granular layers and an sc , with the latter displaying permeability barrier functions . treatment of 3d keratinocytes with pacdase and water - soluble stimulants of keratinocytes , including trypsin , dermatophagoides pteronyssinus class 1 allergen ( der p1 ) , and dermatophagoides farinae allergen ( der f1 ) had no effect on the expression of any of the genes in our dna microarray analysis , indicating that the sc of the 3d keratinocyte culture acts as a permeability barrier . triton x-100 is a detergent that reduces permeability barrier functions , thereby moderately increasing transepidermal water loss and the production of erythema on human skin . in the presence of 0.1% triton x-100 , pacdase markedly enhanced tnf- mrna expression in 3d keratinocytes , an increase not observed in cells treated with triton x-100 alone . tnf- mrna expression was not enhanced by heat - inactivated or mutant pacdase , suggesting that ceramide metabolites induce tnf- mrna expression in keratinocytes . tnf- , a critical cytokine in several dermatological diseases , is secreted by keratinocytes and shown to be involved in the progression of atopic dermatitis ( ad ) . among the metabolites of ceramide , s1p is synthesized from sphingosine by sphingosine kinase ( sphk ) and stimulates 3d keratinocytes through specific receptors . both the specific sphk inhibitor cas 1177741 - 83 - 1 and the s1p receptor antagonist vpc 23019 suppressed the pacdase - induced expression of tnf- mrna in 3d keratinocytes . s1p is generally considered to stimulate cells through plasma membrane g protein - coupled receptors , for example , s1p1s1p5 . however , vpc2301 , a competitive antagonist for s1p1 and s1p3 receptors , inhibited the pacdase - enhanced gene expression not only of tnf- but also of endothelin-1 and il-8 . thus , the s1p - induced production of these inflammatory mediators is mediated by s1p receptors in human primary keratinocytes in a 3d culture system . cdna microarray analysis showed that s1p strongly upregulated the expression of endothelin-1 , cxcl1 , tnf- , -defensin 5 , il-8 , cxcl2 , interferon regulatory factor 1 , gadd45 gamma , and il-23 subunit mrnas . il-8 , cxcl1 , and cxcl2 have been reported to be upregulated in the lesional skin of patients with ad and psoriasis . s1p also enhanced the expression of claudin-4 mrna , which has been observed in more layers of psoriatic than normal epidermis . tnf- can induce the production of endothelin-1 and il-8 by human keratinocytes [ 66 , 67 ] . infliximab , a chimeric igg1 monoclonal antibody against human tnf- , inhibits the tnf--mediated production of il-8 by keratinocytes . the nf-b inhibitor curcumin inhibited pacdase - induced expression of il-8 and endothelin-1 mrnas but not of tnf- mrna . therefore , it is likely that s1p induces tnf- production and release from 3d keratinocytes via s1p receptors , resulting in tnf- induction of cytokine production through nf-b - mediated signal transduction ( figure 3 ) . tnf- is a critical cytokine in psoriatic immunopathology , and the development of an effective strategy is required to counteract its effects . infliximab , which is used to treat patients with plaque psoriasis , psoriatic arthritis , pustular psoriasis ( excluding localized type ) , and psoriatic erythroderma [ 71 , 72 ] , downregulates antiapoptotic proteins in regressing psoriatic skin . the effects of infliximab have also been evaluated in other inflammatory dermatoses and in systemic diseases involving the skin , pityriasis rubra pilaris , pyoderma gangrenosum , and cutaneous sarcoidosis . ad is characterized by a marked reduction in ceramides in the sc of lesional and nonlesional forearms [ 74 , 75 ] and by increased activities of the enzymes ceramidase ( cdase ) . the metabolic conversion of ceramide to s1p has been found to protect keratinocytes against uvb - induced , ceramide - mediated apoptosis . histamine h1-receptor blockers are used to treat all types of itch resulting from serious skin diseases , such as ad , as well as from renal and liver diseases . however , they often lack efficacy in chronic itch , a profound clinical problem that decreases quality of life . nerve density in the epidermis is partly involved in itch sensitization in pruritic skin diseases , such as ad . endothelin-1 has been shown to elicit itch in humans [ 8082 ] . the molecular pathways that contribute to the transduction of itch responses to endothelin-1 do not require either plc3 or trpv1 of neurons , which mediate histamine- and serotonin - induced itch responses , respectively . several reports have described the roles of ceramide metabolites in immunological and inflammatory diseases [ 8488 ] . however , the physiological roles of gsl - enriched microdomains are largely undetermined , although much is known about the organization and functions of laccer - enriched microdomains [ 24 , 89 , 90 ] . the analogous patterns of gsls and motifs of pamps result in the generation of autoantibodies against these gsls , inducing severe autoimmune inflammatory diseases . antibodies against neuronal tissues are involved in immune - mediated neurological disorders , with expression of several of these antibodies found to correlate with the pathophysiology of these diseases [ 92 , 93 ] . therefore , elucidation of their organization and structural specificities , based on interactions between gsls and surrounding molecules , are important for understanding the physiological functions of gls - enriched microdomains and their related diseases . stimulated by the promise of mechanically interlocked molecular architectures with potential employment in future nanotechnological applications such as in the development of molecular machines and switches , the interest being shown in their construction is ever increasing . rotaxane and catenane species can also , however , be designed to function as selective host systems whereby the topologically unique interlocked three - dimensional cavities are exploited to selectively recognise specific guest species . in previous work , we have utilised anion templation to construct a range of rotaxanes and catenanes , which , upon removal of the template , bind anions strongly and selectively in competitive solvent mixtures . furthermore , selective anion binding can be exploited to bring about triggered motion within the interlocked supramolecular architectures , underlining the possible application of these systems in the preparation of molecular switches . to sense and monitor anion binding ( and/or binding - triggered motion ) , it is desirable to integrate a redox- or photoactive reporter group in proximity to the interlocked anion - binding site . examples of interlocked hosts with the capability of sensing anions by electrochemical or optical[3b , 4 , 6 ] methods are rare . herein , we report the preparation of the first anion - templated rotaxane incorporating an optically- and electro - active osmium(ii ) tris(bipyridine ) reporter group . after removal of the anion template , selective anion binding is investigated by monitoring the optical and electronic output from the osmium(ii ) tris(bipyridine ) reporter moiety . to develop and fabricate a prototype molecular sensory system , surface association removes complications associated with brownian motion , typically increases conformational rigidity , and will underpin potential applications of such molecular architectures and , ultimately , device integration.[1b ] to date , only a limited number of surface - bound interlocked structures have been reported.[5b , c , 7 ] hence , we additionally report the anion - templated assembly of these electrochemically active osmium(ii ) bipyridyl rotaxane structures on gold substrates and , after template removal , the specific reporting of anion recruitment from solution ( figure 1 ) . schematic representation of the recognition and sensing of anions for a surface - bound rotaxane . axle in blue , macrocycle in green , with anion binding functionalities shown in red . design of the system : the target design for the mechanically bonded host features the incorporation of the osmium(ii ) tris(bipyridine ) reporter group into the macrocyclic component of a rotaxane molecular framework containing a convergent hydrogen - bond - donor anion - binding interlocked cavity . the macrocycle contains the 4,4-bis(amide)-2,2-bipyridyl motif for coordination to the osmium(ii ) metal centre and electron - rich hydroquinone units to facilitate supplementary secondary aromatic donor acceptor interactions with the electron - deficient positively charged pyridinium axle.[3a ] the choice of the osmium(ii ) bipyridyl reporter group comes from its established electro- and photochemical properties , making the system an attractive probe to sense the anion - binding event . synthesis of the rotaxanes : three distinct osmium(ii ) bipyridyl ( bipy ) rotaxanes , 13 , were prepared by clipping and stoppering anion - templated synthetic methodologies , comprising the same macrocycle component and different axle lengths ( scheme 1 ) . rotaxane 1 was obtained in two steps by a chloride - anion - templated clipping reaction between axle[3a ] components bis(amine ) 8 and 4,4-bis(chlorocarbonyl)-2,2-bipyridine 9 in the presence of et3n in dry dichloromethane ( scheme 2 and scheme s1 in the supporting information ) . the crude metal - free rotaxane intermediate was then treated with [ os(bipy)2cl2 ] , 16 , in an etoh / h2o mixture at reflux to give , after anion exchange using 0.1 m aqueous nh4pf6 , rotaxane 1 in 6 % overall yield . macrocycle 19 was synthesised in 37 % yield by condensation of the bis(amine ) 8 with bis(acid chloride ) 9 in the presence of et3n and template 17 in dry dichloromethane . reaction of macrocycle 19 with 16 in an etoh / h2o mixture at reflux afforded , after anion exchange , macrocycle 4 in 74 % yield . the reaction of carboxy terphenyl amide pyridine derivative 10[3f ] with oxalyl chloride produced the corresponding acid chloride , which upon condensation with 3-bromopropylamine hydrobromide in the presence of et3n in dry dichloromethane gave bis(amide ) 11 . this was converted to its azide derivative 12 by reaction with sodium azide in dimethylformamide . reaction of compound 12 with methyl iodide at reflux , followed by anion exchange using 0.1 m aqueous nh4pf6 , gave the desired axle precursor 13 . an analogous condensation reaction between the acyl chloride derivative of 10 and 4-(azidomethyl)biphenyl-4-yl - methanamine , followed by methylation and anion exchange , produced axle precursor 14 ( scheme 2 ) . rotaxanes 2 and 3 were obtained in 16 and 72 % yield , respectively , by mono stoppering reactions followed by washing with nh4pf6/h2o to remove the chloride template ( see the supporting information , scheme s1 ) . axle precursors 13 and 14 were added to macrocycle 4 leading to chloride - anion - templated pseudo - rotaxane assembly . alkyne cycloaddition ( cuaac ) reactions in dry dichloromethane using alkyne stopper 15 , copper(i ) tetrakis(acetonitrile ) hexafluorophosphate , tris(benzyltriazolylmethyl)amine ( tbta ) and diisopropylethylamine afforded the desired rotaxanes 2 and 3 . rotaxanes 13 were characterised by nmr spectroscopy ( h , c , f and p ) and by mass spectrometry ( maldi - tof ) . h nmr spectra of rotaxanes 13 ( figure 2 ) reveal splitting and an upfield shift of hydroquinone protons of the macrocycle component , owing to aromatic donor acceptor interactions between the electron - rich hydroquinone groups and the electron - deficient pyridinium moiety of the axle , characteristic of an interlocked structure . h nmr spectra ( 500 mhz , [ d6]acetone / d2o ( 7:3 ) , 293 k ) of rotaxanes 1 ( a ) , 2 ( c ) , 3 ( e ) and their chloride complexes 1 cl ( b ) , 2 cl ( d ) , and 3 cl ( f ) after addition of one equivalent of chloride . anion - binding studies in solution : anion binding was probed by using nmr , luminescence and electrochemical methods in assorted solvents as dictated by various factors . these techniques all have dramatically different limits of detection and different requirements from the system . in the case of nmr titration , initial assessments in acetonitrile revealed strong association with anions , compounded by the low solubility of the generated complexes , a fact that necessitated the use of a more competitive aqueous solvent mixture , [ d6]acetone / d2o ( 7:3 ) . luminescence spectroscopy , offering much lower detection limits , presents a quantum yield weighted average ; as a consequence , the response of the observed luminescence to anion concentrations varies between solvents . this is particularly true in the case of osmium tris(bipy ) complexes , in which solvation of the excited state plays an important role in determining the form of the spectrum . small quantities of water were added to the acetonitrile mixtures ; it was found that the addition of 3 % water produced optimal and reproducible changes to the osmium emission spectrum . in the case of electrochemistry , the need for a large potential window to resolve bipy - ligand - based voltammetry and the use of relatively concentrated electrolytes dictated the use of acetonitrile as a solvent system . h nmr investigations : preliminary h nmr anion titration experiments between rotaxanes 1 , 2 and 3 and various anions ( cl , aco and h2po4 ) were performed in competitive [ the addition of one equivalent of tetrabutylammonium ( tba ) chloride resulted in a downfield shift of inner protons ( 0.13 , 0.05 and 0.29 ppm ) and c ( 0.10 , 0.05 and 0.34 ppm ) for rotaxanes 1 , 2 and 3 , respectively , indicative of halide binding within the rotaxane s interlocked binding cavity ( figure 2 ) . in addition , modest upfield perturbations of the macrocyclic hydroquinone protons of the respective rotaxane were observed , g ( 0.02 , 0.02 and 0.06 ppm ) and h ( 0.03 , 0.02 and 0.03 ppm ) for 1 , 2 and 3 , respectively . when oxoanions such as acetate or dihydrogen phosphate where added , only a small perturbation was observed for inner protons . in the case of rotaxane 3 , the protons move upfield , an indication of the oxoanion binding on the periphery of the rotaxane . wineqnmr2 analysis of the binding isotherms with chloride and acetate anions , obtained by monitoring the chemical - shift perturbation of proton of the axle component of the respective rotaxane versus equivalent of anion ( figure 3 ) , determined 1:1 stoichiometric association constants ( table 1 ) . it proved impossible to obtain quantitative binding data from any of the dihydrogen phosphate titration experiments , indicative of a complex equilibrium binding process ( chemical shifts observed were too small , association constants < 100 m , to be sensibly fitted within error ) . a ) chemical - shift perturbation of proton upon addition of cl ( ) , aco ( ) and h2po4 ( ) ( as the tba salt ) to a solution of rotaxane 1 ( unfilled ) , 2 ( half - filled ) and 3 ( filled ) in 7:3 [ d6]acetone / d2o at 293 k. b ) chemical - shift perturbation of proton c upon addition of cl ( as the tba salt ) to a solution of macrocycle 4 in 7:3 [ d6]acetone / d2o ( ) and in 9:1 [ d6]acetone / d2o ( ) at 293 k. symbols represent experimental data ; continuous lines represent calculated curves . association constants [ m ] of rotaxanes 1 , 2 and 3 in 7:3 acetone / d2o and macrocycle 4 in 9:1 acetone / d2o with cl and aco ( as tba salts ) at 293 k [ a ] obtained by monitoring proton . error < 15 % ( for all values ) . [ b ] determined by monitoring proton c. error < 10 % . [ c ] in 9:1 acetone / d2o . the results indicate selectivity towards cl , with only weak and peripheral association with aco and h2po4 . the oxoanions are too large to penetrate the interlocked binding domain , whereas chloride anions are of complementary size and shape leading to strong binding even in this competitive 30 % aqueous solvent mixture . comparing the strength of chloride anion binding for the three rotaxanes , the interlocked host 3 , containing a rigid biphenyl axle linkage , exhibits significantly stronger binding . this may reflect the optimal degree of preorganisation of the interlocked binding domain of the respective rotaxane as determined by the nature of the axle component . the relatively more preorganised binding sites of rotaxanes 1 and 3 , as compared with the flexible propyl - linked axle component present in 2 , potentially enhances chloride binding . steric congestion could be responsible for 1 being a relatively inferior halide - complexing reagent in comparison with 3 . the binding of chloride anions by macrocycle 4 was also investigated ( table 1 and figure 3 b ) . as expected , the binding is very weak in 7:3 acetone / water , as shown by the small downfield shift of protons c , 0.03 ppm after addition of one equivalent of chloride . the h nmr titration experiment was repeated in 9:1 acetone / water , a less competitive solvent mixture , which enabled a 1:1 stoichiometric association constant of 210 m to be determined . analogous titrations with acetate anions revealed only small perturbations of the macrocycle proton , indicative of weak binding . these observations serve to highlight the rotaxanes potency for chloride anion recognition as a consequence of their three - dimensional binding cavities being capable of encapsulation of this guest . macrocycle 4 and the rotaxanes ( 13 ) all display two emission bands in the near - ir region centred at approximately 800 and 925 nm ( figure 4 ) following excitation into metal - to - ligand charge transfer ( mlct ) absorption bands at 430 nm , with the lifetimes of the two bands determined to be 44 and 35 ns at 800 and 950 nm , respectively . a ) titration of rotaxane 1 with tbacl in 97:3 acetonitrile / water , showing emission spectra uncorrected for detector sensitivity ; ( ) increasing chloride anion concentration . b ) binding isotherms and fits from the same titration following the two emission bands . these observations are consistent with those of nozaki and co - workers , who assigned the presence of two bands in the emission spectra of osmium bipyridyl complexes to subtle variations in the osmium environment arising from solvation - induced polarisation of the triplet state . upon titration with tbacl in 97:3 acetonitrile / water , an increase in emission intensity was observed for all four species , with the increases being more pronounced for the higher - energy bands . given the conclusions of nozaki regarding solvent - induced distortions to the triplet - state structure , it is plausible that anion binding will also influence local structure to differing degrees . representative spectra are shown in figure 4 along with the corresponding binding isotherms and fits . association constants were obtained with the dynafit software using a 1:1 binding model ( table 2 ) . association constants [ m ] determined by luminescence titration of macrocycle 4 and rotaxanes 1 , 2 and 3 in 97:3 acetonitrile / water , with chloride , dihydrogen phosphate and acetate anions , as tba salts [ a ] 99 % confidence intervals given in square brackets . a 1:1 binding model acetate and dihydrogen phosphate ( as their tba salts ) were also titrated with rotaxane 1 and the macrocycle 4 for comparison , although with the latter , phosphate anion binding could not be determined owing to precipitation . the data indicate that the binding of chloride anions with the rotaxanes is two orders of magnitude greater than that with the macrocycle . the chloride anion association constants obtained from luminescence measurements for the three rotaxanes confirm the observations from nmr spectroscopy of strong binding of chloride ions by rotaxane 3 . it should be noted that the change in solvent system will also be reflected in differences in binding owing to different solvation of the host . in this relatively uncompetitive solvent system , it is likely that many anions will associate with the cationic receptors ; indeed , studies on dihydrogen phosphate and acetate bear out this hypothesis . it is important to point out that it is likely such optical titrations are notably less reflective of binding selectivity in that they report anion association , be it cavity confined or peripheral . cyclic voltammetric scans ( figure 5 ) of an acetonitrile solution containing macrocycle 4 revealed an electrochemical redox system at 0.605 v vs. ag / ag , assigned to the reversible os(+2/+3 ) redox couple . in addition , the three bipyridyl - ligand - centred redox systems were observed at 1.375 , 1.715 and 1.975 v vs. ag / ag ( labelled x , y , z , respectively ) consistent with previous reports of a tris(bipy ) ruthenium moiety . these assignments were confirmed by equivalent integration of the charges associated with the os(+2/+3 ) redox system and the bipyridyl - ligand - centred redox events , as expected as all are one - electron redox systems on the same species . in accordance with our previous report , the least - cathodic bipy redox couple ( x , figure 5 ) is assigned to the amide - substituted bipyridyl , located next to the anion - binding site ( in light of the electron - withdrawing nature of the carbonyl amide moieties ) . cyclic voltammetric scans of 0.2 mm of macrocycle 4 in 0.15 m tbapf6/ch3cn ; scan rate=0.1 vs. insert : electrochemical titrations of 4 with tbacl monitoring bipy couples x , y and z. titrations of macrocycle 4 with tbacl or tbaoac were carried out to determine the ability of ligand- and metal - centre - based redox potentials to report on anion association . as shown in figure 6 a , cathodic perturbations of approximately 20 mv and 40 mv of the metal - centred osmium - based ( + 2/+3 ) couple are observed upon the addition of 5 molar equivalents of tbacl and tbaoac , respectively . the bipy - ligand - centred responses scale in accordance with their vicinity to the anion - association site , with x showing the strongest perturbations and z the weakest , in the presence of 5-fold excess of cl and aco ( see insert figure 5 , schemes s2 and s3 in the supporting information ) . these observations are fully consistent with both anionic species being bound in the vicinity of the bis(amide ) bipy cavity . dynafit modelling analysis of a simultaneous fit of both the metal - centred os(+2/+3 ) couple and the least - cathodic ligand - centred ( x ) potential perturbations as a function of anion concentration gave 1:1 stoichiometric association constants of 2.510 [ 2.22.710 ] and 1.510 [ 1.12.110 ] m , for chloride and acetate anions , respectively . the larger association constant ( and potential shifts ) observed after addition of tbaoac , compared with tbacl , can be attributed to the stronger association of aco to the amide - based binding site than cl , owing to the relative basicities of the anions . e1/2 of a ) os(+2/+3 ) and b ) bipy couple versus equivalents of tbacl ( ) and tbaaco ( ) added . we assign the higher luminescence and electrochemically defined association constants , in comparison to those obtained by nmr spectroscopy , to the more competitive aqueous solvent system used for the latter , but we acknowledge also that optical and redox transitions may not be exactly equivalent in their response to specific anion association . indeed , optical transition energies are likely to be sensitive to broad electrostatic / dielectric change in the vicinity of relevant chromophore orbitals ; redox signatures are more specifically sensitive to electron density at the bipyridyl and osmium centres and , hence , may be more reflective of specific anion association . resolved fundamental electrochemical characteristics of the rotaxane hosts are similar to those of the macrocycle and are reported in the supporting information ( page 4 ) . unfortunately , a combination of sluggish diffusion and strong physical - adsorption - based electrode fouling precluded the attainment of reliable electrochemical anion - binding data with the rotaxanes . surface immobilisation of the rotaxane : the cui - catalysed huisgen cycloaddition of axles 13 and 14 to prepared alkyne - terminated molecular films on gold ( see the experimental section ) in the presence of templating chloride anions and a 5-fold excess of macrocycle 4 , generates surface - confined rotaxane assemblies ( scheme 3 ) . accompanying ellipsometry - defined increases in film thickness ( ( 1.30.1 ) nm ) are consistent with axle - to - surface orientations being normal or close to normal . control surface analyses , in the absence of cui catalyst , confirm this surface assembly to be specifically click - chemistry driven . the associated template locking of the osmium bipy macrocycle at the surface by this process is confirmed by resolved osmium - based ( + 2/+3 ) electrochemical signatures ( figure 7 ) . an integration of these signals resolves surface macrocycle concentrations of 1.05 and 2.510 mol cm for axles 13 and 14 , respectively , corresponding to 20 and 45 % of a densely packed monolayer , based on a macrocycle footprint of 3.0 nm . surface association is both stable to ultrasonic washing and only observed with the chloride anion template specifically involved ( importantly , no macrocycle - osmium - based ( + 2/+3 ) electrochemical response is observed if the pf6 salts of 13 or 14 are used ) . the linear scaling of os(+2/+3 ) redox peak current with voltage scan rate is further confirmatory of surface entrapment . the surface - density differences between axle - only and rotaxane films are , additionally , resolvable through both electrode access of a solution - phase faradaic redox probe and surface - capacitance analysis ; an axle - only film being significantly more densely packed ( 10 f cm ) than a rotaxane film with a mushroom - shaped component ( 25 and 15 f cm for surfaces prepared with 13 and 14 , respectively ; scheme s4 in the supporting information ) . estimated surface concentration as 50 % of theoretical maximum ( calculated from a molecular footprint of the macrocycle , 3.0 nm ) . distance of faradaic exchange estimated at approximately 2 nm based on localisation of the macrocycle at the pyridinium station and a perpendicular orientation of the axle to the underlying gold surface . insert : potential shifts of surface - bound os(+2/+3 ) couple , after immersion of rotaxane interface in tbaaco / ch3cn and tbacl / ch3cn solutions following washing with nh4pf6/h2o to remove cl template . it is notable that axle 14 generates a significantly higher ( > 200 % ) macrocycle capture on the surface than that observed with axle 13 , consistent with both increased axle rigidity and a higher associated chloride anion template association constant ( table 1 ) . these films constitute not only an addition to the few surface - confined tethered interlocked systems but , to the best of our knowledge , are also the first incorporating a redox - active osmium(ii ) bipy reporter motif . we have also investigated the possibility that surface - presented vacant interlocked cavities ( generated by using axle 14 ) can provide a means of selectively recruiting and electrochemically sensing chloride anions . the chloride anion template was initially removed by washing with copious amounts of 0.1 m nh4pf6/h2o . pleasingly , subsequent halide anion recruitment is both detectable and selective , exhibiting no cathodic perturbation upon immersion in a 50 m solution of acetate anions ( the observed 2 mv shift is less than the 3 mv error of the reference electrode ) , but a 14 mv cathodic shift upon immersion in a chloride - anion - containing solution of the same concentration(with both solutions being formed from the tba salt in anhydrous ch3cn , figure 7 ) . no potential shift was observed when the cavity was not depopulated prior to immersion in the chloride solution , as expected for a cavity exhibiting 1:1 binding stoichiometry . these results are consistent with those presented in table 1 and confirm the selectivity of the three - dimensional cavity towards chloride anion binding . furthermore , the smaller potential shift observed here upon binding of chloride anions , in comparison with that observed of the macrocycle , is attributed to the electron - withdrawing nature of the positively charged pyridinium motif of the axle component , mitigating donation of electron density to the osmium centre , and is indicative of the interlocked surface - bound structure . interlocked structures can be engineered to bind specific guests within the topologically constrained three - dimensional cavities created during their template - driven syntheses . this binding ability , when coupled to the signal - transduction capabilities associated with appended reporter groups , make catenanes and rotaxanes highly promising candidates for the development of molecular sensors . we have reported herein the solution and surface synthesis of a number of anion - templated rotaxanes incorporating the photo- and electroactive tris(bipy ) osmium moiety . upon removal of the chloride anion template , h nmr titration of the solution rotaxanes with a number of anions ( chosen on the basis of their contrasting size , shape and basicity ) showed selectivity for chloride anions over acetate anions and dihydrogen phosphate oxoanions , observations broadly supported by associated perturbations in osmium luminescence or redox character . rotaxane immobilisation onto an alkyne - modified gold electrode substrate by copper(i)-catalysed huisgen cycloaddition produced molecular films capable of responding electrochemically and selectively to chloride anions . this work clearly demonstrates the successful application of self - assembled monolayers to the electrochemical sensing of halide ions . general procedure : commercially available solvents and chemicals were used without further purification unless otherwise stated . where dry solvents were used , they were degassed with nitrogen , dried by passing through an mbraun mpsp-800 column and then used immediately . h , c , f and p nmr spectra were recorded on a varian mercury - vx 300 , and a bruker avii500 spectrometer . synthesis : macrocycle 19,[6b ] axle 5,[3a ] bis(amine ) 8 , thread 17,[3a ] and axle 14 have been synthesized according to reported procedures . the syntheses of 11 , 12 and 13 along with electrochemical , surface analysis and luminescence anion titration details are given in the supporting information . rotaxane 1 : in a 50 ml round - bottom flask , 2,2-bipyridine-4,4-dicarboxylic acid ( 60 mg , 245 mol , 1.2 equiv ) was suspended in 10 ml of thionyl chloride , a drop of dmf was added and the solution was heated at reflux under n2 for 16 h. after removal of the solvent , the residue was dissolved in 10 ml of dry dichloromethane and added dropwise to a 50 ml dry dichloromethane solution containing bis(amine ) 8 ( 86 mg , 204 mol , 1 equiv ) , axle 5 ( 220 mg , 204 mol , 1 equiv ) and triethylamine ( 71 l , 510 mol , 2.5 equiv ) . the reaction mixture was stirred at room temperature ( 213 c ) for 2 h , washed with 10 % hcl(aq . ) after removal of the solvent , the residue was dissolved in 10 ml of acetonitrile , filtered and solvent removed in vacuo to give 121 mg of a crude mixture containing rotaxane and macrocycle 19 . this crude material ( 121 mg , 71 mol , 1 equiv ) was suspended in 20 ml of a 4:1 etoh / h2o mixture and [ os(bipy)2cl2 ] 16 ( 82 mg , 142 mol 2 equiv ) was added . after removal of the solvent , the crude product was purified by preparative tlc ( sio2 , ch3cn / h2o / kno3(sat . rotaxane 1 ( 30 mg , 6 % ) was obtained as a brown solid after anion exchange to the hexafluorophosphate salt by washing a dichloromethane solution of the rotaxane with 0.1 m nh4pf6(aq . ) h nmr ( 500 mhz , [ d6]acetone / d2o ( 7:3 ) ) : =9.40 ( 2 h , s ; py ) , 9.33 ( 1 h , s ; py ) , 9.29 ( 2 h , s ; arhc ) , 8.75 ( 2 h , d , j=8.4 hz ; bipy ) , 8.71 ( 2 h , d , j=8.4 hz ; bipy ) , 8.05 ( 2 h , d , j=6.2 hz ; arha ) , 8.02 ( 2 h , t , j=8.1 hz ; bipy ) , 7.90 ( 2 h , t , j=8.1 hz ; bipy ) , 7.78 ( 2 h , d , j=5.6 hz ; bipy ) , 7.71 ( 2 h , d , j=5.6 hz ; bipy ) , 7.66 ( 2 h , dd , j=6.1.4 hz , j=1.7 hz ; arhb ) , 7.59 ( 4 h , s , j=8.9 hz ; arh ) , 7.46 ( 2 h , t , j=6.7 hz ; bipy ) , 7.067.27 ( 28 h , m ; arhstopper and bipy ) , 6.50 ( 4 h , m , j=8.9 hz ; arhg ) , 6.39 ( 4 h , m , j=9.1 hz ; arhh ) , 4.61 ( 3 h , s ; h ) , 3.92 ( 4 h , m ; ch2 ) , 3.86 ( 4 h , m ; ch2 ) , 3.79 ( 4 h , m ; ch2 ) , 3.77 ( 4 h , s ; ch2 ) , 3.54 ( 4 h , m ; ch2 ) , 1.18 ppm ( 36 h , s ; tbuh ) ; c nmr ( 125 mhz , [ d6]acetone ) : =164.0 , 160.7 , 159.7 , 159.5 , 152.4 , 151.7 , 149.5 , 147.9 , 144.7 , 138.9 , 138.8 , 132.3 , 131.7 , 131.4 , 129.4 , 129.3 , 128.4 , 127.4 , 126.9 , 125.7 , 125.7 , 125.3 , 123.2 , 121.1 , 116.5 , 115.4 , 71.4 , 70.9 , 67.5 , 64.7 , 40.4 , 34.9 , 31.7 ppm ; f nmr ( 282.5 mhz , [ d6]acetone ) : =72.4 ppm ( d , j=707 hz ; pf6 ) ; p nmr ( 121.6 mhz , [ d6]acetone ) : =144.3 ppm ( sept , j=707 hz ; pf6 ) ; ms ( maldi - tof ) : m / z calcd for [ c128h130f18n11o10osp3 ] [ mh3(pf6 ) ] : 1085.98 ; found : 1085.98 . rotaxane 2 : in a 50 ml round - bottom flask , osmium macrocycle 4 ( 100 mg , 70 mol , 2 equiv ) and thread 13 ( 26 mg , 35 mol , 1 equiv ) were slowly stirred for 1 h in 25 ml of dry dichloromethane . after removal of the solvent , the residue was dissolved in 25 ml of dry and degassed dichloromethane . stopper 15 ( 19 mg , 35 mol , 1 equiv ) , cu(ch3cn)4pf6 ( 2.6 mg , 7 mol , 0.2 equiv ) , tbta ( 3.7 mg , 7 mol , 0.2 equiv ) and diisopropylethylamine ( dipea ) ( 12 l , 70 mol , 2 equiv ) were successively added and the reaction mixture stirred at room temperature ( 213 c ) for 3 d. then , 10 ml of kno3(sat . after removal of the solvent , the crude product was purified by preparative tlc ( sio2 , ch3cn / h2o / kno3(sat . rotaxane 2 ( 16 mg , 16 % ) was obtained as a brown solid after anion exchange to the hexafluorophosphate salt by washing a chloroform solution of the rotaxane with 0.1 m nh4pf6(aq . ) h nmr ( 500 mhz , [ d6]acetone ) : =10.06 ( 1 h , s ; nhd ) , 9.42 ( 1 h , s ; pyg ) , 9.36 ( 3 h , m ; pyb / b , arhc and arhc ) , 9.20 ( 1 h , s ; pyb / b ) , 8.82 ( 2 h , d , j=7.5 hz ; bipy ) , 8.77 ( 2 h , d , j=8.2 hz ; bipy ) , 8.74 ( 2 h , d , j=8.2 hz ; bipy ) , 8.36 ( 1 h , s ; nhd ) , 8.20 ( 2 h , m ; arha ) , 8.04 ( 1 h , s ; hc ) , 7.968.03 ( 8 h , m ; bipy ) , 7.78 , ( 2 h , d , j=6.1 hz ; arhbj ) , 7.74 ( 2 h , d , j=5.8 hz ; arhe ) , 7.51 ( 4 h , m ; bipy ) , 7.147.39 ( 29 h , m ; arhstopper ) , 6.90 ( 2 h , d , j=8.8 hz ; arhe ) , 6.63 ( 4 h , s ; arhg ) , 6.47 ( 4 h , s ; arhh ) , 5.03 ( 2 h , s ; hl ) , 4.65 ( 3 h , s ; ha ) , 4.44 ( 2 h , t , j=6.8 hz ; hnj ) , 4.04 ( 4 h , m ; och2 ) , 3.99 ( 4 h , m ; och2 ) , 3.91 ( 8 h , s ; nch2 and och2 ) , 3.63 ( 4 h , m ; och2 ) , 3.48 ( 2 h , m ; hr ) , 2.19 ( 2 h , m ; hk ) , 1.29 ( 18 h , s ; tbuh ) , 1.27 ppm ( 27 h , s ; tbuh ) ; c nmr ( 125 mhz , [ d6]acetone ) : =164.4 , 160.8 , 159.8 , 159.7 , 159.6 , 157.4 , 153.3 , 152.5 , 152.4 , 152.0 , 151.7 , 149.6 , 149.3 , 148.0 , 145.3 , 144.8 , 144.2 , 142.3 , 140.8 , 138.9 , 132.9 , 132.4 , 131.8 , 131.5 , 131.4 , 129.6 , 129.4 , 128.5 , 127.2 , 126.9 , 125.7 , 125.4 , 125.2 , 125.2 sic , 125.1 , 123.4 , 121.0 , 120.9 , 116.6 , 115.5 , 115.5 sic , 114.3 , 71.5 , 70.9 , 68.5 , 67.5 , 67.4 , 64.8 , 64.0 , 62.2 , 55.0 , 50.2 , 48.5 , 40.3 , 36.2 , 34.9 , 31.7 ppm ; f nmr ( 282.5 mhz , [ d6]acetone ) : =72.5 ppm ( d , j=708 hz ; pf6 ) ; p nmr ( 121.6 mhz , [ d6]acetone ) : =144.3 ppm ( sept , j=709 hz ; pf6 ) ; ms ( maldi - tof ) : m / z calcd for [ c138h147f18n14o11osp3 ] [ m(pf6 ) ] : 2658.03 ; found : 2658.26 . rotaxane 3 : in a 50 ml round - bottom flask , osmium macrocycle 4 ( 100 mg , 70 mol , 2 equiv ) and thread 14 ( 31 mg , 35 mol , 1 equiv ) were slowly stirred for 1 h in 25 ml of dry dichloromethane . after removal of the solvent , the residue was dissolved in 25 ml of dry and degassed dichloromethane . stopper 15 ( 19 mg , 35 mol , 1 equiv ) , cu(ch3cn)4pf6 ( 2.6 mg , 7 mol , 0.2 equiv ) , tbta ( 3.7 mg , 7 mol , 0.2 equiv ) and dipea ( 12 l , 70 mol , 2 equiv ) were successively added and the reaction mixture was stirred at room temperature ( 213 c ) for 3 d. then , 10 ml of kno3(sat . after removal of the solvent , the crude product was purified by preparative tlc ( sio2 , ch3cn / h2o / kno3(sat . rotaxane 3 ( 74 mg , 72 % ) was obtained as a brown solid after anion exchange to the hexafluorophosphate salt by washing a chloroform solution of the rotaxane with 0.1 m nh4pf6(aq . ) ( 815 ml ) and h2o ( 215 ml ) . h nmr ( 500 mhz , [ d6]acetone ) : =9.92 ( 1 h , s ; nh ) , 9.44 ( 1 h , s ; py ) , 9.34 ( 1 h , s ; py/ ) , 9.27 ( 1 h , s ; arhc / c ) , 9.23 ( 1 h , s ; py/ ) , 9.12 ( 1 h , s ; arhc / c ) , 8.78 ( 4 h , d , j=8.5 hz ; bipy ) , 8.36 ( 1 h , s ; nh ) , 8.18 ( 2 h , d , j=6.1 hz ; arha ) , 8.17 ( 1 h , s ; h ) , 8.03 ( 4 h , m ; bipy ) , 7.87 ( 4 h , m ; bipy ) , 7.80 ( 2 h , m ; arhb ) , 7.70 ( 2 h , s ; arh ) , 7.48 ( 4 h , m ; bipy ) , 7.097.42 ( 37 h , m ; arhstopper and arhbiphenyl ) , 6.92 ( 2 h , d , j=9.0 hz ; arh ) , 6.61 ( 4 h , m ; arhg ) , 6.41 ( 4 h , m ; arhh ) , 5.67 ( 2 h , s ; h ) , 5.16 ( 2 h , s ; h ) , 4.67 ( 3 h , s ; h ) , 4.08 ( 4 h , m ; och2 ) , 4.04 ( 4 h , m ; och2 ) , 3.94 ( 4 h , m ; och2 ) , 3.87 ( 8 h , m ; nch2 and och2 ) , 3.71 ( 2 h , s ; h ) , 1.30 ( 18 h , s ; tbuh ) , 1.28 ppm ( 27 h , s ; tbuh ) ; c nmr ( 125 mhz , [ d6]acetone ) : =163.8 , 162.4 , 160.3 , 159.4 , 159.1 , 157.1 , 152.8 , 152.2 , 151.9 , 151.4 , 151.3 , 151.2 , 149.1 , 148.8 , 147.6 , 146.4 , 144.9 , 144.3 , 141.8 , 140.3 , 138.5 , 137.6 , 135.8 , 132.4 , 132.0 , 131.3 , 131.1 , 131.0 , 129.2 , 129.1 , 129.0 , 128.7 , 128.1 , 127.6 , 127.5 , 127.0 , 126.5 , 125.3 , 125.0 , 124.7 , 123.2 , 122.7 , 120.5 , 116.3 , 116.2 , 115.0 , 113.9 , 71.0 , 70.4 , 68.0 , 67.2 , 67.0 , 64.4 , 63.4 , 61.9 , 54.6 , 53.6 , 49.7 , 39.8 , 35.8 , 34.6 , 34.5 , 31.3 ppm;f nmr ( 282.5 mhz , [ d6]acetone ) : =72.5 ppm ( d , j=708 hz ; pf6 ) ; p nmr ( 121.6 mhz , [ d6]acetone ) : =144.3 ppm ( sept , j=709 hz ; pf6 ) ; ms ( maldi - tof ) : m / z calcd for [ c149h153f18n14o11osp3 ] [ m(pf6 ) ] : 2796.07 ; found : 2796.10 . macrocycle 4 : in a 250 ml round - bottom flask , macrocycle 19 ( 168 mg , 270 mol , 1 equiv ) was suspended in 100 ml of a 4:1 etoh / h2o mixture and [ os(bipy)2cl2 ] 16 ( 153 mg , 142 mg , 2 equiv ) was added . the brown solution was left to cool to room temperature ( 213 c ) and then filtered through a celite bed before the solvent was removed in vacuo . the brown residue was then redissolved in 20 ml of h2o and nh4pf6(s ) was added to the solution until precipitation ceased . the precipitate was collected by vacuum filtration and dried under vacuum to give macrocycle 4 as a black solid ( 282 mg , 198 mol , 74 % ) . h nmr ( 300 mhz , [ d6]acetone ) : =9.14 ( 2 h , s ; arhc ) , 8.82 ( 4 h , m ; bipy ) , 8.51 ( 2 h , t , j=5.3 hz ; nhd ) , 8.19 ( 2 h , d , j=5.6 hz ; arha ) , 8.05 ( 4 h , t , j=7.9 hz ; bipy ) , 7.97 ( 4 h , t , j=6.0 hz ; bipy ) , 7.80 ( 2 h , dd , j=6.1 hz , j=1.7 hz ; arhb ) , 7.53 ( 2 h , t , j=6.8 hz ; bipy ) , 7.47 ( 2 h , t , j=6.8 hz ; bipy ) , 6.88 ( 8 h , m ; arhg , h ) , 4.14 ( 4 h , t , j=5.3 hz ; ch2 ) , 4.06 ( 4 h , t , j=4.5 hz ; ch2 ) , 3.733.85 ( 8 h , m ; ch2 ) , 3.67 ppm ( 4 h , s ; ch2 ) ; c nmr ( 75.5 mhz , [ d6]acetone ) : =163.6 , 160.8 , 159.7 , 153.9 , 152.4 , 152.2 , 151.7 , 142.4 , 138.8 , 129.4 , 127.5 , 125.6 , 122.7 , 116.5 , 116.3 , 71.4 , 70.4 , 68.9 , 67.4 , 40.5 ppm ; f nmr ( 282.5 mhz , [ d6]acetone ) : =72.5 ppm ( d , j=708 hz ; pf6 ) ; p nmr ( 121.6 mhz , [ d6]acetone ) : =144.3 ( sept , j=709 hz ; pf6 ) ; ms ( maldi ) : m / z calcd for [ c54h52f12n8o8osp2 ] [ m(pf6)2 ] : 1132.35 ; found : 1132.38 . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

glycosphingolipids ( gsls ) are composed of hydrophobic ceramide and hydrophilic sugar chains . gsls cluster to form membrane microdomains ( lipid rafts ) on plasma membranes , along with several kinds of transducer molecules , including src family kinases and small g proteins . however , gsl - mediated biological functions remain unclear . lactosylceramide ( laccer , cdw17 ) is highly expressed on the plasma membranes of human phagocytes and mediates several immunological and inflammatory reactions , including phagocytosis , chemotaxis , and superoxide generation . laccer forms membrane microdomains with the src family tyrosine kinase lyn and the gi subunit of heterotrimeric g proteins . the very long fatty acids c24:0 and c24:1 are the main ceramide components of laccer in neutrophil plasma membranes and are directly connected with the fatty acids of lyn and gi . these observations suggest that the very long fatty acid chains of ceramide are critical for gsl - mediated outside - in signaling . sphingosine is another component of ceramide , with the hydrolysis of ceramide by ceramidase producing sphingosine and fatty acids . sphingosine is phosphorylated by sphingosine kinase to sphingosine-1-phosphate , which is involved in a wide range of cellular functions , including growth , differentiation , survival , chemotaxis , angiogenesis , and embryogenesis , in various types of cells . this review describes the role of ceramide moiety of gsls and its metabolites in immunological and inflammatory reactions in human . we report the preparation of [ 2]rotaxanes containing an electrochemically and optically active osmium(ii ) bipyridyl macrocyclic component mechanically bonded with cationic pyridinium axles . such interlocked host systems are demonstrated to recognise and sense anionic guest species as shown by 1h nmr , luminescence and electrochemical studies . the rotaxanes can be surface assembled on to gold electrodes through anion templation under click copper(i)-catalysed huisgen cycloaddition conditions to form rotaxane molecular films , which , after template removal , respond electrochemically and selectively to chloride .

fatty acids ( fas ) , the components of phospholipids in organelle and cellular membranes , play important biological roles by maintaining or processing membrane protein function or fluidity.1 in addition , fas modulate vascular inflammation , a key mechanism of atherosclerosis , cerebral small vessel pathologies , and stroke , by altering intracellular signal transduction or controlling lipid mediators such as prostaglandins , thromboxanes , or leukotrienes.2 among fas , 3-polyunsaturated fas ( 3-pufas ) , such as eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , are potent anti - inflammatory molecules . epa and dha decrease expression of receptors for chemoattractants on blood inflammatory cells and prohibit migration of neutrophils or monocytes . therefore , 3-pufas may protect from atherosclerotic changes.3,4 several clinical studies have emphasized the role of fas in the risk or occurrence of stroke5 or cardiovascular disease6 ; however , the effects due to the composition of fas on stroke or cardiovascular disease remain controversial . high - dose 3-pufas has been reported to have beneficial effects on cardiac or sudden death.7 high levels of plasma 3-pufas can decrease the risk of myocardial infarction.8 in terms of stroke , low levels of circulating 3-pufas in the blood is a risk factor for ischemic and hemorrhagic stroke.9 a decreased proportion of linoleic acid is also associated with ischemic stroke.10 compared to normal controls , stroke patients with moderate - to - severe intracranial arterial stenosis or occlusion had decreased levels of dha.11 on the other hand , a recent meta - analysis revealed that the evidence for the beneficial effects of 3-pufas is insufficient in adults with peripheral arterial disease associated with poor cardiovascular outcome.12 to date , little information is available on the relationship between the composition of fas and prognosis of stroke . therefore , we investigated whether the composition of fas was associated with stroke severity on hospital admission and functional outcomes at 3-months follow - up of patients with acute non - cardiogenic ischemic stroke . between september 2007 and may 2010 , we prospectively enrolled patients diagnosed with a first - episode of ischemic stroke and admitted to our hospital within 7 days after onset of symptoms . the patient 's demographic information as well as past medical , medication and familial history ; brain imaging studies ( computerized tomography [ ct ] and/or magnetic resonance imaging [ mri ] ) ; vascular imaging studies ( digital subtraction angiography , ct angiography , or mr angiography ) ; chest radiography ; 12-lead electrocardiography ; electrocardiography monitoring during a median time period of 3 days at the intensive stroke care unit ; transthoracic echocardiography ; and routine blood test data were collected.13 patients were preferentially excluded if they did not agree to provide blood samples for our study , or if they were taking lipid - lowering agents such as statins , niacin , fenofibrate , or 3-pufas as components of health foods and supplements.11 of a total of 401 subjects were enrolled , however those , who on the basis of the trial of org 10,172 in acute stroke treatment classification system,14 had moderate or high risk cardiac sources of embolism ( n=127 ) were excluded . patients who had undetermined stroke subtype ( n=45 : negative evaluation , n=19 : two or more causes identified ) or those who had rare causes of stroke subtype ( n=10 ) , such as moyamoya disease , arterial dissection , or venous thrombosis were also excluded from this study . moreover , patients who received incomplete vascular imaging work - up ( n=2 ) and those who had transient ischemic attacks with negative diffusion - weighted images ( n=42 ) were not enrolled . for extracranial arteries , the degree of arterial stenosis was measured according to the published method used in the north american symptomatic carotid endarterectomy trial.15 for intracranial arteries , the degree of arterial stenosis was measured based on the methods used in the warfarin - aspirin symptomatic intracranial disease study.16 vascular images were evaluated by two independent vascular neurologists , who were blinded to the clinical information . inter - observer agreement on the presence of more than 50% stenosis and/or occlusion was excellent ( kappa=0.95 ) . the severity of neurologic deficits was determined using the national institutes of health stroke scale ( nihss ) on admission.17 a recurrent ischemic stroke was considered in the presence of acute onset of focal neurological signs of more than 24 hours duration with evidence of a new ischemic lesion on ct or mri scan , or when new lesions were absent but the clinical syndrome was consistent with stroke from admission to 3 months after the index stroke . functional outcomes were assessed using the modified rankin scale ( mrs ) 3 months after the index stroke . blood samples for lipid profiles were collected from the patients within 24 hours of admission and at a fasting state of more than 12 hours . they were centrifuged to separate plasma or serum from the whole blood and then were stored at -70 until analysis could be performed . the methods for measuring fa composition have been described previously.11 briefly , plasma total lipids were extracted according to the folch method18 and the phospholipid fraction was isolated by thin layer chromatography using a development solvent composed of hexane , diethyl ether , and acetic acid ( 80:20:2 ) . the phospholipid fractions were then methylated to fa methyl esters ( fames ) by the lepage and roy method.19 the fames of individual fas of phospholipids were separated by gas chromatography using a model 6890 apparatus ( agilent technologies , palo alto , ca , usa ) and a 30 m omegawaz tm 250 capillary column ( supelco , bellefonte , pa , usa).20 peak retention times were obtained by comparison with known standards ( 37 component fame mix and pufa-2 , supelco ; glc37 , nucheck prep , elysian , mn , usa ) and analyzed with chemstation software ( agilent technologies ) . the average of duplicate measurements for each sample was calculated.11,21 plasma phospholipid fas were expressed as the percentage of total fas . the sum ( ) of 16:0 palmitic acid , 18:0 stearic acid , 20:0 arachidonic acid , and 22:0 behenic acid was defined as the saturated fatty acids ; 16:1 palmitoleic acid , 18:1 9 oleic acid , and 22:1 erucic acid were defined as the monounsaturated fatty acids ; 18:2 6 linolenic acid , 18:3 6 -linolenic acid , 20:3 6 dihomo--linolenic acid , and 20:4 6 arachidonic acid were defined as the 6-pufas ; and 18:3 3 -linolenic acid , 20:3 3 5 - 8 - 11-eicosatrienoic acid , 20:5 3 epa , and 22:6 3 dha were reported as the 3-pufas . hypertension was defined as having resting systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg on repeated measurements , or receiving treatment with anti - hypertensive medications . diabetes mellitus was diagnosed when the patient had fasting blood glucose level 7.0 mmol / l , or was being treated with oral hypoglycemic agents or insulin . patients were defined as smokers , if they were smokers at the stroke event or if they had stopped smoking within 1 year before the stroke event . body mass index was estimated by dividing body weight by height ( kg / m ) . the presence of coronary artery disease was defined as a patient history of unstable angina , myocardial infarction , or angiographically confirmed coronary artery disease . all statistical analyses were performed using the windows spss software package ( version 18.0 , chicago , il , usa ) . independent t test , mann - whitney u test , one - way analysis of variance ( anova ) with bonferroni post hoc analysis , and kruskal - wallis test were used to compare the continuous variables . categorical variables were compared using the chi - square test or fisher 's exact test . continuous variables were expressed as meansstandard deviations ( sd ) or as medians and interquartile ranges ( iqr ) . univariate and multivariate linear regression analyses were performed to determine the factors associated with nihss at admission . functional outcome was dichotomized into good outcome ( mrs <3 ) or poor outcome ( mrs 3 ) . univariate and multivariate binary logistic regression analyses were performed to determine the predictive factors for functional outcome . to assess the goodness of fit of the logistic regression model , cox & snell r square was calculated and the hosmer - lemeshow test was performed . between september 2007 and may 2010 , we prospectively enrolled patients diagnosed with a first - episode of ischemic stroke and admitted to our hospital within 7 days after onset of symptoms . the patient 's demographic information as well as past medical , medication and familial history ; brain imaging studies ( computerized tomography [ ct ] and/or magnetic resonance imaging [ mri ] ) ; vascular imaging studies ( digital subtraction angiography , ct angiography , or mr angiography ) ; chest radiography ; 12-lead electrocardiography ; electrocardiography monitoring during a median time period of 3 days at the intensive stroke care unit ; transthoracic echocardiography ; and routine blood test data were collected.13 patients were preferentially excluded if they did not agree to provide blood samples for our study , or if they were taking lipid - lowering agents such as statins , niacin , fenofibrate , or 3-pufas as components of health foods and supplements.11 of a total of 401 subjects were enrolled , however those , who on the basis of the trial of org 10,172 in acute stroke treatment classification system,14 had moderate or high risk cardiac sources of embolism ( n=127 ) were excluded . patients who had undetermined stroke subtype ( n=45 : negative evaluation , n=19 : two or more causes identified ) or those who had rare causes of stroke subtype ( n=10 ) , such as moyamoya disease , arterial dissection , or venous thrombosis were also excluded from this study . moreover , patients who received incomplete vascular imaging work - up ( n=2 ) and those who had transient ischemic attacks with negative diffusion - weighted images ( n=42 ) were not enrolled . for extracranial arteries , the degree of arterial stenosis was measured according to the published method used in the north american symptomatic carotid endarterectomy trial.15 for intracranial arteries , the degree of arterial stenosis was measured based on the methods used in the warfarin - aspirin symptomatic intracranial disease study.16 vascular images were evaluated by two independent vascular neurologists , who were blinded to the clinical information . inter - observer agreement on the presence of more than 50% stenosis and/or occlusion was excellent ( kappa=0.95 ) . the severity of neurologic deficits was determined using the national institutes of health stroke scale ( nihss ) on admission.17 a recurrent ischemic stroke was considered in the presence of acute onset of focal neurological signs of more than 24 hours duration with evidence of a new ischemic lesion on ct or mri scan , or when new lesions were absent but the clinical syndrome was consistent with stroke from admission to 3 months after the index stroke . functional outcomes were assessed using the modified rankin scale ( mrs ) 3 months after the index stroke . blood samples for lipid profiles were collected from the patients within 24 hours of admission and at a fasting state of more than 12 hours . they were centrifuged to separate plasma or serum from the whole blood and then were stored at -70 until analysis could be performed . the methods for measuring fa composition have been described previously.11 briefly , plasma total lipids were extracted according to the folch method18 and the phospholipid fraction was isolated by thin layer chromatography using a development solvent composed of hexane , diethyl ether , and acetic acid ( 80:20:2 ) . the phospholipid fractions were then methylated to fa methyl esters ( fames ) by the lepage and roy method.19 the fames of individual fas of phospholipids were separated by gas chromatography using a model 6890 apparatus ( agilent technologies , palo alto , ca , usa ) and a 30 m omegawaz tm 250 capillary column ( supelco , bellefonte , pa , usa).20 peak retention times were obtained by comparison with known standards ( 37 component fame mix and pufa-2 , supelco ; glc37 , nucheck prep , elysian , mn , usa ) and analyzed with chemstation software ( agilent technologies ) . the average of duplicate measurements for each sample was calculated.11,21 plasma phospholipid fas were expressed as the percentage of total fas . the sum ( ) of 16:0 palmitic acid , 18:0 stearic acid , 20:0 arachidonic acid , and 22:0 behenic acid was defined as the saturated fatty acids ; 16:1 palmitoleic acid , 18:1 9 oleic acid , and 22:1 erucic acid were defined as the monounsaturated fatty acids ; 18:2 6 linolenic acid , 18:3 6 -linolenic acid , 20:3 6 dihomo--linolenic acid , and 20:4 6 arachidonic acid were defined as the 6-pufas ; and 18:3 3 -linolenic acid , 20:3 3 5 - 8 - 11-eicosatrienoic acid , 20:5 3 epa , and 22:6 3 dha were reported as the 3-pufas . hypertension was defined as having resting systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg on repeated measurements , or receiving treatment with anti - hypertensive medications . diabetes mellitus was diagnosed when the patient had fasting blood glucose level 7.0 mmol / l , or was being treated with oral hypoglycemic agents or insulin . patients were defined as smokers , if they were smokers at the stroke event or if they had stopped smoking within 1 year before the stroke event . body mass index was estimated by dividing body weight by height ( kg / m ) . the presence of coronary artery disease was defined as a patient history of unstable angina , myocardial infarction , or angiographically confirmed coronary artery disease . all statistical analyses were performed using the windows spss software package ( version 18.0 , chicago , il , usa ) . independent t test , mann - whitney u test , one - way analysis of variance ( anova ) with bonferroni post hoc analysis , and kruskal - wallis test were used to compare the continuous variables . categorical variables were compared using the chi - square test or fisher 's exact test . continuous variables were expressed as meansstandard deviations ( sd ) or as medians and interquartile ranges ( iqr ) . univariate and multivariate linear regression analyses were performed to determine the factors associated with nihss at admission . functional outcome was dichotomized into good outcome ( mrs <3 ) or poor outcome ( mrs 3 ) . univariate and multivariate binary logistic regression analyses were performed to determine the predictive factors for functional outcome . to assess the goodness of fit of the logistic regression model , cox & snell r square was calculated and the hosmer - lemeshow test was performed . the demographic data of study subjects and comparative analysis according to functional outcome at 3 months after index stroke are summarized in table 1 . of all patients , there were 60 ( 38.5% ) patients with large artery atherosclerosis stroke subtype and 96 ( 61.5% ) patients with small vessel occlusions . the meanssd of proportions of epa and dha were 2.00.7 and 8.91.4 , respectively . in the case of 3-pufa , considering stroke subtypes , there was no difference between large artery atherosclerosis and small vessel occlusion stroke subtypes in terms of the proportion of epa , dha , or 3-pufas ( supplementary table 1 ) . of the 156 patients , 122 ( 78.2% ) patients had good functional outcome with meanssd of epa and dha proportions of 2.10.7 and 9.11.3 , respectively . the remaining 34 ( 21.8% ) patients had poor outcome with a relatively smaller proportion of epa ( 1.80.6 , p=0.032 ) and dha ( 8.11.3 , p=0.001 ) . the proportion of 3-pufa in patients with poor outcome was significantly lower than that in patients with good outcome ( 10.81.6 vs. 12.21.9 , p=0.001 ) ( table 1 ) . after adjusting for factors including age , sex , and variables with p<0.1 in the univariate analysis ( stroke subtypes , hemoglobin , high density lipoprotein , high sensitivity c - reactive protein , fasting glucose , 16:0 palmitic acid , and saturated fatty acids ) , lower proportion of epa and dha were independently associated with stroke severity on admission ( : -0.751 , standard error ( se ) : 0.376 , p=0.048 for epa , : -0.610 , se : 0.215 , p=0.005 for dha ) . moreover , the 3-pufa was significantly associated with stroke severity on admission ( : -0.462 , se : 0.156 , p=0.004 ) ( table 2 ) ( fig . 1 ) . considering stroke subtypes , dha and 3-pufa were correlated with stroke severity on admission , in both large artery atherosclerosis ( even though it showed tendency for 3-pufa , p=0.065 ) and small vessel occlusion , however epa ( supplementary table 2 ) did not appear to be correlated in multivariate linear regression analysis . there were six recurrent stroke cases and events , and epa was relatively lower in the recurrent group compared to the non - recurrent group ( 1.60.2 vs. 2.00.7 , p=0.006 ) . however , the proportions of dha and 3-pufas were not different between the two groups ( supplementary table 3 ) . regarding functional outcome at three months after index stroke , a lower proportion of dha ( odds ratio ( or ) : 0.20 , 95% confidence interval ( ci ) : 0.04 - 0.88 , p=0.033 ) and 3-pufa ( or : 0.22 , 95% ci : 0.05 - 0.84 , p=0.028 ) showed a significant relationship with poor functional outcome . however , epa was not independently associated with poor functional outcome ( or : 0.60 , 95% ci : 0.12 -2.93 , p=0.533 ) in multivariate analysis after adjusting for age , sex , smoking status , nihss score , stroke subtypes , or 16:0 palmitic acid ( table 3 ) . considering stroke subtypes , a lower proportion of dha and 3-pufas was independently associated with poor functional outcome in both the large artery atherosclerosis subtype ( or : 0.62 , 95% ci : 0.42 - 0.93 , p=0.023 for dha , or : 0.65 , 95% ci : 0.47 - 0.90 , p=0.011 for 3-pufa ) and the small vessel occlusion subtype ( or : 0.49 , 95% ci : 0.28 - 0.85 , p=0.012 for dha , or : 0.64 , 95% ci : 0.42 - 0.98 , p=0.044 for 3-pufa ) ( supplementary table 4 ) . the demographic data of study subjects and comparative analysis according to functional outcome at 3 months after index stroke are summarized in table 1 . of all patients , there were 60 ( 38.5% ) patients with large artery atherosclerosis stroke subtype and 96 ( 61.5% ) patients with small vessel occlusions . the meanssd of proportions of epa and dha were 2.00.7 and 8.91.4 , respectively . in the case of 3-pufa , considering stroke subtypes , there was no difference between large artery atherosclerosis and small vessel occlusion stroke subtypes in terms of the proportion of epa , dha , or 3-pufas ( supplementary table 1 ) . of the 156 patients , 122 ( 78.2% ) patients had good functional outcome with meanssd of epa and dha proportions of 2.10.7 and 9.11.3 , respectively . the remaining 34 ( 21.8% ) patients had poor outcome with a relatively smaller proportion of epa ( 1.80.6 , p=0.032 ) and dha ( 8.11.3 , p=0.001 ) . the proportion of 3-pufa in patients with poor outcome was significantly lower than that in patients with good outcome ( 10.81.6 vs. 12.21.9 , p=0.001 ) ( table 1 ) . after adjusting for factors including age , sex , and variables with p<0.1 in the univariate analysis ( stroke subtypes , hemoglobin , high density lipoprotein , high sensitivity c - reactive protein , fasting glucose , 16:0 palmitic acid , and saturated fatty acids ) , lower proportion of epa and dha were independently associated with stroke severity on admission ( : -0.751 , standard error ( se ) : 0.376 , p=0.048 for epa , : -0.610 , se : 0.215 , p=0.005 for dha ) . moreover , the 3-pufa was significantly associated with stroke severity on admission ( : -0.462 , se : 0.156 , p=0.004 ) ( table 2 ) ( fig . 1 ) . considering stroke subtypes , dha and 3-pufa were correlated with stroke severity on admission , in both large artery atherosclerosis ( even though it showed tendency for 3-pufa , p=0.065 ) and small vessel occlusion , however epa ( supplementary table 2 ) did not appear to be correlated in multivariate linear regression analysis . there were six recurrent stroke cases and events , and epa was relatively lower in the recurrent group compared to the non - recurrent group ( 1.60.2 vs. 2.00.7 , p=0.006 ) . however , the proportions of dha and 3-pufas were not different between the two groups ( supplementary table 3 ) . regarding functional outcome at three months after index stroke , a lower proportion of dha ( odds ratio ( or ) : 0.20 , 95% confidence interval ( ci ) : 0.04 - 0.88 , p=0.033 ) and 3-pufa ( or : 0.22 , 95% ci : 0.05 - 0.84 , p=0.028 ) showed a significant relationship with poor functional outcome . however , epa was not independently associated with poor functional outcome ( or : 0.60 , 95% ci : 0.12 -2.93 , p=0.533 ) in multivariate analysis after adjusting for age , sex , smoking status , nihss score , stroke subtypes , or 16:0 palmitic acid ( table 3 ) . considering stroke subtypes , a lower proportion of dha and 3-pufas was independently associated with poor functional outcome in both the large artery atherosclerosis subtype ( or : 0.62 , 95% ci : 0.42 - 0.93 , p=0.023 for dha , or : 0.65 , 95% ci : 0.47 - 0.90 , p=0.011 for 3-pufa ) and the small vessel occlusion subtype ( or : 0.49 , 95% ci : 0.28 - 0.85 , p=0.012 for dha , or : 0.64 , 95% ci : 0.42 - 0.98 , p=0.044 for 3-pufa ) ( supplementary table 4 ) . our study revealed that 3-pufas , especially dha , were associated with stroke severity on hospital admission and poor functional outcome even after adjusting for the nihss score , which is considered a strong predictive factor for stroke outcome . for example , treatment with dha - albumin complex in animal studies decreased brain injury after a transient and permanent focal cerebral ischaemia.22 dha showed anti - inflammatory and neuroprotective effects by decreasing oxidative stress via activation of nuclear factor e2-related factor 2 and heme oxygenase-1 expression and by attenuating c - jun phosphorylation , or the activating protein-1 signaling pathway.23 consistently , studies in humans have produced similar results . one previous study in 281 japanese patients with acute ischemic stroke diagnosed within 24 hours of onset , showed that the epa / arachidonic acid ( aa ) ratio , dha / aa ratio , and the epa+dha / aa ratio were independently and negatively associated with early neurological deterioration.24 furthermore , a population - based study in the u.s.a , which included 2,692 elderly adults without prevalence of stroke or cardiovascular disease , revealed that higher plasma 3-polyunsaturated fas ( epa , dha , and total 3 fas ) were associated with lower mortalities.25 overall , these previous studies showed that 3-pufas ( especially epa and dha ) were related to vascular outcome , which is in line with our study results . however , the reason why epa was not independently associated with poor functional outcome in our study population remains to be elucidated . dha exerts vasodilating properties by stimulating nitric oxide release in the vascular endothelium , and hence may also be responsible for decreasing heart rate variability and potentially for dyslipidemia improvement , effects not observed by epa.26 loss of nitric oxide control , heart rate variation , and dyslipidemia may be related to poor vascular disease outcome,27 and our study results can be explained by the correlation observed with dha , and not epa , and with functional outcome . in addition , the difference in race and staple food of the study population could represent another possible explanation . our results demonstrated a relationship between 3-pufas and stroke severity on hospital admission and poor functional outcomes , in both the large artery atherosclerosis and the small vessel occlusion stroke subtypes . 3-pufas give rise to anti - inflammatory molecules ( resolvins and protectins ) through lipoxygenase or cyclo - oxygenase pathways.28,29 resolvins or protectins play an important role in the resolution of inflammation , which decreases atherosclerotic changes and tissue injuries . because cerebral small vessel pathologies such as lacunar infarction , white matter changes , and cerebral microbleeds are linked to inflammatory reactions30 and increase arterial stiffness31 caused by progressive atherosclerosis , these anti - inflammatory and anti - atherogenic effects of 3-pufas could explain the results observed in our study . moreover , because progressive cerebral atherosclerosis is associated with a poor stroke outcome,32 our finding relative to the association between 3-pufas and stroke prognosis may be valid . in addition , the atherosclerotic plaque stabilization effect of 3-pufas may be an important mechanism . a previous study performed on patients awaiting carotid endarterectomy showed that plaques from patients treated with fish oil ( 1.4 g 3-polyunsaturated fas per day ) had a well - formed thick fibrous cap ( i.e. less vulnerable ) atheroma compared to that in patients of the placebo group.33 pathologically , infiltration of macrophages was less severe in patients treated with fish oil.33 furthermore , another prospective study confirmed that patients treated with fish oil had lower plaque inflammation and instability.34 because the vulnerability of the atherosclerotic plaque is a very important determinant of thrombosis - related stroke , as well as the degree of arterial stenosis , our results relative to the relationship between stroke outcome and 3-pufas is within expectation . lastly , a recent study revealed that 3-pufas enhanced cerebral angiogenesis in animal models,35 and because increased angiogenesis could augment brain repair and improve long - term functional recovery after cerebral infarction , these findings may support our study.35 one limitation of our study is that the blood samples were obtained from acute stroke patients on admission . therefore , the fatty acids and their composition were not serially assessed during the time course of the stroke . moreover , even though we prospectively enrolled our study subjects , our study design is mainly cross - sectional . in addition , the short - term of observation with a small sample size is another limitation of this study . further studies with a long - term follow - up and larger population size are needed . finally , our study design was not that of a randomized control study . furthermore , because stroke severity on admission correlated with both lower proportion of 3-pufas and functional outcome at 3 months , there could be a bias due to the interaction between severity on admission and 3-pufas . that is , there may be a possibility that the functional outcome at 3 months is only weakly associated with 3-pufas . our results demonstrate that 3-pufa levels correlate with stroke severity at admission and functional outcomes at 3 months . 3-pufas may be considered potential blood biomarkers for prognosis of acute non - cardiogenic ischemic stroke patients . comparison of proportions of 3-pufas according to stroke subtypes the relationship between fatty acids composition and stroke severity on admission according to stroke subtypes comparison of functional outcome based on occurrence of recurrent stroke the relationship between fatty acids composition and poor functional outcome according to stroke subtype prediction of mandibular growth pattern ( mgp ) plays an important role in orthodontic treatment planning ; it is critical when managing the development of dentofacial structures . different methods have been introduced for predicting mgp and assessing , symphyseal morphology is one of them . ricketts and others stated that morphology of the symphysis may be used to predict the direction of mandibular growth . he found that forward inclination of the condylar head was associated with forward mandibular rotation , along with a greater curvature of the mandibular canal compared with mandibular contour . a tendency toward backward mandibular rotation was associated with a pronounced apposition below the symphysis with more overall concavity of the inferior mandibular border . jarabak 's cephalometric analysis predicted the direction of mandibular growth by a facial polygon , including the saddle angle ( n - s - ar ) , articular angle ( s - ar - go ) , and gonial angle ( ar - go - me ) ; a sum of these three angles greater than 396 is predictive of a posterior mgp while a sum less than 396 was associated with anterior mgp . also a ratio of posterior ( s - go ) to anterior face height ( n - me ) of 56 - 62% indicated a posterior mgp , whereas a ratio of 65 - 80% indicated an anterior growth tendency . although many cephalometric measurements have been introduced , it is still very difficult to accurately predict the direction of mandibular growth . although , skieller 's four variables accounted for 86% of the variability of changes in the direction of mandibular growth ( mandibular inclination , intermolar angle , shape of the inferior border of the mandible , and inclination of the symphysis ) ; however , it accounted for only 8% in lee 's study . thus , he concluded that predicting the direction of mandibular growth is very perplexing and problematic . assessed symphyseal height , depth ratio ( h / d ) and angle . in his study , the morphology of the symphysis was found to be associated with the direction of mandibular growth . a mandible with an anterior growth direction was associated with a small height , large depth , small ratio , and large angle of the symphysis . conversely , a posterior growth direction was associated with a large height , small depth , large ratio , and small angle of the symphysis . the purpose of this study was to evaluate symphyseal morphology ( height , depth , and height / depth ) in patients aged between 9 and 14 years with different sagittal and vertical mgps . lateral cephalometric radiographs and charts of adolescents aged between 9 and 14 years were used in this study . the first part of the study was conducted on 60 normal cephalograms taken from normal individuals according to normal occlusion defined by moyer 's and appropriate facial proportions were approved by two orthodontists . the samples had no history of orthodontic treatment or history of systemic or developmental diseases . to find normal value for vertical growth pattern of mandible vertical indices , that is , the sum of bijork angles , gonial angle and sn - man angle were used . wylie 's analysis was also used for assessing sagittal growth pattern of mandible , which included mandibular length and lower mandibular length . the second part of the study was to define case groups , which included cl ii vertical , cl iii vertical , cl ii horizontal , and cl iii horizontal patients with normal maxillae . the four case groups were those who were out of this range classified as horizontal or vertical growing patterns according to vertical indices and as cl ii or cl iii skeletal growing patterns according to wylie indices . there were 15 samples in each group that were matched to normal groups according to sex and cervical maturation stage ( cvms ) . baccetti 's method for evaluation of the cervical maturation stage the symphyseal measurement included height , depth , and ratio ( h / d ) measured according to the aki et al . linear measurements used the tangent drawn at point b and parallel and perpendicular lines drawn to this tangent . the method of measuring height and depth of the symphysis is shown in figure 2 . cephalometric measurements used to quantify symphysis morphology all radiographs were taken with the same panoramic radiographic device ( planmeca proline x , helsinki , finland ) . all of them were scanned and digitized ( dpi 300 ) , and the measurements were made using dolphin imaging software , version 10 ( patterson dental supply , usa ) for symphyseal parameters , real size tracings were printed by dolphin imaging software and measured using a grid . normal data distribution was evaluated by one sample k - s test and the levene test was performed to indicate variance equality between groups . after confirmation of normality of the groups and equation of their variances , the two - way analysis of variance ( anova ) model was assessed by adjusted chi - square for analysis of data ( p < 0.001 ) , and the comparison of cases with the normal group was performed using the dunnett method . for evaluation of intraobserver reliability , intraclass correlation coefficient ( icc ) was assessed and the reliability was approved ( icc coefficiency was between 0.0726 and 0.0871 ) . normal data distribution was evaluated by one sample k - s test and the levene test was performed to indicate variance equality between groups . after confirmation of normality of the groups and equation of their variances , the two - way analysis of variance ( anova ) model was assessed by adjusted chi - square for analysis of data ( p < 0.001 ) , and the comparison of cases with the normal group was performed using the dunnett method . for evaluation of intraobserver reliability , intraclass correlation coefficient ( icc ) was assessed and the reliability was approved ( icc coefficiency was between 0.0726 and 0.0871 ) . evaluation of 60 normal radiographs with the baccetti method showed that 21 were in stage iii , 29 were in stage ii , and the remaining were in stage i. the case groups also were selected from the patients in stage iii and matched to 15 samples with normal radiographs , which were in the same stage . mean and standard deviation of the sagittal and vertical parameters are shown in table 1 . mean and standard deviation of mandibular parameters of sagittal and vertical analysis table 2 lists the height and depth of the symphysis based on sex and growth pattern in study groups . comparison of each case group with normal group showed only statistically significant differences between the normal group and cl ii horizontal and cl iii vertical groups in height ( p < 0.001 ) . mean and standard deviation of symphyseal height and depth in the normal group and four subgroups based on gender comparison of each case group with the normal group showed only statistically significant difference between it and the cl iii horizontal and cl ii vertical groups in depth ( p < 0.001 ) and no significant difference relevant to sex . table 3 shows the symphysis ratio relevant to sex and growth pattern . mean and standard deviation of symphyseal ratio in the normal group and four subgroups based on gender the intraobserver reliability was assessed by icc of reliability . evaluation of 60 normal radiographs with the baccetti method showed that 21 were in stage iii , 29 were in stage ii , and the remaining were in stage i. the case groups also were selected from the patients in stage iii and matched to 15 samples with normal radiographs , which were in the same stage . mean and standard deviation of the sagittal and vertical parameters are shown in table 1 . table 2 lists the height and depth of the symphysis based on sex and growth pattern in study groups . comparison of each case group with normal group showed only statistically significant differences between the normal group and cl ii horizontal and cl iii vertical groups in height ( p < 0.001 ) . mean and standard deviation of symphyseal height and depth in the normal group and four subgroups based on gender comparison of each case group with the normal group showed only statistically significant difference between it and the cl iii horizontal and cl ii vertical groups in depth ( p < 0.001 ) and no significant difference relevant to sex . mean and standard deviation of symphyseal ratio in the normal group and four subgroups based on gender the intraobserver reliability was assessed by icc of reliability . quantification of skeletal data has been shown to be an effective and reliable method of demonstrating variation in human growth as well as for monitoring and interpreting the growth of various skeletal elements in the living . morphological changes in the mandibular body had been studied in previous researches , and it was found that the gonial angle became significantly smaller after the third molar eruption . also , the posterior mandibular body length had a linear correlation with gonial angle . is symphysis morphology . due to ease in selection of landmarks for evaluating symphysis morphology in cephalograms the characteristics of the normal subjects were mentioned in other studies . despite wide variations in the size and shape of the human face , head , and body , , significant relationships were found between the measures of mandibular incisor crowding and basal bone dimensions in female subjects . except for the vestibular part of cancellous bone thickness , all mandibular incisor bone measurements were greater in the male subjects than in the female subjects . we matched our study groups according to the baccetti 's skeletal age , in order to lower the developmental age effect on the mandibular growth . the samples were also matched based on gender . in this way , the confounding effect of gender and skeletal age was minimized on mandibular growth . it was also found that there was a sexual dimorphism on the morphology of the symphysis , with the mean symphyseal ratio being higher in the female samples than in the male samples . this difference has been indicated in all studies on mandibular growth , because bone deposition in the pogonion region is an x - linked trait . it was noticed that with horizontal growth patterns of the mandible whether in cl ii or cl iii , the symphyseal ratio was higher than in the normal group . in cases with vertical growth patterns the height of the symphysis was greatest in cl iii vertical and the least in clii horizontal groups , with no significant difference in other subgroups . regarding the depth of the symphysis , the greatest depth was measured in cl iii horizontal and the least was indicated in the cl ii vertical group . the size and shape of the mandibular symphysis is an important consideration in evaluation of orthodontic patients . with a prominent symphysis , more protrusion of the incisors is esthetically acceptable and therefore a greater chance that a nonextraction approach to treatment may be considered . conversely , in patients with greater symphyseal height and small chin , an extraction approach is preferred for compensation of arch length discrepancies . this concept was confirmed by the results of our study because we measured the deepest depth in horizontal growth patterns ; therefore , we can use nonextraction approach in these individuals . furthermore in vertical growth patterns it is better to extract teeth because of the decreased symphyseal depth . did a study to determine whether symphysis morphology could be used as a predictor of the direction of mandibular growth and to assess growth changes of the symphysis . they determined the direction of mandibular growth based on vertical parameters and divided it into anterior and posterior growth patterns . consequently , they subdivided the size of the symphysis into large , small , and medium . symphysis morphology was found to be associated with the direction of mandibular growth , especially in male subjects with symphysis ratio having the strongest relationship in adults . a mandible with an anterior growth direction was associated with a small height , large depth , small ratio , and large angle of the symphysis . conversely , a posterior growth direction was associated with a large height , small depth , large ratio , and small angle of the symphysis . symphyseal dimensions continued to change until adulthood with male subjects having a greater and later change compared with female subjects . in addition , the ratio was low in horizontal growth ( anterior growth direction ) and the depth was greater . it seems that based on both studies , the symphysis morphology is different in various vertical growth patterns of the mandible . in summary , we can conclude that female adolescents had greater symphyseal ratio in comparison to male adolescents . also , symphyseal depth differed significantly in vertical and horizontal growth patterns in comparison to normal groups and symphysis ratio ( height / depth ) was strongly related to the vertical pattern of mandibular growth in both genders .

background and purposealterations in blood fatty acid ( fa ) composition are associated with cardiovascular diseases . we investigated whether plasma fa composition was related to stroke severity and functional outcome in acute ischemic stroke patients.methodswe prospectively enrolled 156 patients with first - episode cerebral infarction , within 7 days of symptom onset . the proportion of fas was analyzed using gas chromatography , and the summation of the omega-3 polyunsaturated fatty acids ( 3-pufa ) , 18:3 3 -linolenic acid , 20:3 3 eicosatrienoic acid , 20:5 3 eicosapentaenoic acid ( epa ) , and 22:6 3 docosahexaenoic acid ( dha ) was reported as 3-pufas . stroke severity was assessed using the national institutes of health stroke scale ( nihss ) score on admission . poor functional outcome was defined by modified rankin scale ( mrs ) 3 at three months after the index stroke.resultslower proportions of epa ( =-0.751 ) , dha ( =-0.610 ) , and 3-pufas ( =-0.462 ) were independently associated with higher nihss score , after adjusting for stroke subtype , hemoglobin , high density lipoprotein , high sensitivity c - reactive protein , fasting glucose , 16:0 palmitic acid , and saturated fatty acids . moreover , a lower proportion of dha ( odds ratio [ or ] : 0.20 , 95% confidence interval [ ci ] : 0.04 - 0.88 ) , and 3-pufas ( or : 0.22 , 95% ci : 0.05 - 0.84 ) showed an independent relationship with poor functional outcome after adjusting for age , sex , smoking status , nihss score , stroke subtype , and 16:0 palmitic acid.conclusionsour results demonstrate that 3-pufas correlated with stroke severity on admission and functional outcomes at 3 months . 3-pufas are potential blood biomarkers for prognosis of acute non - cardiogenic ischemic stroke patients . background : this study sought to assess symphyseal morphology in adolescents with different mandibular growth patterns ( mgps ) in order to see if a relation exists.materials and methods : in this study the symphyseal parameters ( height , depth , and ratio ) of normal subjects were compared with four groups with malocclusion ( cl iii vertical , cl ii vertical , cl iii horizontal , and cl ii horizontal ) . these groups ( 15 samples each ) were matched ( for sex and cervical maturation stage [ cvms ] ) based on their cephalograms and patient charts . growth patterns were differentiated by seven vertical parameters and the wylie analysis . after confirmation of normality of the groups and similarity of their variances the two - way analysis of variance ( anova ) was used for analysis of data assessed by adjusted chi - square ( p < 0.001 ) . the comparison of cases with the normal group was performed by the dunnett method . intraclass correlation coefficient ( icc ) was used for evaluation of intraobserver reliability.results:we found the symphyseal ratio to have a significant correlation with the mgp ( p < 0.001 ) . the symphyseal ratio ( height / depth ) was small in a mandible with vertical growth pattern cl ii or cl iii . conversely , a horizontal growth pattern of a cl ii or cl iii mandible was associated with a larger ratio of the symphysis in comparison with the normal group . the symphyseal ratio was also found to be greater in females.conclusion:the symphyseal ratio was found to be strongly associated with the mgp .

established in 2003 , erste foundation has a history stretching back 190 years to the year 1819 when it was founded as the erste sterreichische spar casse in vienna . rooted in our history as a social enterprise and financial service provider , erste foundation recognises that contemporary society faces huge challenges and that for a new and united europe to work , integration is crucial and that means uniting the economic , cultural and social capital of our region . we develop ideas and concepts to increase social participation and to ensure that no - one is left out , whatever their circumstances which in turn creates stable , effective and fairer societies . we want to work against prejudice and nationalism , to integrate thinking and living across borders , and make these experiences accessible , particularly to the young generation . we intend to play an active role in giving people opportunities to increase their understanding of each other . therefore , erste foundation has supported research projects that concern themselves with the effects of societal transformation processes : in particular we have been looking at the effects that demographic changes will have on the long - term care needs of elderly people in central and eastern europe . from this , we intend to develop policy recommendations for decision makers in civil society , economy and politics . in modern nursing , nurses are expected to deliver holistic care , taking into account patients biological , psychosocial , and spiritual needs . however , formal nursing programs in iran follow a biomedical approach that focuses mainly on medical problems.[35 ] these formal education programs focus on the transfer of knowledge and the mastery of techniques related to patient physical care ; however , students also learn informally through hidden curricula of nursing education . direct observation of situations , such as birth , death , loss , and suffering results in emotional learning . additionally , students gain experience through interactions with each other , teachers , and senior professionals , known as peer learning , which studies have found can have life - long effects on students . the limited scope of formal nursing education in iran may result in informal learning experience for nursing students that differs from those of other countries . if nursing education recognizes this informal learning and its achievements may be able to take advantages of these opportunities to improve students competency . informal or indirect learning can occur as a function of observing , retaining , and replicating behaviors during educational experiences . the aim of this qualitative study was to explore the perceived informal learning achievements in undergraduate nursing programs from the iranian student perspective . this study was designed to answer the question : what are undergraduate nursing student perceptions of the informal learning that occurs during nursing studies ? this study was conducted over a 2-year period ( 2009 - 2010 ) using a qualitative content analysis . the participants were 14 nursing students in their fourth year of study at x university who were recruited through a purposeful sampling method . they were at their final stage of clinical apprenticeship so had passed major part of their education and could share their achievements of learning experiences , including informal ones . sampling was done according to the maximum variant approach in terms of gender , age , marital status , geographical location ( local or not local ) , student work experience , and level of academic achievement ( e.g. , good , average , or superior ) . each interview began with the open question : apart from the theoretical and practical knowledge you gained during four years of nursing study , what other changes do you think you have experienced during your study ? answers were explored more deeply with relevant probing statements or questions , such as : please tell me more about that , how did that make you feel ? please describe an actual experience you have had that will help me understand what it meant to you . the interviews lasted an average of 70 min , usually in a single session . each interview was transcribed verbatim , and then converted to rich text format for use in maxqda software , which facilitated data management . data were analyzed through a constant comparison analysis method using strauss and corbin coding techniques to develop the categories . data were analyzed initially by the main author , then the emerging codes were revised independently by 2 other members of the research team . throughout coding and category development , the analysis process and the accuracy of codes and categories were discussed and checked in regular sessions ; discrepancies were resolved through continuing discussion and analysis . lincoln and guba ( 1985 ) criteria were used to address trustworthiness ( p.296 ) . dependability was gained by precise documentation of data , methods , and decisions about the research . credibility was established through participant confirmation of our understanding , prolonged engagements with participants , a peer check , and an external check . throughout the study , data were managed by maxqda and all analysis precisely documented to maintain an audit trail . student participation was voluntary . written information about the purpose of the study and confidentiality management was delivered to all participants and written consent was obtained for the study , including permission for recording the entire interview for transcribing . the 14 participants consisted of 3 male and 11 female nursing students , age 21 - 23 years , in the last semester of their studies . based on students experiences , 5 categories were extracted : personal maturity and emotional growth , social development , closeness to god , alterations in value systems , and ethical and professional commitments [ table 1 ] . although some participants described the process leading to their internal changes as challenging and difficult , almost all of them stated that it facilitated their growth as a whole person . students achievements of informal learning during nursing study this category consisted of 2 subcategories : personal maturity ( consisting of self - awareness and self - discovery , accelerated mental maturity , and more open - mindedness ) and emotional growth ( consisting of greater patience , appreciation , kindness , and compassion , and altruism ) . the participants viewed their 4 years in nursing school as an opportunity for gaining a deeper self - awareness and self - knowledge . they believed that the duties of nursing practice , such as frequent personal interactions and development of relationships , have great influence on deep reflection about humanity as well as their personal beliefs . in contrast , the importance of rigorous assessment throughout nursing studies resulted in regular self - assessment in their daily lives . these helped them obtain more realistic knowledge about their personal strengths , weaknesses , and talents . some participants described this awareness as self - discovery by saying : i learned about myself based on what they taught us about humans in the program . i understood myself , in the university setting , through the eyes of my friends , who criticized me . another significant change reported by students was an increasing maturity of mind that resulted in more rational behavior . the frequent exposure to life - and - death situations in nursing helped promote maturity . one student said : my personality developed at this school . at first , i behaved very childishly . there were a series of realities of which i was wholly ignorant ; i acquired them here in the school . they also attributed this increase in maturity to multiple factors , including interactions with instructors , senior students , and people with various personalities , as well as engagement in difficult situations . one participant said : this maturity has been partly influenced by interactions with professional instructors and encounters with classmates as well as senior students i improved little by little through experiencing critical situations , seeing the darker side of life , the troubles and pains of other people , all of which i was unaware of in my previous relaxed lifestyle . the other significant experience reported by participants was increasing open - mindedness . with respect to the principles of fundamental nursing classes , they learned to focus on a patient as a human being , regardless of nationality , ethnicity , religious beliefs , or financial or sociocultural status . as a result , students reported that they were able to accept people by adopting a nonjudgmental manner . one student , who was caring for people with various religious beliefs , described her primary concern about encountering clients with different beliefs as a challenging experience and explained how she successfully overcame those conflicts by overlooking religious prejudices . she said : at first i thought i had treated them differently ; well , one patient was muslim , the other held another belief . however , after a while , i realized that they are ultimately human , and my job was not to judge the belief system of people . then the differences were no longer serious or problematic and i found myself adapting easily by getting closer to them . i am very grateful to be able to communicate well with various religious minorities despite so many differences . some of them said that they learned patience from patients who were suffering from chronic illnesses . when they saw how these patients worked to manage the disease and its disabling consequences , they realized the triviality of their own problems . i had patients who underwent dialysis every day , yet were quite happy , spoke with joy , laughed , and induced others to be in a good mood . in comparison with them , how could i concern myself with minor issues while i was in good health physically and emotionally ? i felt embarrassed when i saw them struggle steadfastly with such pain , illness , and deprivation . other participants believed that the patience resulting from the nature of discipline made them stronger to handle difficulties . one participant stated : nowadays the problems do not exhaust my spirit . since in nursing practice you see the pains and troubles of other people , a series of problems are no longer considered problems , or perhaps your experiences and abilities have improved . you should be more patient , in order to help those people who need you and have their hope invested in you . they said seeing patients suffer made them realize the importance of the gifts they took for granted , especially the gift of health : now i see patients with their problems and realize the gifts i have in my life , which i did nt appreciate before . they also explained that nursing made them similar to mothers - kind and ready to help and show compassion . now they feel a stronger sense of love and concern for humankind as well as the ability to love and care for others . one of them indicated : studying in the nursing field gives you the sense of being a mother . they teach us that we must be able to establish good communication , and understand others and support them . one participant stated : generally , my morale and personality is such that i really like people . it seems that these traits were developed for inner satisfaction and for the affection you can induce in others . the second category derived from the interviews was related to becoming more social and expert in interpersonal relationships . some of the participants , especially those who had limited communication skills before entering the program , acknowledged nursing studies as a turning point that improved their social relationships . they asserted that gaining communication skills with patients in clinical settings as fundamental to improving their interpersonal proficiency by saying : nursing improved my communication with others . i , who had a habit of building fences around me , was forced out of that closed space . during this period , my communication skills got much stronger , since they are required and necessary skills in nursing . encouraging and developing communication with patients required students to exploit strategies , such as developing cultural and ideological common ground , respecting others interests and wishes , and adapting themselves to others . gradually they noticed they were applying these advanced communication skills in their personal life . now , i can look at the subject from his / her point of view . i take the initiative and try to begin from a point that is welcomed by the other party . another factor that contributed to students social development was existing in a social environment larger than that of high school , which involved interaction with a heterogeneous population from various towns , ethnic groups , and cultures , which was regarded as expanding the friends circle by participants : i do nt limit myself to making friends with people of my own type ; i always search for new thoughts and new ideas with their own experiences . nonlocal participants , who resided in the student dormitory during their studies , explained the significant influence of living in the dormitory on their social growth and development as : when you become a college student and live in the dormitory with other students coming from different places and cultures with various upbringings , you are obliged to encounter them , and automatically learn how to adapt yourself and in fact be flexible . since all participants were muslim , a belief in god was one fundamental characteristic . however , most of them stated that they experienced a different type of connection with god during their nursing studies . some of them attributed this new belief to gaining a deeper knowledge about the intricacies of the human body and about human beings in general during the nursing program : i understood the complexities of this perfect system ( the human body ) , how accurately it has been designed , and how many problems are caused by a small deviation . we were told that the patients do nt respond to any treatment , but they did and i saw with my own eyes the effect of the prayers that the person accompanying the patient offered from the depths of the heart , and how it worked . i touched god s power and presence . others stated they felt closer to god through nursing practice because they were satisfying god s wishes by caring for the most vulnerable . this optimism significantly improved the quality of their prayers and replaced traditional ceremonies with enthusiastic worship and inner dialog with god . this type of close connection with god led to a pleasant feeling of dependency , which made them calmer when confronting difficulties : now i feel that god is looking at me , for my interest in caring for his people . i tell myself since i serve god s people sincerely ; certainly he would also pay attention to me ... i have noticed that whenever i have troubles , they are resolved at once . i sense some resolution and interpret it as god s promise that , if you help someone , god will help you in return . participants believed that confrontation with the light as well as the dark side of life in nursing expanded their world view through a change in focus from living a superficial life to a life more concerned with serious and substantive matters beyond their calendar age . one participant explained : in nursing , you see the beginning of life and the termination of it . you are faced with certain facts that make other issues trivial and insignificant in life while they are still important for your peers . these challenging confrontations acted as awakening experiences , allowing students to obtain a new perspective about life and death that stimulated changes in their value systems . as a result they described these alterations as achieving a deeper and more realistic point of view of life : now my objectives for the future have changed in principle . whenever i see patients who have only a few days to live , i no longer waste my time . before , i had a child - like view of life as a bowl of cherries with no worries , but now i know that life has its own hardships , which are considered a part of life . moreover , most of them asserted that they feel they were chosen by god for this profession and they appreciated it as an opportunity for growth and transcendence . i sensed that i was invited by god to take a right path that changed my destiny forever . i began taking steps on a road that i was supposed to begin with and i interpreted this as special attention to me on god s part ... i believe that we were summoned by god . nurses were invited for their kind heart , for their sympathy , and for their ability to provide relaxation to patients . if you knew this from the beginning and you appreciated being invited , any experience along the way would become important and valuable to you . most participants admitted that they feel a strong commitment to their work and try to be conscientious in the care of their patients . they believed that this inner commitment had a stronger influence than any rules or external controls . some of them attributed this inner commitment to their increased closeness to god and consequent sense of being observed constantly by god . they were delighted by this connection and attention so they tried to sustain this perceived attention by working ethically and conscientiously . even though i can work unsterile and the patient may not know it , i notice what i do and god does , too . however , your ethical commitment forces you to work better . to me , respecting the patient s rights is more important than anything else . i always tell myself to imagine that the patient is one of your dear siblings . i always try to hold that thought and work according to such feelings to do my best . in addition , participants attributed this commitment to other reasons , such as respecting patient s rights and dignity to enjoy humanistic and proper care as a human being , responding their inner desire to do their best as a nurse , and trying to improve nursing image in society . one participant related her sense of ethical and professional commitment to satisfaction of her inner philanthropic desire : to me , it does not matter whether someone see or appreciate my efforts or ethically working . doing my best firstly as a human , secondly as a nurse satisfies me , so it is enough to follow my inner voice and desire to work as well as i can to help a needy patient to make him / her condition better . this study was designed to answer the question : what are undergraduate nursing student perceptions of the informal learning that occurs during nursing studies ? this study was conducted over a 2-year period ( 2009 - 2010 ) using a qualitative content analysis . the participants were 14 nursing students in their fourth year of study at x university who were recruited through a purposeful sampling method . they were at their final stage of clinical apprenticeship so had passed major part of their education and could share their achievements of learning experiences , including informal ones . sampling was done according to the maximum variant approach in terms of gender , age , marital status , geographical location ( local or not local ) , student work experience , and level of academic achievement ( e.g. , good , average , or superior ) . each interview began with the open question : apart from the theoretical and practical knowledge you gained during four years of nursing study , what other changes do you think you have experienced during your study ? answers were explored more deeply with relevant probing statements or questions , such as : please tell me more about that , how did that make you feel ? please describe an actual experience you have had that will help me understand what it meant to you . each interview was transcribed verbatim , and then converted to rich text format for use in maxqda software , which facilitated data management . data were analyzed through a constant comparison analysis method using strauss and corbin coding techniques to develop the categories . data were analyzed initially by the main author , then the emerging codes were revised independently by 2 other members of the research team . throughout coding and category development , the analysis process and the accuracy of codes and categories were discussed and checked in regular sessions ; discrepancies were resolved through continuing discussion and analysis . lincoln and guba ( 1985 ) criteria were used to address trustworthiness ( p.296 ) . dependability was gained by precise documentation of data , methods , and decisions about the research . credibility was established through participant confirmation of our understanding , prolonged engagements with participants , a peer check , and an external check . throughout the study , data were managed by maxqda and all analysis precisely documented to maintain an audit trail . student participation was voluntary . written information about the purpose of the study and confidentiality management was delivered to all participants and written consent was obtained for the study , including permission for recording the entire interview for transcribing . the 14 participants consisted of 3 male and 11 female nursing students , age 21 - 23 years , in the last semester of their studies . based on students experiences , 5 categories were extracted : personal maturity and emotional growth , social development , closeness to god , alterations in value systems , and ethical and professional commitments [ table 1 ] . although some participants described the process leading to their internal changes as challenging and difficult , almost all of them stated that it facilitated their growth as a whole person . this category consisted of 2 subcategories : personal maturity ( consisting of self - awareness and self - discovery , accelerated mental maturity , and more open - mindedness ) and emotional growth ( consisting of greater patience , appreciation , kindness , and compassion , and altruism ) . the participants viewed their 4 years in nursing school as an opportunity for gaining a deeper self - awareness and self - knowledge . they believed that the duties of nursing practice , such as frequent personal interactions and development of relationships , have great influence on deep reflection about humanity as well as their personal beliefs . in contrast , the importance of rigorous assessment throughout nursing studies resulted in regular self - assessment in their daily lives . these helped them obtain more realistic knowledge about their personal strengths , weaknesses , and talents . some participants described this awareness as self - discovery by saying : i learned about myself based on what they taught us about humans in the program . i understood myself , in the university setting , through the eyes of my friends , who criticized me . another significant change reported by students was an increasing maturity of mind that resulted in more rational behavior . the frequent exposure to life - and - death situations in nursing helped promote maturity . one student said : my personality developed at this school . at first , i behaved very childishly . there were a series of realities of which i was wholly ignorant ; i acquired them here in the school . they also attributed this increase in maturity to multiple factors , including interactions with instructors , senior students , and people with various personalities , as well as engagement in difficult situations . one participant said : this maturity has been partly influenced by interactions with professional instructors and encounters with classmates as well as senior students i improved little by little through experiencing critical situations , seeing the darker side of life , the troubles and pains of other people , all of which i was unaware of in my previous relaxed lifestyle . the other significant experience reported by participants was increasing open - mindedness . with respect to the principles of fundamental nursing classes , they learned to focus on a patient as a human being , regardless of nationality , ethnicity , religious beliefs , or financial or sociocultural status . as a result , students reported that they were able to accept people by adopting a nonjudgmental manner . one student , who was caring for people with various religious beliefs , described her primary concern about encountering clients with different beliefs as a challenging experience and explained how she successfully overcame those conflicts by overlooking religious prejudices . she said : at first i thought i had treated them differently ; well , one patient was muslim , the other held another belief . however , after a while , i realized that they are ultimately human , and my job was not to judge the belief system of people . then the differences were no longer serious or problematic and i found myself adapting easily by getting closer to them . i am very grateful to be able to communicate well with various religious minorities despite so many differences . almost all of the participants indicated that increasing patience was their most significant change . some of them said that they learned patience from patients who were suffering from chronic illnesses . when they saw how these patients worked to manage the disease and its disabling consequences , they realized the triviality of their own problems . i had patients who underwent dialysis every day , yet were quite happy , spoke with joy , laughed , and induced others to be in a good mood . in comparison with them , how could i concern myself with minor issues while i was in good health physically and emotionally ? i felt embarrassed when i saw them struggle steadfastly with such pain , illness , and deprivation . other participants believed that the patience resulting from the nature of discipline made them stronger to handle difficulties . since in nursing practice you see the pains and troubles of other people , a series of problems are no longer considered problems , or perhaps your experiences and abilities have improved . you should be more patient , in order to help those people who need you and have their hope invested in you . they said seeing patients suffer made them realize the importance of the gifts they took for granted , especially the gift of health : now i see patients with their problems and realize the gifts i have in my life , which i did nt appreciate before . they also explained that nursing made them similar to mothers - kind and ready to help and show compassion . now they feel a stronger sense of love and concern for humankind as well as the ability to love and care for others . one of them indicated : studying in the nursing field gives you the sense of being a mother . they teach us that we must be able to establish good communication , and understand others and support them . one participant stated : generally , my morale and personality is such that i really like people . it seems that these traits were developed for inner satisfaction and for the affection you can induce in others . the second category derived from the interviews was related to becoming more social and expert in interpersonal relationships . some of the participants , especially those who had limited communication skills before entering the program , acknowledged nursing studies as a turning point that improved their social relationships . they asserted that gaining communication skills with patients in clinical settings as fundamental to improving their interpersonal proficiency by saying : nursing improved my communication with others . i , who had a habit of building fences around me , was forced out of that closed space . during this period , my communication skills got much stronger , since they are required and necessary skills in nursing . encouraging and developing communication with patients required students to exploit strategies , such as developing cultural and ideological common ground , respecting others interests and wishes , and adapting themselves to others . gradually they noticed they were applying these advanced communication skills in their personal life . now , i can look at the subject from his / her point of view . i take the initiative and try to begin from a point that is welcomed by the other party . i moved away from a partial and limited understanding of others and i feel that i am growing in regards to social relations in general . another factor that contributed to students social development was existing in a social environment larger than that of high school , which involved interaction with a heterogeneous population from various towns , ethnic groups , and cultures , which was regarded as expanding the friends circle by participants : i tried to experience all group types . i do nt limit myself to making friends with people of my own type ; i always search for new thoughts and new ideas with their own experiences . nonlocal participants , who resided in the student dormitory during their studies , explained the significant influence of living in the dormitory on their social growth and development as : when you become a college student and live in the dormitory with other students coming from different places and cultures with various upbringings , you are obliged to encounter them , and automatically learn how to adapt yourself and in fact be flexible . since all participants were muslim , a belief in god was one fundamental characteristic . however , most of them stated that they experienced a different type of connection with god during their nursing studies . some of them attributed this new belief to gaining a deeper knowledge about the intricacies of the human body and about human beings in general during the nursing program : i understood the complexities of this perfect system ( the human body ) , how accurately it has been designed , and how many problems are caused by a small deviation . we were told that the patients do nt respond to any treatment , but they did and i saw with my own eyes the effect of the prayers that the person accompanying the patient offered from the depths of the heart , and how it worked . i touched god s power and presence . others stated they felt closer to god through nursing practice because they were satisfying god s wishes by caring for the most vulnerable . this optimism significantly improved the quality of their prayers and replaced traditional ceremonies with enthusiastic worship and inner dialog with god . this type of close connection with god led to a pleasant feeling of dependency , which made them calmer when confronting difficulties : now i feel that god is looking at me , for my interest in caring for his people . i tell myself since i serve god s people sincerely ; certainly he would also pay attention to me ... i have noticed that whenever i have troubles , they are resolved at once . i sense some resolution and interpret it as god s promise that , if you help someone , god will help you in return . participants believed that confrontation with the light as well as the dark side of life in nursing expanded their world view through a change in focus from living a superficial life to a life more concerned with serious and substantive matters beyond their calendar age . one participant explained : in nursing , you see the beginning of life and the termination of it . you are faced with certain facts that make other issues trivial and insignificant in life while they are still important for your peers . these challenging confrontations acted as awakening experiences , allowing students to obtain a new perspective about life and death that stimulated changes in their value systems . as a result they described these alterations as achieving a deeper and more realistic point of view of life : now my objectives for the future have changed in principle . whenever i see patients who have only a few days to live , i no longer waste my time . before , i had a child - like view of life as a bowl of cherries with no worries , but now i know that life has its own hardships , which are considered a part of life . moreover , most of them asserted that they feel they were chosen by god for this profession and they appreciated it as an opportunity for growth and transcendence . i sensed that i was invited by god to take a right path that changed my destiny forever . i began taking steps on a road that i was supposed to begin with and i interpreted this as special attention to me on god s part ... i believe that we were summoned by god . nurses were invited for their kind heart , for their sympathy , and for their ability to provide relaxation to patients . if you knew this from the beginning and you appreciated being invited , any experience along the way would become important and valuable to you . most participants admitted that they feel a strong commitment to their work and try to be conscientious in the care of their patients . they believed that this inner commitment had a stronger influence than any rules or external controls . some of them attributed this inner commitment to their increased closeness to god and consequent sense of being observed constantly by god . they were delighted by this connection and attention so they tried to sustain this perceived attention by working ethically and conscientiously . even though i can work unsterile and the patient may not know it , i notice what i do and god does , too . however , your ethical commitment forces you to work better . to me , respecting the patient s rights is more important than anything else . i always tell myself to imagine that the patient is one of your dear siblings . i always try to hold that thought and work according to such feelings to do my best . in addition , participants attributed this commitment to other reasons , such as respecting patient s rights and dignity to enjoy humanistic and proper care as a human being , responding their inner desire to do their best as a nurse , and trying to improve nursing image in society . one participant related her sense of ethical and professional commitment to satisfaction of her inner philanthropic desire : to me , it does not matter whether someone see or appreciate my efforts or ethically working . doing my best firstly as a human , secondly as a nurse satisfies me , so it is enough to follow my inner voice and desire to work as well as i can to help a needy patient to make him / her condition better . since students who felt more personal growth were more eager to participate in the study , it is possible that other students experiences have been ignored unintentionally by the researchers . according to the results of this study , the undergraduate nursing students achieved knowledge and insights that were not provided by the formal , biomedical - centered , nursing program . these included the following : personal maturity and emotional development , social development , greater closeness to god , an alteration in value systems , and ethical and professional commitment . these are nontechnical professional competencies that are important to the goals of the nursing curriculum . although these are not included in a specific lesson , they are beyond theoretical knowledge and the technical skills and could be considered as the soul of nursing education . the results indicate that nursing students develop important qualities through a hidden curriculum that help fulfill the mission of nursing education in creating ethical , spiritual , legal , and professional values . in this study , one of the most important categories was personal growth , which included self - awareness , self - knowledge , and self - discovery due to frequent interactions with patients and other people and reflection on the experiences of these relationships . the meaning of this category was compatible with that of a previous study , indicating a self - awareness theme related to experiences of being and presence . participants in the previous study also experienced nurse - patient interactions that resulted in self - awareness . the participants who increased self - awareness did so through reflection , and found meaning in being a nurse . chiu the outcome of university courses are not limited to professional development ; they also promoted the personal growth of nurses . being a nurse requires compassion , respect , and humanity . in the present study , participants described nursing as an opportunity to gain empathy and other humanistic feelings . they appreciated the characteristics , such as patience , thankfulness , kindness , compassion , and concern about humankind , that could help them achieve emotional growth . emotional growth means the increasing capacity to recognize one s own emotions and allow them to mature . wilson , stated that nursing students develop emotional competence by critical reflection through classroom sessions and clinical experiences . the participants in the present study also indicated that reflection occurred outside formal sessions in the classroom . because nursing is a profession involving difficult situations , nursing students need preparation to mature enough to successfully handle their professional role , especially during a crisis . studies reveal that nurses younger than 30 years experience greater burnout than their older counterparts . such findings support the idea that emotional issues should be addressed in nursing curricula to nurture a healthier and more competent nursing workforce . the participants in the present study also emphasized improvement in interpersonal and communication skills as a result of their nursing studies . they believed that nursing taught them to put themselves in the patient s place and see through their eyes , which increased the awareness of patients feelings , perceptions , and needs . these critical abilities are referred to as social growth qualities , gained through interactions with professors , classmates , roommates , and patients while out of the classroom . although our participants were muslims , who must maintain a respectful distance from members of the opposite sex that can interfere with some nursing tasks , findings indicated that even the strongly religious female participants did not have problems caring for male patients . another aspect of nursing training that affected students in this study was their relationship with god . from an islamic perspective additionally , nursing is considered a divine profession in iran . in this ideologic context , almost all of the study participants appreciated nursing as a call from god toward transcendence . ravari et al . also reported that the perception of nursing as an opportunity to worship god is a common belief among iranian nurses , which provided their career a transcendental meaning . the study participants reported changes in their value systems , such as finding a deeper meaning beyond the superficial activities of everyday life and more lasting goals and values . university studies provided an awakening experience , and inspired students to re - evaluate and redefine their personal values . since universities are expected to help students achieve a broader perspective , nursing education should help promote student receptivity of human values compatible with their professional mission . indicated that empathizing with patients and caring about their feelings initiates an ethical reflection process in nursing students that may eventually lead to the development of a set of personal values . the last category revealed that , although the participants were just starting their career , they gained a sense of professional and ethical commitment . in a similar study by rahimaghaee et al , participant views about commitment went beyond work commitment to include an inner commitment related to a belief in god s supervision of their lives , including their careers . additionally , le duc and kotzer , found that even novice nurses can hold professional values , indicating that nursing education can initiate a sense of professional commitment in students . according to some nursing theorists , including watson , nursing schools should acknowledge the hidden learning that occurs outside the classroom can foster and facilitate development of students as whole human beings , not just professional nurses . according to watson , curriculum development should combine and integrate the cognitive with education about beauty , art , ethics , intuition , esthetics , and spirituality of human - to - human interactions within nursing . the nursing discipline needs to incorporate this type of curriculum to improve nursing student s personal development . during their education , this aspect of nursing was reported by paterson and zedrad ( 2007 ) in their humanistic nursing theory . the findings of this study revealed that nursing education , especially clinical training with its patient - student interactions , provides an excellent opportunity for student personal growth that is less likely in other majors . through their studies , nursing students do not gain expertise in theoretical and technical matters alone , but also fulfill their potential as a whole person . nursing education can prepare students to deliver holistic care only if they consider all aspects of the students as humans . the ability to provide holistic care of patients requires treating each nursing student as a whole person through both formal and informal education . this idea agrees with theories of student development that consider college education as involving the whole person and as a process involving interaction between intellectual development and interpersonal competence . therefore , recognizing and encouraging covert aspects of nursing education as well as overt ones provide more opportunities for student development , not only in cognitive and technical domains but also in affective ones . this can help bring nursing education closer to the ideal of comprehensiveness that enables nurses to care holistically for patients .

introductionestablished in 2003 , erste foundation has a history stretching back 190 years to the year 1819 when it was founded as the erste sterreichische spar casse in vienna . rooted in our history as a social enterprise and financial service provider , erste foundation recognises that contemporary society faces huge challenges and that for a new and united europe to work , integration is crucial and that means uniting the economic , cultural and social capital of our region.descriptionwe develop ideas and concepts to increase social participation and to ensure that no - one is left out , whatever their circumstances which in turn creates stable , effective and fairer societies.european integration is important to us . we want to work against prejudice and nationalism , to integrate thinking and living across borders , and make these experiences accessible , particularly to the young generation.projectswe intend to play an active role in giving people opportunities to increase their understanding of each other . therefore , erste foundation has supported research projects that concern themselves with the effects of societal transformation processes : in particular we have been looking at the effects that demographic changes will have on the long - term care needs of elderly people in central and eastern europe . from this , we intend to develop policy recommendations for decision makers in civil society , economy and politics . background : nursing education is both formal and informal . formal education represents only a small part of all the learning involved ; and many students learn more effectively through informal processes . there is little information about nursing student informal education and how it affects their character and practice.materials and methods : this qualitative study explores undergraduate nursing student perceptions of informal learning during nursing studies . data were gathered through semi - structured interviews with a sample of undergraduate nursing students ( n = 14 ) . strauss and corbin s constant comparison analysis approach was used for data analysis.results:the categories that emerged included personal maturity and emotional development , social development , closeness to god , alterations in value systems , and ethical and professional commitment.conclusion:findings reveal that nursing education could take advantage of informal learning opportunities to develop students nontechnical skills and produce more competent students . implications for nursing education are discussed .

the in vitro ageing of human diploid fibroblasts ( hdfs ) , first described by hayflick and moorhead , has become a classical experimental model to study cellular ageing . hdfs have a limited ability to divide when cultured in vitro . normally after about 50 cell divisions , hdfs enter a state of irreversible proliferative arrest , termed as replicative senescence or cellular senescence . cells with less than 10 passages were considered young cells with high proliferative ability while cultures at 1020 passages have entered an intermediate state or pre - senescence , and cultures of over 25 passages with no detectable doubling in cell numbers for 2 weeks were considered as senescent cells . senescent cells have been shown to accumulate with age in human tissues and , thus , have been suggested to contribute to organismal ageing . reactive oxygen species ( ros ) were implicated in replicative senescence and ageing . during physiological metabolism , endogenous ros which include superoxide anion , hydrogen peroxide , hydroxyl radicals , and singlet oxygen accumulation of oxidatively damaged cellular macromolecules is suggested to account for the free radical theory of ageing . upon entering the state of senescence the cell size or volume is increased with accumulation of cellular debris and intracellular vesicles , many of which are lysosomes . it has been reported that senescent fibroblasts became flattened and more irregular in shape with increased expression of senescence marker such as senescence associated -galactosidase ( sa--gal ) . both in vitro and in vivo studies have shown the increase in percentage of cells positive for sa--gal with cumulative population doublings ( cpds ) and age . nevertheless , the mechanisms that are responsible for the continuous cell growth and increased in sa--gal expression in senescent cells have not been well elucidated . cell cycle checkpoints sense damage of the dna structure and elicit complex cellular repair response . the checkpoints maintain cell cycle arrest while the repair takes place followed by cell cycle progression once repair is completed . cells undergo permanent cell cycle arrest and apoptosis if the dna can not be repaired adequately . a permanent cell cycle arrest occurs with cells remaining in g0/g1 phase during senescence . besides oxidative dna damage accumulation , telomere shortening has been widely considered as another important mechanism to trigger replicative senescence in human fibroblasts . significant telomere shortening occurred as a consequence of prolonged h2o2 treatment but not in acute treatment . . showed that telomere shortening was largely dependent on the interplay of oxidative stress and antioxidant defence rather than the cell divisions . a comparison of the telomere shortening rates in different hdfs under different conditions revealed that the ratio between oxidative damage and antioxidative defence was quantitatively the most important determinant of telomere shortening . to prevent progressive dna loss with subsequent rounds of dna replication , many cells maintain their telomeres by the action of telomerase , a specialized rna - protein complex that uses its rna component as a template for the extension of the telomere by its reverse transcriptase subunit . in the absence of an adequate repair system , accumulated dna damage caused mutagenic alterations resulting in cancer , ageing , and abnormalities in the nervous system . accumulating evidence demonstrated that vitamin e , the most potent lipid peroxyl radical scavenger , significantly reduced free radical - induced chromosomal damage . vitamin e has been shown to reduce h2o2-induced oh generation and subsequent dna base pair modification in human oral epithelial cells and h2o2-induced dna strand breaks in human skin cell line vh10 . measurement of dna repair ability tested in lymphocytes indicated that vitamin e increased the removal rate of damaged dna compared to cells that are not treated with vitamin e . natural vitamin e comprises of eight different isomers ; they are - , - , - , and -tocopherols and - , - , - , and -tocotrienols . based on its lipophilicity , vitamin e is considered to be the major chain - breaking antioxidant preventing the propagation of oxidative stress , especially in biological membranes . vitamin e specifically -tocopherol has been reported to be able to modulate signal transduction and gene expression via its antioxidant and non - antioxidant properties . recently , the different isomers of tocotrienol have gained increasing scientific interest due to their eminent antioxidant effects and a non - antioxidant activity profile that differs from tocopherols . tocotrienols are found in abundance in rice bran , palm oil , oat , and barley . tocotrienol - rich fraction consists of -tocopherol and four isomers of tocotrienols ( , , , and ) , all of which are potent membrane - soluble antioxidants . the differences in the properties of tocotrienol have suggested protective effects in diseases such as cardiovascular disease and cancer in experimental animals . in an animal model of ageing , tocotrienol extended lifespan by 19% while reducing protein carbonylation , a particularly toxic oxidation process indicative of ageing . since oxidants contribute to the ageing process and ageing - related diseases in many species , possible protection from ageing by antioxidants such as tocotrienol and tocopherol is suggested . even though beneficial effects of vitamin e have been established in ageing , ongoing studies are being done to determine mechanisms involved and actual response of different stages of cellular ageing . in view of this , this study was designed to evaluate the molecular mechanism of tocotrienol - rich fraction in possibly modulating different stages of cellular ageing in hdfs by determining the changes in molecular markers of cell - ageing sa--gal , dna damage , telomere shortening , and cell cycle progression in young , presenescent , and senescent hdfs . this research has been approved by the ethics committee of national university of malaysia ( approval project code : ff-218 - 2008 ) . primary hdfs were derived from the foreskins of three 9- to 12-year - old boys after circumcision . the samples were aseptically collected and washed several times with 75% alcohol and phosphate buffered saline ( pbs ) containing 1% antibiotic - antimycotic solution ( paa , austria ) . after removing the epidermis , the pure dermis was cut into small pieces and transferred into a falcon tube containing 0.03% collagenase type i solution ( worthington biochemical corporation , usa ) . pure dermis was digested in the incubator shaker at 37c for 612 h. then , cells were rinsed with pbs before being cultured in dulbecco modified eagle medium ( dmem ) containing 10% fetal bovine serum ( fbs ) ( paa , austria ) and 1% antibiotic - antimycotic solution at 37c in 5% co2 humidified incubator . after 56 days , the cultured hdfs were harvested by trypsinization and culture - expand into new t25 culture flasks ( nunc , denmark ) with expansion degree of 1 : 4 . when the subcultures reached 8090% confluence , serial passaging was done by trypsinization and the number of population doublings ( pds ) was monitored until hdfs reached senescence . for subsequent experiments , cells were used at either passage 4 ( young cell , population doubling ; pd < 12 ) , passage 15 ( presenescent cell , 30 < pd < 40 ) , and passage 30 ( senescent cell , pd > 55 ) . cell viability study was performed using mts assay . briefly , stock solutions of trf gold tri e 50 ( golden hope bioganic sdn bhd , malaysia ) was freshly prepared in 100% ethanol ( 1 : 1 ) and kept at 20c for not more than one month . serial dilutions of trf at concentrations of 0.1 , 0.2 , 0.3 , 0.4 , and 0.5 mg / ml were prepared in culture medium mixed with 50% ethanol ( 1 : 1 ) . , the medium was replaced with new medium containing the various concentrations of trf and incubated for 24 h at 37c , in 5% co2 . after incubation , 20 l mts was added and cells were further incubated for 2 h. the absorbance of mts formazan formed was measured at 490 nm with a microtiter plate reader ( veramax molecular devices , usa ) . the viability assay was performed to obtain the optimum dose of trf treatment for subsequent experiments . in the subsequent experiments , treated hdfs were incubated with 0.5 mg / ml trf for 24 h while untreated cells were cultured in dulbecco modified eagle medium ( dmem ) containing 10% fetal bovine serum ( fbs ) ( paa , austria ) . sa--gal staining was performed with a senescent cell staining kit ( sigma , usa ) according to the manufacturer 's instructions . a total of 1 10 cells in the trf - treated and control groups were seeded in six - well plates and incubated with fixation buffer ( 2% formaldehyde/0.2% glutaraldehyde ) for 67 mins at room temperature . cells were then rinsed three times with pbs and incubated with 5-bromo-4-chloro-3-indolyl -d - galactopyranoside at 1 mg / ml in a buffer containing 40 mm citric acid / phosphate ( ph 6.0 ) , 5 mm k3fecn6 , 5 mm k4fecn6 , 150 mm nacl , and 2 mm mgcl2 for 4 h at 37c in the absence of co2 . blue staining was visible after incubation , and the percentage of blue cells observed in 100 cells under a light microscope was calculated . telomere assay was performed using telotaggg telomere length assay kit ( roche , usa ) that determined telomere length using terminal restriction fragment principle . three g of dna was digested with 20 units each of hinfi and rsai for 2 h at 37c . fractionated dna fragments were transferred to nylon membranes hybond - n ( amersham , uk ) by an alkaline transfer technique using capillary blotting . the blotted dna fragments were hybridized to a digoxigenin ( dig)-labeled probe specific for telomeric repeats ( ttaggg ) for 3 h at 42c with gentle agitation with a dig - specific antibody covalently coupled to alkaline phosphatase . finally , the immobilized telomere probe was visualized by alkaline phosphatase metabolizing cdp - star , a highly sensitive chemiluminescence substrate . the average terminal restriction fragment length was determined by comparing signals relative to a nuclear weight standard on x - ray film . the average terminal restriction fragment length of each sample was obtained by scanning the exposed x - ray film and quantifying using imagemaster total lab software . detection of telomerase activity using the telomeric repeat amplification protocol ( trap ) in cultured cells involves the addition of ttaggg repeats by telomerase to an oligonucleotide ( ts ) and the subsequent pcr amplification of these extension products with both the forward ( ts ) and reverse ( cx ) primers . the trapeze telomerase detection kit ( chemicon , usa ) was used as recommended by the manufacturer with minor modifications . the lysis buffer contained 1% nonidet p-40 and 0.25 mm sodium deoxycholate to increase the efficiency of extraction . cells were lysed , then left on ice for 30 min , and centrifuged at 14,000 g for 20 min at 4c . the extracted protein concentration was determined from bovine serum albumin ( bsa ) standard plot of od595 versus g bsa based on bradford method . for the pcr reaction , the total counts in the sample was added to 48 l reaction mixture and 2 units of taq dna polymerase ( promega , usa ) . after incubation at room temperature for 30 min for the telomerase extension reaction , samples were heated to 92c for 3 min to inactivate telomerase followed by pcr amplification . pcr products were electrophoresed on 10% polyacrylamide gel , and the gel was further analysed and quantitated using the imagemaster total lab software . telomerase activity was calculated as the ratio of the intensity of telomerase ladders to the intensity of the 36-bp internal standard . dna damage was assessed using single - cell gel electrophoresis assay ( scge ; comet assay ) . the alkaline version of comet assay was performed according to the method described by singh et al . . cells were embedded in 65 l 0.6% low - melting point agarose layer ( boehringer mannheim , germany ) ( dnase free , rnase free ) on agarose - coated frosted slides . the cells were lysed in a buffer containing 2.5 m nacl , 100 mm edta , 10 mm tris , 1% n - laurylsarcosine , 1% triton , and 2% dimethylsulphoxide , at ph 10 , for 1 h at 4c . thereafter , the cells were exposed to a strong alkaline solution ( 300 mm naoh , 1 mm edta , ph 13 ) for 25 min in an electrophoresis chamber . electrophoresis was performed for 20 min at 25 v and 200 ma . the slides were then neutralized ( 0.4 m tris , ph 7.5 ) and stained with ethidium bromide ( 20 g / ml ) . comets were analysed by fluorescence microscopy ( carl zeiss , germany ) with visual inspection of tail length of nuclei . the cell nuclei were classified into five categories : ( 0 ) undamaged ( nuclei without comet tail ) , ( 1 ) low damaged ( nuclei with comet tails up to two fold longer than nucleus diameter ) , ( 2 ) damaged ( nuclei with comet tail two to three - fold longer than nucleus diameter ) , ( 3 ) highly damaged ( nuclei with comet tails three fold longer than nucleus diameter ) , and ( 4 ) severely damaged ( cell nuclei was almost not visible with long and dispersed comet tails ) . at least 300 cells per slide were counted , and two slides were prepared for each treatment . a total damage score was determined by multiplying the number of cells assigned to each grade of damage by the numeric value of the grade according to methods describe by heaton et al . . total dna damage score was calculated as follows : ( 1)total dna damage=[(0n0)+(1n1)+(2n2 ) + ( 3n3)+(4n4 ) ] , where n0 = cells with score 0 , n1 = cells with score 1 , n2 = cells with score 2 , n3 = cells with score 3 , and n4 = cells with score 4 . untreated control and trf - treated hdfs were subcultured in 10 cm tissue culture dishes . after 24 h incubation , cells were harvested and prepared using cycletest plus dna reagent kit ( becton dickinson , usa ) according to the manufacturer 's instruction . cell cycle status was analyzed by facs caliber flow cytometer ( becton dickinson , usa ) using propidium iodide ( pi ) as a specific fluorescent dye probe . each experiment was carried out in triplicates with at least 3 independent cultures with comparable results . comparison between groups were made by anova and student 's t - test ( two - tailed ) . figure 1 shows the percentage of viable fibroblast cells after incubated with trf at various concentrations ( 0.10.5 mg / ml ) for 24 h. the percentage of viable cells was significantly increased ( p < .05 ) with trf treatment at 0.5 mg / ml ( figure 1(a ) ) for young hdfs . for presenescent hdfs , the percentage of viable cells was significantly increased ( p < .05 ) with trf incubation at concentrations of 0.3 mg / ml , 0.4 mg / ml , and 0.5 mg / ml ( figure 1(b ) ) while the percentage of viable cells for senescent hdfs was significantly increased ( p < .05 ) with trf treatment at all concentrations ( figure 1(c ) ) . therefore , trf at concentration 0.5 g / ml was used for the subsequent experiments for all types of hdfs . however , with senescence , the original fibroblastic shape was lost and hdfs became larger and flattened with accumulation of cytoplasmic granular inclusions ( figures 2(a)2(c ) ) . the morphology of trf - treated hdfs resembled that of young cells with more spindle shaped cells present ( figures 2(d)2(f ) ) . positive blue stain of sa--gal appeared mainly in hdfs at passage 30 suggesting the presence of senescent cells ( figure 3 ) . quantitative analysis showed the percentage of cells positive for sa--gal staining was increased ( p < .05 ) in senescent cells compared to young and presenescent hdfs . incubation of senescent cells with 0.5 mg / ml trf significantly decreased ( p < .05 ) the percentage of positive sa--gal stained cells compared to untreated control ( figure 4 ) . damaged dna was higher in senescent hdfs compared to young and presenescent cells ( p < .05 ) which was decreased with trf - treatment ( p < cell cycle progression analysis showed that the cell population in the s phase was lower ( p < .05 ) in senescent hdfs compared to young hdfs . treatment with trf significantly increased ( p < .05 ) cells in the s phase and g2/m phase for all stages of cellular senescence of hdfs . in contrast , cell populations in g0/g1 phase decreased significantly ( p < .05 ) with trf treatment in young , presenescent , and senescent hdfs ( figure 6 ) . figure 7(a ) shows the representative southern blot analysis of hdfs at various passages with trf treatment . telomere length in senescent hdfs was significantly decreased compared to untreated young hdfs ( p < .05 ) . similar trf treatment had no effect on telomere length in young hdfs ( figure 7(b ) ) . figure 7(c ) shows the representative pcr analysis for telomerase activity of hdfs at various passages with trf treatment . treatment with trf significantly increased the telomerase activity in senescent hdfs ( p < .05 ) whereby no effects was observed in young hdfs ( figure 7(d ) ) . the present study evaluated the effects of tocotrienol - rich fraction ( trf ) in possibly modulating cellular ageing in hdfs . our results showed that when hdfs reached senescence , there were clear changes in cell morphology , decreased cell proliferation and increased senescence associated -galactosidase activity . typical morphology of senescent cells observed in this study has also been reported with multiple passages of cells . granular cytoplasmic and vacuoles accumulation are common features of senescent cells accounting for the large cells . senescent cells are also filled with large and numerous lysosomes that contain hydrolytic enzymes which function in ingesting and digesting organelles that are aged or damaged . the increased sa--gal positive cells which was observed with senescence of hdfs can be attributed to increased in lysosomal content . increased autophagy may be associated with an increase in lysosomal mass and sa--gal in in vitro ageing . in addition , it is possible to find a correlation between the increase in sa--gal and appearance of the senescent morphotypes . increase in sa--gal activity in senescent hdfs and stress - induced premature senescence ( sips ) is an irreversible process upon subculture , suggesting that cellular ageing results in irreversible nonmitotic growth . however , incubation with trf was shown to decrease the percentage of cells positive for sa--gal suggesting a reversal of cellular ageing of hdfs . this effect is possibly due to the antioxidant property of trf which consists of -tocopherols and all isomers of tocotrienols . previous study has shown that tocotrienols have high antioxidant activity and that the uptake of tocotrienols from the culture medium by cultured cells was more efficient than tocopherols . our data on cells viability study showed trf treatment increased the percentage of viable cells with increasing concentrations of trf . the percentage of viable cells was highest at the maximum concentration studied , that is , 0.5 mg / ml indicating trf promoted cells propagation and viability in all stages of cellular ageing of hdfs . oxidative stress caused damage to dna which increased significantly in senescent cells , especially in postmitotic tissues and the levels correlated with ageing and age - related diseases . reported that age - related increased in the levels of oxidative dna base damage was a significant contributor to many age - related pathological diseases . if accumulation of damaged dna exceeds its elimination by dna repair mechanism , cellular senescence , or apoptosis takes place and contributes to the ageing process . comparison of damaged dna in different treatment groups of hdfs in the present study showed that total dna damage was significantly increased in senescent cells while less damaged dna was observed in young and presenescent hdfs . ros caused chronic and persistent damage to dna , causing double strand breaks that remain unrepaired which tend to accumulate in senescent cells indicating a possible cause of ageing in mammals . increased in ros - induced dna damage was reported to be correlated with cell cycle arrest . similarly , our data showed significant decreased in s phase cells and increased in damaged dna in senescent hdfs indicating less cell proliferation and greater dna damage as cells aged . it is highly probable that protection against dna damage with trf treatment in senescent hdfs could be attributed to either prevention of oxidative stress - induced dna damage or enhancement of dna repair mechanism . vitamin e , particularly -tocopherol , has been suggested to inhibit activation of endonuclease that can be triggered by intracellular oxidative stress and enhanced dna repair by increasing the rate of removal of damaged dna . previous study reported that vitamin e reduced h2o2-induced ho generation and subsequent dna base pair modification in human oral epithelial cells besides decreasing h2o2-induced dna strand breaks in human skin cell line vh10 . cellular senescence in response to dna damage or degradation would make the cells 's progeny nonviable and might indicate a decline in renewal capability of cells as they reach an irreversible growth arrest . our data showed no significant difference in g0/g1 or g2/m phase with cellular ageing of hdfs , while the s phase cells decreased significantly . the reduction in s phase cells with senescence can be attributed to slow rate of proliferation as cells undergoing ageing . the decreased and increased cells in g0/g1 phase and s phase , respectively , were observed with trf treatment at various stages of cellular ageing indicating inhibition of growth arrest and enhancement of cell replication . this observation indicated trf was able to promote cell progression , and increased cells replicative capacity . decreased level of total dna damage and increased percentage of cells in s phase in trf - treated senescent cells indicated a potential protective role of trf against dna strand breaks and cell cycle arrest . it has been clearly demonstrated that cell replication caused telomere shortening due to the inability of dna polymerase to completely replicate the 3-ends of lagging strands in the majority of normal human somatic cells which lack telomerase activity . our data in the present study suggested that both telomere shortening and decreased telomerase activity might be the contributing factors in cellular ageing . it has been reported that due to inherent limitations in the mechanics of dna replication , telomeres shorten at each cell division , and in the absence of telomerase , when telomere shortening reaches a critical limit , cells are susceptible to chromosomal aberrations such as end - to - end fusion and aneuploidy . in such a situation , treatment with trf , however , protected against telomere shortening in senescent hdfs with concomitant increased in telomerase activity . our previous study had shown that both -tocotrienol and -tocopherol protected against oxidative stress - induced telomere shortening in hdfs derived from differently aged individuals . the restoration of telomerase activity induced by -tocopherol caused significant increased in telomere length especially in skin fibroblast obtained from old donor [ 26 , 41 ] . in summary , trf treatment exerted better protection in senescent hdfs when compared to young and presenescent hdfs . possibly higher requirement for antioxidants is indicated in senescent hdfs as the uptake of trf and its utilization in senescent hdfs is enhanced for protection against ros related damage . therefore , we concluded that tocotrienol - rich fraction delays or prevents cellular ageing particularly in senescent hdfs as shown by the elongated telomere length , decreased levels of damaged dna , and progression of cell cycle to s phase besides increased cell propagation in all stages of cellular ageing of hdfs with trf treatment . young , healthy organisms strive to maintain their proteome in a functional state through the tight control of rates of protein synthesis , folding , and degradation . extensive quality - control systems are set up throughout the cell to prevent and manage protein damage . as the organism ages , these control mechanisms become less efficient , leading to a disruption in protein homeostasis ( balch et al . , 2008 , david , 2012 ) . aging is the main risk factor for a variety of neurodegenerative diseases where specific proteins accumulate as pathological aggregates . recently , there has been considerable interest in investigating widespread protein aggregation in the absence of disease . multiple studies have demonstrated that several hundred proteins become highly detergent - insoluble in aged animals ( ayyadevara et al . , 2016 , david , 2012 , david et al . , 2010 , demontis and perrimon , 2010 , reis - rodrigues et al . , 2012 , , 2016 , walther et al . , 2015 ) . computational analysis of the insoluble proteome indicates an overrepresentation of proteins with functional and structural similarities ( david et al . , 2010 ) . the examination of some of these proteins in vivo reveals their assembly into large the discovery of endogenous age - dependent protein aggregation in model organisms gives us the unprecedented opportunity to dissect the intrinsic cellular machineries responsible for preventing protein aggregation without using ectopically expressed human disease - associated proteins . at this time , very little is known concerning the regulation of widespread protein insolubility with age and its consequences for the health of the organism . interestingly , several studies show that protein insolubility is modified in long - lived animals with reduced insulin / insulin growth factor ( igf)-1 daf-2 signaling , but it remains unclear to which extent ( david et al . , 2010 , demontis and perrimon , 2010 , walther et al . , 2015 ) . a growing number of familial and sporadic forms of neurodegenerative diseases show pathological inclusions caused by abnormal aggregation of rna - binding proteins ( rbps ) . the first rbps identified in these inclusions were tar dna binding protein of 43 kda ( tdp-43 ) and fused in sarcoma ( fus ) , associated with amyotrophic lateral sclerosis ( als ) and frontotemporal lobar degeneration ( ftld ) ( arai et al . , 2006 , neumann et al . , 2009 , neumann et al . , 2006 ) . since then additional rbps such as taf15 , ewsr1 , hnrnpa2b1 , hnrnpa1 , and hnrnpa3 all of the known rbps associated with dementia contain a low - complexity ( lc ) prion - like domain enriched in glycines and uncharged polar amino acids , and similar to the sequences driving yeast prion aggregation ( alberti et al . lc prion - like domains are also present in key rbps that mediate the assembly of rna granules by liquid - liquid phase separation ( lin et al . , 2015 , significantly , a small proportion of liquid droplets made by rbps transform into solid aggregates over time in vitro ( lin et al . , 2015 , , 2015 , murakami et al . , 2015 , patel et al . , 2015 ) . for clarity , we will use the term aggregation only when referring to the formation of non - dynamic rbp aggregates . an important question is whether the special assembly properties of rbps puts them at risk of aggregating during aging in a multicellular organism and not just in the context of disease . interestingly , several rbps with lc prion - like domains were identified in the insoluble proteome of aged animals ( david et al . , 2010 ) . overall , it is imperative to know the causes and consequences of wild - type rbp aggregation during aging in order to fully understand rbp aggregation in neurodegenerative diseases . furthermore , it is likely that the organism has evolved specific mechanisms to control liquid droplet protein aggregation . in the current study , we chose to focus on key rbps responsible for stress granule formation . stress granules are a specific type of rna granule that protect the cell by sequestering mrna from the translational machinery during periods of stress . importantly , stress granule proteins are often found to co - localize with pathological inclusions of tdp-43 and fus ( bentmann et al . whether these stress granule proteins are innocent bystanders transiently interacting with tdp-43 and fus or whether they co - aggregate and accelerate disease - associated rbp aggregation remains intensely debated ( bentmann et al . , 2013 , li et al . , 2013 ) . we show that key stress - granule - related rbps ( sgrbps ) accumulate in aberrant stress granule - like puncta and in large solid aggregates in aged c. elegans . proteomic analysis revealed that long - lived animals with reduced daf-2 signaling preferentially abrogate the insolubility of rna granule components . importantly , sgrbp aggregates are associated with reduced animal size , motility , and lifespan . we show that sgrbp aggregation is triggered at an earlier age by their co - aggregation with other misfolded proteins , a process that is prevented by daf-16 in daf-2 mutants . in addition , the proteostasis network established by heat shock transcription factor 1 ( hsf-1 ) during development is required to maintain dynamic stress granule proteins throughout the animal s life . to identify and quantify changes in aggregation - prone proteins in animals with reduced daf-2 signaling , we performed an in - depth proteomic analysis of the insoluble proteome from both control and long - lived animals ( figure 1a ; table s1 ) . because protein misfolding and aggregation is highly abundant in aged c. elegans gonads and masks changes in other somatic tissues ( david et al . , 2010 , goudeau and aguilaniu , 2010 , zimmerman et al . , 2015 ) , we used a gonad - less mutant to focus our analysis on protein insolubility in non - reproductive tissues . we isolated large aggregates that are pelleted by low centrifugal forces ( 20,000 g ) and insoluble in 0.5% sds . in three biological replicates , we identified 260 insoluble proteins , of which 186 were highly prone to aggregate with age in control animals ( figure 1b ) . the strong correlations between the control replicates ( r = 0.77 , r = 0.85 , and r = 0.67 ) and long - lived replicates ( r = 0.87 , r = 0.86 . and r = 0.82 ) attest to the quality of the quantification and experimental reproducibility ( figures s1a and s1b ) . none of these insoluble proteins was more prone to aggregate with age in the long - lived animals as compared with controls . these results are consistent with previous observations ( david et al . , 2010 , demontis and perrimon , 2010 ) . surprisingly , a recent proteomic study showed that endogenous protein insolubility is higher in daf-2 mutants than in wild - type animals ( walther et al . , 2015 ) . to account for procedural differences , we performed the extraction following the less stringent extraction protocol from walther et al . ( 2015 ) . by omitting sds and using ultracentrifugation at 500,000 g , walther et al . analyzed highly insoluble large aggregates ( as in this study ) , as well as smaller and more soluble aggregates . however , we did not observe a general change in the action of daf-2 signaling on protein insolubility with age after using the less stringent extraction protocol ( figure s1c ) . next , we asked whether the inconsistencies between the studies could be related to protein aggregation in the gonad and indeed , we found that long - lived animals with gonads have proportionally more insoluble proteins compared with wild - type animals with gonads ( figure s1d ) . these results suggest that aggregation in the gonad masks the protective effect of reduced daf-2 signaling in somatic tissues . importantly , we confirmed this protective action of reduced daf-2 signaling with several candidates ( see below ) . taken together , these data demonstrate that reduced daf-2 signaling promotes protein solubility in most tissues with age . to investigate specifically changes in insolubility regulated by reduced daf-2 signaling , we sorted proteins prone to aggregate with age in control animals ( > 2-fold ) by their fold change in insolubility with age in the long - lived conditions . we restricted our analysis to 87 proteins that aggregated less with age ( < 0.83-fold ) and 52 proteins that continued to aggregate with age ( > 1.2-fold ) in the long - lived animals ( figure 1b ; table s1 ) . previous bioinformatics analysis of aggregation - prone proteins revealed an enrichment in both -sheet propensity and aliphatic amino acids ( david et al . , 2010 ) . a similar enrichment in the whole insoluble proteome was identified in this study ( figures s1e and s1f ) . intriguingly , when examining the two groups of insoluble proteins that were differentially regulated by reduced daf-2 signaling , we found a segregation of the two properties . proteins with abrogated aggregation with the daf-2 rnai treatment were highly enriched in aliphatic amino acids , in particular alanine , glycine , and valines , but not in sheets ( figures 1c , 1d , and s1 g ) . conversely , proteins that still aggregate in the daf-2( ) condition had a significant propensity to form sheets but were only modestly enriched in aliphatic amino acids ( figures 1c and 1d ) . next , we searched for functional differences between the two groups differentially regulated by reduced daf-2 signaling . strikingly , ribosomal proteins and rna granule components , including stress granule and p - granule rbps , were highly overrepresented among the proteins that were prevented from aggregating with age in the long - lived animals ( tables s2a and s3 ) . conversely , chaperones and vitellogenin yolk proteins were overrepresented among proteins that were still prone to aggregate with age in daf-2( ) conditions ( table s2b ) . among the rbps , four are predicted to have lc prion - like domains ( alberti et al . , 2009 ) : pab-1 , fib-1 , hrp-1 , and car-1 ( figure s2a ) . by western blot , we confirmed that reduced daf-2 signaling abrogated their aggregation with age ( figures 1e and s2b ) . in addition , we evaluated two proteins without rna - binding or lc prion - like domains , par-5 and daf-21 , quantified by mass spectrometry as more insoluble with age in both control and long - lived animals . we confirmed that par-5 and daf-21 continued to aggregate with age in long - lived animals ( by > 7-fold and 10-fold , respectively ) , albeit to a reduced extent compared with controls ( figure s2c ) . of note , changes in aggregation were not correlated with changes in total protein levels ( figure s3 ) . a previous study found that the chemical b - isox exclusively causes rna granules to precipitate out from whole - cell lysates by inducing their assembly into a hydrogel ( han et al . proteins precipitated by b - isox were also enriched in aliphatic amino acids ( in particular glycine and to a lesser extent alanine and valine ) and not in the propensity to form sheets ( figures 1c , 1d , and s1 g ) . we checked for proteins in common with our study and found a very significant overlap between proteins precipitated by b - isox and proteins no longer aggregating in the long - lived conditions ( figure 1f ; table s4 ) . together , these results indicate that different types of rna granule components including key rbps responsible for their assembly become insoluble with age , and long - lived animals with reduced daf-2 signaling are highly successful in preventing their aggregation . interestingly , higher levels of aliphatic amino acids in rna granule components could help their assembly and/or drive their age - dependent aggregation . to investigate further the aggregation of key rbps with lc prion - like domains and to understand the mechanisms involved , we generated c. elegans strains expressing pab-1 and tiar-2 in the pharyngeal muscles , fused to fluorescent tags . pab-1 and tiar-2 are the c. elegans homologs of human polyadenylate - binding protein 1 ( pabp-1 ) and t - cell - restricted intracellular antigen-1 ( tia-1 ) , two prominent rbps that localize to stress granules and are also minor components of pathological inclusions that occur in als and fltd ( bentmann et al . , 2012 ) . both of these rbps harbor lc prion - like domains ( figure s2a ) ( alberti et al . , 2009 ) . exposing cells to stressors such as heat induces rbps with lc prion - like domains to form liquid droplets ( molliex et al . , 2015 , patel et al . , 2015 ) . similarly , heat shock in c. elegans caused pab-1 and tiar-2 to form stress granules ( figures 2a and 2c ; figure s4a ) ( murakami et al . , 2012 , rousakis et al . , 2014 ) . when co - expressed , pab-1 and tiar-2 localized to the same stress granules ( figure s4b ) . consistent with the dynamic nature of stress granules , these puncta were no longer observed 24 hr after the heat shock ( figures 2a and 2c ) . using antibodies , we observed a similar change in pattern with endogenous pab-1 , indicating the effect is not merely due to overexpression or the fluorescent tag ( figure s4c ) . as an additional control , we showed that kinase kin-19 , which is not an rbp and lacks a lc prion - like domain , does not localize to stress granules upon heat shock ( figure s4d ) . with age , we observed a striking change in the distribution pattern of these key stress granule rbps . whereas the majority of pab-1 and tiar-2 proteins were diffusely localized in non - stressed young animals , we found that both stress granule components accumulated in distinct puncta in aged animals ( figures 2b and 2d ; figure s4f ) this change was specific for pab-1 and tiar-2 as the fluorescent tags alone ( venus or tagrfp ) remained diffuse with age ( figure s4e ) ( david et al . , 2010 ) . the results were not simply caused by overexpression because endogenous pab-1 formed similar puncta with age , in the different head regions where it is natively expressed ( figure s4 g ) . for both pab-1 and tiar-2 , we observed a significant increase with age in the number of worms with large puncta that are visible by low - magnification microscopy ( figures 2e and 2f ; figure s4h ) . interestingly , imaging at high magnification revealed that tiar-2 in aged animals was localized predominantly in small puncta highly reminiscent of stress granules assembled during heat shock , whereas pab-1 accumulated to a greater extent in large puncta that were not normally observed upon heat shock in young animals ( figures 2b , 2d , single - plane insets , 3a , 3c , and 3d ) . quantification confirmed that stress granules induced during heat shock and aberrant tiar-2 stress granule - like puncta formed during aging had similar sizes ( figure 3b ) . remarkably , when co - expressed , pab-1 preferentially co - localized within tiar-2-positive age - dependent stress - granule - like puncta ( figure 2 g ) , suggesting that interactions between rbps change their aggregation patterns . a hallmark of protein aggregation associated with disease is the immobility of proteins within the aggregates . to evaluate this aspect , we monitored fluorescence recovery after photobleaching ( frap ) in large pab-1 and tiar-2 puncta ( figures 3e3i ) . all large tiar-2 puncta and half of the large pab-1 puncta in aged animals showed no fluorescence recovery , demonstrating that these are solid aggregates ( figures 3e3i ) . because of the small size of the age - dependent stress - granule - like puncta , it was not possible to assess the mobility of tiar-2 or pab-1 in these structures . consequently , we used the large puncta as a readout for sgrbp aggregation in subsequent experiments . the consequences of age - dependent protein aggregation for the animal s health are poorly understood . here , we evaluated the impact of the aggregation of a key stress granule component during aging . surprisingly , pab-1 overexpression was protective as animals , grown at 15c , 20c , or 25c all their life , lived longer than non - transgenic animals ( table s5 ) . however , this effect is likely due to higher levels of functional pab-1 and not related to protein aggregation because pab-1 does not aggregate in animals at 15c ( figure s5a ) . in order to distinguish effects specifically related to pab-1 aggregation , we separated pab-1 transgenics at day 7 into three groups depending on their aggregation levels . we found that animals with pab-1 aggregation were significantly smaller in size compared with animals without pab-1 aggregation ( figure 4a ; figure s5b ) . in addition , smaller animals were visibly less motile ( movie s1 ) . importantly , mildly stressed animals with the highest levels of aggregation died earlier ( figure 4b ; table s5 ) . our proteomic study revealed that reduced daf-2 signaling efficiently prevents the insolubility of stress granule components with age . we confirmed that long - lived daf-2 mutants greatly delay the formation of both stress - granule - like structures and large aggregates of pab-1 and tiar-2 ( figures 5a and 5b ; figures s6a and s6b ) . to gain insight into the mechanisms controlling protein aggregation , we investigated the role of the transcription factor hsf-1 activated by reduced daf-2 signaling ( hsu et al . , 2003 , hsp40 modulate stress granule dynamics in saccharomyces cerevisiae and/or in cell culture ( cherkasov et al . because chaperone expression is controlled by hsf-1 in c. elegans , we speculated that hsf-1 may regulate sgrbp aggregation . indeed , impairing hsf-1 activity in both daf-2( ) and wild - type backgrounds caused severe pab-1 aggregation already in young adults as well as in aged individuals , an effect that was reversed by overexpressing hsf-1 ( figures 5c5e ; figures s6c and s6d ) . interestingly , hsf-1 in daf-2 mutants did not control the aggregation of the kinase kin-19 , which was previously shown to misfold and form solid aggregates with age ( figure 5 g ) ( david et al . , 2010 ) . these results suggest that hsf-1 regulates different types of endogenous protein aggregation with age to different extents . it was previously shown that to assure daf-2( ) longevity , hsf-1 is most highly expressed and acts mainly during development ( volovik et al . , 2012 ) . we observed that impairing hsf-1 activity by rnai during adulthood had no effect on pab-1 aggregation ( figure 5f ; figure s6e ) . conversely , reducing hsf-1 activity by rnai during development caused pab-1 aggregation in young adults , albeit mainly in the anterior bulb ( figure 5f ) . the same chaperones discovered to regulate stress granule dynamics in other model systems could also play a role in preventing sgrbp aggregation . in yeast the hsp40 proteins sis1 and ydj1 were shown to co - localize with stress granules and to play a role in stress granule disassembly ( walters et al . , 2015 ) . we evaluated worm strains overexpressing yellow fluorescent protein ( yfp)-tagged dnj-13 and dnj-19 , the worm orthologs of sis1 and ydj1 , together with tagrfp::pab-1 . we observed occasional co - localization of both chaperones with both heat - induced pab-1 stress granules and age - dependent large and stress - granule - like pab-1 puncta ( figures s6 g and s6h ) . these findings were confirmed in single tagrfp::pab-1 transgenics using antibodies against dnj-13 and dnj-19 ( data not shown ) . however , overexpression of dnj-19 and dnj-13 did not significantly reduce pab-1 aggregation with age ( figures s6f and s6i ) , indicating that these chaperones may not modulate sgrbp aggregation . however , we observed no co - staining with either pab-1 stress granules induced by heat shock or pab-1 puncta formed during aging . therefore , it is possible that hsp110 does not modulate stress granule dynamics and sgrbp aggregation in c. elegans . finally , we investigated the role of hsp70 on sgrbp aggregation and found that inhibition of hsp70 by rnai did not alter pab-1 aggregation ( table s6 ) . of note , the later results could be caused by redundancy between chaperone functions . collectively , these results reveal that hsf-1 is an important regulator of sgrbp aggregation throughout life , and it contributes to maintaining dynamic stress granule proteins in long - lived daf-2 mutants . next , we examined the role of the transcription factor daf-16 activated by reduced daf-2 signaling ( lin et al . 1997 ) . in daf-2 mutants , daf-16 protected against pab-1 aggregation in aged animals ( figures 5c and 5d ) importantly , daf-2 mutants use daf-16 to control different types of age - dependent protein aggregation because delayed aggregation of the kinase kin-19 was also dependent on daf-16 ( figure 5 g ) . recent work performed in s. cerevisiae and drosophila reveals that stress granules dynamically interact with misfolded proteins ( cherkasov et al . , 2013 , kroschwald et al . , 2015 ) . we speculated that widespread protein misfolding and aggregation occurring with age could promote sgrbp aggregation . in support of this hypothesis , we observed the co - localization of pab-1 and kin-19 in large immobile aggregates in double transgenics ( figures 6a and 6b ) . next , we evaluated the rate of pab-1 and kin-19 aggregation in the double transgenics relative to their aggregation rates in single transgenics ( figures 6c and 6d ) . in the single transgenics , importantly , when both pab-1 and kin-19 were co - expressed , the presence of misfolded kin-19 triggered more abundant pab-1 aggregation at an earlier age ( figure 6c ) . conversely , kin-19 aggregation was slightly impeded by pab-1 overexpression ( figure 6d ) , indicating that sgrbp aggregation does not cross - seed kin-19 aggregation . notably , pab-1 aggregation was not accelerated by the co - expression of a fluorescent tag alone ( figure 6e ) . furthermore , we did not observe a general induction of pab-1 stress granules in young double - transgenic animals expressing both kin-19 and pab-1 ( figure s6j ) . these data strongly suggest that pab-1 aggregation is not simply the consequence of generalized cellular stress induced by kin-19 overexpression . rather , the co - localization of kin-19 and pab-1 in the same aggregates as well as the earlier and accelerated aggregation of pab-1 is consistent with a seeding mechanism related to kin-19 aggregation . taken together , we interpret these findings to imply that the accumulation of misfolded proteins with age acts as a seed for sgrbp aggregation . therefore , the overall reduction in widespread protein aggregation in long - lived daf-2( ) conditions as evidenced by our proteomic analysis , at least in part through increased daf-16 activity , could be an effective strategy to prevent sgrbp aggregation . several experimental manipulations have been shown to extend the lifespan of c. elegans and protect against proteotoxicity ( kenyon , 2010 , taylor and dillin , 2011 ) . we found that both dietary restriction mimicked in eat-2 mutant animals as well as inhibition of mitochondrial function achieved by targeting cyc-1 with rnai strongly limited pab-1 aggregation with age ( figures 7a and 7b ) . we show that a wide variety of rna granule components become highly insoluble with age in c. elegans . together with previous in vitro and cell culture results , our findings demonstrate that the capacity of rbps to cycle between assembled and disassembled states can become a liability in aging organisms . already in young animals , maintaining sgrbp dynamics necessitates an active control system established by hsf-1 . the accumulation of other misfolded proteins during age acts as a seed for the aggregation of key sgrbps . significantly , one of the main outcomes of the longevity program initiated by reduced daf-2 signaling while responding to widespread protein aggregation is the preservation of rbp solubility with age . in this study , we have examined in detail the aggregation pattern of pab-1 and tiar-2 , two key rbps with lc prion - like domains that are important for the formation of stress granules . during the aging process , both proteins spontaneously assembled into small puncta similar to liquid droplets induced during stress and into larger aggregates . significantly , upon co - expression , both pab-1 and tiar-2 co - localized in these age - related stress - granule - like structures . because our proteomic analysis revealed a number of stress granule components in the insoluble proteome , it is likely that secondary stress granule proteins are also incorporated . therefore , an attractive hypothesis is that these small puncta represent stress granules formed as a response to stress related to aging . the inherent aggregation propensity of sgrbps would induce at least some of these droplets to undergo the irreversible transition into a solid state . these stabilized stress granules could then grow into large aggregates as we observed with pab-1 , or simply accumulate with age as seen with tiar-2 . an important question remains how the inherent propensity of rna granule components to aggregate with age could influence pathogenesis in neurodegenerative diseases . one possibility is that inherent rbp aggregation impacts cellular health and thereby indirectly accelerates pathology . we observed reduced lifespan and a striking decrease in size and mobility of animals with higher levels of pab-1 aggregation . as yet , it remains unclear whether reduced fitness is a cause or consequence of pab-1 aggregation . in support of a gain of function related to sgrbp aggregation , two rare diseases are caused by mutated pabpn1 and tia-1 , the human homologs of pab-1 and tiar-2 , which accumulate in pathological aggregates ( brais et al . our proteomic analysis of aging c. elegans highlighted three other rbps with lc prion - like domains that are highly prone to aggregate with age : hrp-1 , fib-1 , and car-1 . the aggregation of the human homologs of hrp-1 , hnrnp - a1 , and hnrnp - a3 was recently discovered to cause multisystem proteinopathy and als / ftld ( kim et al . indeed , a loss in nucleolar protein fib-1 function caused by its aggregation with age could impair ribosomal biogenesis ( tollervey et al . the mammalian lsm14b and lsm14a homologs of car-1 localize to p bodies ( eulalio et al . , 2007 ) , and aggregation of key p - body components could impair non - sense - mediated decay . therefore , it will be important to investigate the consequences of fib-1 and car-1 aggregation on cellular health . apart from accelerating pathology indirectly by reducing cellular health , the presence of stress granule proteins in pathological protein aggregates is emerging as a common denominator in different types of neurodegenerative diseases including als , ftld , alzheimer s disease , and huntington s disease ( aulas and vande velde , 2015 , bentmann et al . the inherent aggregation propensity of stress granule proteins demonstrates that they are unlikely to be transient interacting partners in pathological aggregates . it remains to be determined whether age - related sgrbp aggregation acts as a seed for disease - associated protein aggregation . overall , the role of stress granules in neurodegenerative diseases is clearly highly complex because there is evidence supporting the recruitment of disease - associated proteins to stress granules and vice versa . interestingly , recent cell culture data show that the assembly of stress granules caused by disease - associated protein aggregation in turn promotes pathological aggregation ( vanderweyde et al . , 2016 ) . the age - dependent aggregation of sgrbps and prevalence of stress granule components in neurodegenerative diseases underline their relevance as therapeutic targets . one successful strategy would be to prevent the initial assembly of stress granules ( kim et al . , 2014 ) . , it will be important to understand how longevity pathways relying on dietary restriction or defective mitochondrial respiration efficiently prevent sgrbp aggregation . in the case of reduced daf-2 signaling , sgrbp aggregation is suppressed by at least two mechanisms : daf-16 activation prevents cross - seeding by delaying the accumulation of misfolded aggregation - prone proteins , and increased activity of hsf-1 during development assures enhanced sgrbp proteostasis throughout adulthood . to date , cross - seeding of rbp aggregation has been observed only with the huntingtin protein containing an expanded polyglutamine repeat region ( furukawa et al . , 2009 ) . it will be important to investigate by which means endogenous aggregation - prone proteins lacking motifs similar to the lc prion - like domain would cross - seed rbps aggregation . overall , both of these strategies used to restore the dynamic nature of stress granule proteins could also directly prevent disease - associated rbp aggregation . a list of strains , strain maintenance , rnai treatment , and lifespan assays is described in the supplemental experimental procedures . worms were either fixed for imaging analysis directly after heat shock or allowed to recover on normal growth ( ng ) plates kept at 20c before fixation . images were taken of synchronized live c. elegans using a leica fluorescence microscope m165 fc with a planapo 2.0 objective and leica dfc310 fx camera . body length was determined for each worm using fiji software ( schindelin et al . , 2012 ) . significance was evaluated by unpaired , two - tailed t test using graphpad prism 6 . worms were examined with a leica sp8 confocal microscope . fixation and immunostaining protocol including imaging parameters representative confocal images are displayed as maximum z stack projection unless mentioned otherwise . in a population of synchronized live c. elegans , aggregation levels were determined using a leica fluorescence microscope m165 fc with a planapo 2.0 objective . animals overexpressing pmyo-2::venus::tiar-2 were divided into two categories : animals with up to 10 ( low aggregation ) or more than 10 venus::tiar-2 puncta ( intermediate aggregation ) in the anterior and posterior pharyngeal bulb . animals overexpressing pmyo-2:tagrfp : pab-1 were divided into three categories : animals with up to 10 ( low aggregation ) or more than 10 ( intermediate aggregation ) tagrfp::pab-1 puncta in the posterior bulb , or more than 10 ( high aggregation ) tagrfp : pab-1 puncta in the anterior bulb . animals expressing pkin-19::kin-19::meos or pkin-19::kin-19::venus were divided into less than 10 puncta ( low aggregation ) , between 10 and 100 puncta ( intermediate aggregation ) , and more than 100 puncta in the anterior bulb ( high aggregation ) . to compare aggregation levels between kin-19::venus and tagrfp::pab-1 , we evaluated tagrfp::pab-1 puncta formation only in the anterior bulb and with the same counting scheme as for kin-19::venus . when comparing different strains , counting was done in a blind fashion . for statistics , two - tailed fisher s exact test significance of high + intermediate against low aggregation levels was calculated unless indicated otherwise . frap analysis was performed as previously described ( david et al . , 2010 ) using the leica sp8 confocal microscope with the harmonic compound , plan , apochromatic ( hc pl apo ) cs2 63 1.30 glycerol objective and photomultiplier tube ( pmt ) detector . further experimental details are described in the supplemental experimental procedures . to obtain large synchronized populations of aged animals and quantify protein aggregation only in the somatic tissues , we used temperature - induced sterile gon-2 mutants as previously described ( david et al . , 2010 ) . to induce longevity , we subjected animals to daf-2 rnai and control animals to gfp rnai from the last larval stage l4 onward . animals aged at 25c were collected at day 3 of adulthood ( young ) and when half the control animals had died between days 14 and 18 ( aged ) . isolation of large sds insoluble aggregates for immunoblot and mass spectrometry analysis were performed as previously described ( david et al . , 2010 ) . the mass spectrometry proteomics data have been deposited to the proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository ( vizcano et al . , 2014 ) with the dataset identifier pxd003451 . aliphatic amino acid residues were defined as a , g , i , l , and v. secondary structure content was predicted using psipred v2.6 ( jones , 1999 ) . the p values were calculated using the unequal variance t test compared with the background set of all proteins detected by mass spectrometry ( n = 5,637 ) . additional details and functional analysis are described in the supplemental experimental procedures . m.c.l . , n.g . , j.c.t . , and d.c.d . designed and performed experiments .

this study determined the molecular mechanisms of tocotrienol - rich fraction ( trf ) in preventing cellular senescence of human diploid fibroblasts ( hdfs ) . primary culture of hdfs at various passages were incubated with 0.5 mg / ml trf for 24 h. telomere shortening with decreased telomerase activity was observed in senescent hdfs while the levels of damaged dna and number of cells in g0/g1 phase were increased and s phase cells were decreased . incubation with trf reversed the morphology of senescent hdfs to resemble that of young cells with decreased activity of sa--gal , damaged dna , and cells in g0/g1 phase while cells in the s phase were increased . elongated telomere length and restoration of telomerase activity were observed in trf - treated senescent hdfs . these findings confirmed the ability of tocotrienol - rich fraction in preventing hdfs cellular ageing by restoring telomere length and telomerase activity , reducing damaged dna , and reversing cell cycle arrest associated with senescence . summarylow - complexity prion - like domains in key rna - binding proteins ( rbps ) mediate the reversible assembly of rna granules . individual rbps harboring these domains have been linked to specific neurodegenerative diseases . although their aggregation in neurodegeneration has been extensively characterized , it remains unknown how the process of aging disturbs rbp dynamics . we show that a wide variety of rna granule components , including stress granule proteins , become highly insoluble with age in c. elegans and that reduced insulin / insulin - like growth factor 1 ( igf-1 ) daf-2 receptor signaling efficiently prevents their aggregation . importantly , stress - granule - related rbp aggregates are associated with reduced fitness . we show that heat shock transcription factor 1 ( hsf-1 ) is a main regulator of stress - granule - related rbp aggregation in both young and aged animals . during aging , increasing daf-16 activity restores dynamic stress - granule - related rbps , partly by decreasing the buildup of other misfolded proteins that seed rbp aggregation . longevity - associated mechanisms found to maintain dynamic rbps during aging could be relevant for neurodegenerative diseases .

hearing loss is a significant public health concern given the deleterious effects that untreated hearing impairment may have on overall physical and cognitive wellbeing.1 , 2 the hearing health foundation reports that nearly 50 million americans have hearing loss.3 sensorineural hearing losses generally have a highfrequency component . this frequency region is essential for good speech understanding in complex listening environments , particularly in noise.4 , 5 individuals with substantial , bilateral highfrequency hearing loss experience hearing difficulties in most aspects of life : at home , on the phone , at work , and in social situations . they can be highly frustrated because existing hearing aid technology can not overcome the problems of reduced word understanding in quiet and noise.6 , 7 , 8 due to their communication problems , they may become isolated , withdrawing from family , colleagues , and friends . with severe hearing loss , areas of minimal or nonfunctioning hair cells or auditory neurons are often present , resulting in cochlear dead regions where vibrations of the basilar membrane are not detected via inner hair cells or neurons in that region . frequencies falling in a dead region are detected via apical or basal spread of vibrations to other cochlear places . therefore , hearing loss at a given frequency may be greater than indicated by the audiometric threshold.9 typically , acoustic amplification of dead regions does not improve speech understanding and may worsen it.10 , 11 individuals with this hearing loss profile may be candidates for electric plus acoustic stimulation in the same ear . treatment options for individuals with bilateral , severe skislope hearing loss have been limited to stateofthe art amplification , including frequency lowering,12 in an effort to improve speech intelligibility . these attempts often end with the rejection of hearing aids due to the lack of benefit , leaving the individual with no other alternatives . studies have shown that an implant with a shorter electrode array provides beneficial electric stimulation for high frequencies while preserving acoustic lowfrequency hearing , resulting in improved speech understanding.13 , 14 recently , lenarz et al . described results from a european multicenter study using the cochlear ltd . , sydney , australia , nucleus hybrid l24 implant.15 we report results of the clinical trial leading to u.s . food and drug administration approval of the firstofitskind combined electric and acoustic ( hybrid ) implant system to address the substantial hearing difficulties of individuals not benefitting from amplification and not eligible for a standard cochlear implant ( ci ) . this was a prospective , singlearm , multicenter trial to determine the safety and effectiveness of the hybrid system . subjects were implanted at 10 clinical sites in the united states and served as their own controls in all test conditions . the protocol was approved by the us food and drug administration and relevant institutional review boards , and all participants gave written informed consent . the ear selected for implantation had severe ( 75 db hl averaged over 2000 , 3000 , 4000 hz ) highfrequency sensorineural hearing loss and relatively good lowfrequency hearing ( 60 db hl at 125 , 250 , and 500 hz ) . in addition , an aided consonantnucleusconsonant ( cnc ) monosyllabic word score of 10% through 60% using an appropriately fit hearing aid was required . aided word recognition in the contralateral ear was required to be similar or better than the ear to be treated , but not better than 80% . those with durations of severe or profound hearing loss greater than 30 years and/or onset of hearing loss less than 2 years were excluded . the protocol included acoustic thresholds measured for each ear preoperatively and postoperatively , at device activation , and 3 , 6 , and 12 months postactivation . consonantnucleusconsonant words were presented at 60 dba ; azbio sentences in noise were presented at 60 dba in 10talker babble noise at + 5 db signaltonoise ratio . to evaluate effectiveness of the hybrid system as used routinely , speech perception outcomes were analyzed in the everyday listening condition , which is listening through the hybrid system in combination with acoustic hearing in the opposite , unimplanted ear . to gain insight into how hearing impacts quality of life , the validated speech , spatial , and qualities of hearing questionnaire ( ssq)16 was administered as a selfassessment of hearing within three domains : hearing speech in various environments , spatial hearing , and sound qualities . a device use questionnaire was administered that addressed overall satisfaction with the hybrid system relative to hearing aids . surgery for the hybrid l24 implant ( cochlear ) is a modification of that for standard cis , similar to the description by gantz et al.13 ; details are provided in the nucleus hybrid l24 implant surgeon 's guide ( cochlear).17 after the postauricular incision , the surgeon creates a well bed on the skull posterior to the mastoid and opens the facial recess ( posterior tympanotomy ) widely to provide good visibility of the round window niche in the middle ear . although the hybrid implant electrode may be inserted through the round window or cochleostomy , in this trial all electrodes were inserted through a small cochleostomy created just inferior to the round window . after performing the cochleostomy , the surgeon opens the endosteum of the cochlea with a pick just prior to inserting the electrode array . the array is slowly inserted 16 mm into the scala tympani instead of the 19 to 24 mm that are more typical for standard cis . the 16mm straight electrode is very thin and has 22 halfband modiolarfacing electrode contacts to stimulate the basal region of the cochlea , with the intent to maintain apical cochlear structures responsible for lowfrequency hearing . coprimary efficacy hypotheses were that outcomes on cnc words ( 100 recorded words administered)18 and azbio sentences in noise ( 40 recorded sentences administered)19 presented through the hybrid implant system ( cochlear ) would be significantly better at 6 months postimplantation than preoperative performance using a hearing aid . the sample size of 50 subjects exceeded the minimum requirement for 90% statistical power , ensuring adequate power . mean differences for subjects on the cnc word and azbio sentence recognition scores preoperatively and at the 6month endpoint were analyzed using paired t tests . if there was evidence that assumptions did not hold , a wilcoxon signed rank test was used . secondary efficacy objectives compared individual preoperative performance with a hearing aid to performance at the 6month endpoint on cnc words and phonemes and azbio sentences . although no formal hypothesis test was conducted for these endpoints , success would be achieved if over 75% of subjects showed equal or better performance from preoperative to postoperative scores using the binomial model.20 the primary safety objective was to describe the safety of implantation with the hybrid system . the primary safety endpoint was defined as any surgical and/or devicerelated event , reported as the number and proportion of individuals experiencing an adverse event . coprimary efficacy hypotheses were that outcomes on cnc words ( 100 recorded words administered)18 and azbio sentences in noise ( 40 recorded sentences administered)19 presented through the hybrid implant system ( cochlear ) would be significantly better at 6 months postimplantation than preoperative performance using a hearing aid . the sample size of 50 subjects exceeded the minimum requirement for 90% statistical power , ensuring adequate power . mean differences for subjects on the cnc word and azbio sentence recognition scores preoperatively and at the 6month endpoint were analyzed using paired t tests . if there was evidence that assumptions did not hold , a wilcoxon signed rank test was used . secondary efficacy objectives compared individual preoperative performance with a hearing aid to performance at the 6month endpoint on cnc words and phonemes and azbio sentences . although no formal hypothesis test was conducted for these endpoints , success would be achieved if over 75% of subjects showed equal or better performance from preoperative to postoperative scores using the binomial model.20 the primary safety objective was to describe the safety of implantation with the hybrid system . the primary safety endpoint was defined as any surgical and/or devicerelated event , reported as the number and proportion of individuals experiencing an adverse event . mean age was 64.1 years ( standard deviation [ sd ] = 14.7 years ) , ranging from 23 to 86.2 years at implantation . there was a 50/50 split for gender , and 52% of right ears were implanted . mean duration of overall hearing loss was 28.1 years , and mean duration of severetoprofound highfrequency loss was 13.1 years . hearing loss etiologies were : unknown ( 50% ) , noise exposure ( 22% ) , and familial ( 20% ) . individual cases ( 8% ) were related to ototoxic drugs , autoimmune ear disease , high fever / infection , and noise exposure / viral . hl = hearing loss ; lf = low frequency ; pta = pure tone average ; sd = standard deviation . table 2 provides a summary of primary outcomes ( cnc words and azbio sentences in noise for the implanted ear ) . when testing the implanted ear , the contralateral ear was plugged to mitigate its contribution to the speech scores . for cncs , subjects experienced a significant ( p < 0.001 ) improvement of 35.8 ( sd = 27.7 ) percentage points with the hybrid device over a hearing aid preoperatively . similarly , for azbio sentences , they experienced a significant ( p < 0.001 ) improvement of 32.0 ( sd = 29.4 ) percentage points . one subject missed 6month assessments , and data were imputed based on the 3month evaluation . < 0.001 one subject missed 6month assessments and data were imputed based on the 3month evaluation . table 3 presents secondary objective outcomes based on binomial comparisons of preoperative to postoperative changes for cnc words and azbio sentences for the implanted ear at the 6month endpoint . the secondary endpoint objectives were met : over 75% of the subjects demonstrated equal or improved performance on cnc words , phonemes , and azbio sentences with the hybrid implant relative to performance with a hearing aid . specifically , 96% and 92% of subjects performed equal or better on cnc words and phonemes , respectively , and 90% on azbio sentences . furthermore , 82% and 86% showed improved performance on cnc words and phonemes , respectively , and 74% improved on sentences . the consistency of the primary endpoints for the treated ear was examined across subject subgroups defined by baseline characteristics : gender , age , duration of hearing loss , duration of severetoprofound highfrequency hearing loss , etiology , and baseline speech perception scores . results indicated that baseline characteristics gender , age , and duration of hearing loss were the main factors in terms of speech perception outcomes . this was not the case for duration of severetoprofound highfrequency hearing loss , etiology , and baseline speech scores . mean benefit scores ( i.e. , improvement ) for females were significantly greater than males for cnc words ( females : 48.8% ; males : 25.7% ) and azbio tests ( females : 42.6% ; males : 23.5% ) ( p = 0.002 and 0.02 , respectively . ) subjects under the median implantation age of 68 years showed significantly greater benefit for cncs ( < 68 years : 46.6% ; > 68 years : 27.8% ) ( p = 0.01 ) but not azbio sentences ( < 68 years : 41.0% ; > 68 years : 25.0% ) the mean benefit for subjects below the median hearing loss duration of 23.5 years was significantly better ( p = 0.01 ) than for hearing loss durations above 23.5 years for cncs ( < 23.5 years : 46.2% ; > 23.5 years : 27.5% ) but not azbio sentences ( < 23.5 years : 40.7% ; > 23.5 years : 24.7% ) ( p = 0.05 ) , although the trend favored shorter durations . mean differences for cnc words and azbio sentences in noise at 6months postactivation , using the implant and contralateral hearing aid , were preoperatively compared to bilateral amplification . for cncs , subjects ( n = 49 ) showed significant ( p < 0.001 ) improvement of 34.7 percentage points ( sd = 17.4 ) compared to bilateral amplification . for azbio sentences , subjects ( n = 49 ) showed significant ( p < 0.001 ) improvement of 33.0 percentage points ( sd = 23.5 ) compared to bilateral amplification . no subject showed a significant decrement preoperatively to postoperatively on either measure . at the 6month endpoint , all subjects performed equal or better than preoperatively with bilateral amplification with hearing aids . fortyeight subjects completed the ssq preoperatively using hearing aids and after 6 months using the hybrid system in the everyday listening condition . for the speech hearing scale , subjects improved significantly ( p < 0.001 ) , showing a mean change score of 2.2 ( sd = 1.8 ) . on the spatial hearing scale , there was a significant ( p < 0.003 ) mean change score of .9 ( sd = 2.0 ) ; on the sound quality scale , subjects experienced significantly ( p < 0.001 ) improved mean change of 1.3 ( sd = 2.0 ) . of the 48 subjects who completed the device use survey , four ( 8% ) were the type and frequency of events were consistent with those reported in cochlear implantation ( e.g. , electrode open or short circuits , postoperative dizziness , changes in tinnitus ) or other mastoid operations ; no unanticipated adverse events were reported . fifty events were medical / surgical in nature and included instances of increased tinnitus , vertigo , and other symptoms associated with a mastoidectomy with facial recess approach used in cochlear implantation . it should be noted that the nine adverse events reporting of dizziness , imbalance , and vertigo were likely reported by a few patients and not nine separate patients ; one could have symptoms of dizziness , imbalance , and vertigo . unlike prior ci trials , this was the first to quantify changes in residual hearing ; any changes in preoperative to postoperative hearing sensitivity were measured throughout the study period . changes resulting in profound ( > 90 db hl ) hearing loss were reported as anticipated adverse events . at 6months postactivation , 66% of subjects ( 33 of 50 ) retained functional acoustic sensitivity determined by a 5frequency pure tone average ( 125 , 250 , 500 , 750 , 1000 hz ) of a severe degree or better ( 90 db hl ) . the degree of hearing loss and the number of subjects in each hearing loss category and their postintervention outcomes are depicted in figure 2a and b. in addition , the amount of residual hearing and the number of subjects in each category and their postintervention outcomes are depicted in figure 3a and 3b . subjects with aidable , residual hearing performed better that those without aidable , residual hearing . however , even if subjects had no residual , aidable hearing , they performed better in the ci electriconly condition than preoperatively with hearing aids . regarding 17 subjects who did not maintain functional acoustic hearing , five chose to have the hybrid implant explanted and replaced with a standard ci . improved speech perception of varying degrees was observed compared to that obtained preoperatively with a hearing aid and at the most recent hybrid evaluation prior to revision surgery . ( a ) cnc word scores for subjects with < 10 , 1020 , 2030 , and > 30 db of hearing loss at 6 months postcochelar implant activation . ( b ) azbio + 5 db signaltonoise ratio scores for subjects with < 10 , 1020 , 2030 , and > 30 db of hearing loss at 6 months postcochlear implant activation . the number of subjects in each category of hearing loss is shown . [ color figure can be viewed in the online issue , which is available at www.laryngoscope.com . ] ( a ) the cnc word scores for subjects in each category of lowfrequency hearing loss . ( b ) the azbio + 5 db signal to noise ratio scores for subjects in each category of lowfrequency hearing loss . [ color figure can be viewed in the online issue , which is available at www.laryngoscope.com . apart from cases of profound hearing loss , all but two events ( one sound quality issue and one decreased performance ) were resolved as of database closure . association of baseline characteristics with adverse events , including profound hearing loss , was examined by univariate cox proportional hazards regression models . baseline characteristics evaluated included age at implantation , hearing loss duration , severetoprofound hearing loss duration , etiology , and preoperative speech perception . none were found to be significantly associated with either outcome of an adverse event or profound hearing loss . mean age was 64.1 years ( standard deviation [ sd ] = 14.7 years ) , ranging from 23 to 86.2 years at implantation . there was a 50/50 split for gender , and 52% of right ears were implanted . mean duration of overall hearing loss was 28.1 years , and mean duration of severetoprofound highfrequency loss was 13.1 years . hearing loss etiologies were : unknown ( 50% ) , noise exposure ( 22% ) , and familial ( 20% ) . individual cases ( 8% ) were related to ototoxic drugs , autoimmune ear disease , high fever / infection , and noise exposure / viral . hl = hearing loss ; lf = low frequency ; pta = pure tone average ; sd = standard deviation . table 2 provides a summary of primary outcomes ( cnc words and azbio sentences in noise for the implanted ear ) . when testing the implanted ear , the contralateral ear was plugged to mitigate its contribution to the speech scores . for cncs , subjects experienced a significant ( p < 0.001 ) improvement of 35.8 ( sd = 27.7 ) percentage points with the hybrid device over a hearing aid preoperatively . similarly , for azbio sentences , they experienced a significant ( p < 0.001 ) improvement of 32.0 ( sd = 29.4 ) percentage points . one subject missed 6month assessments , and data were imputed based on the 3month evaluation . < 0.001 one subject missed 6month assessments and data were imputed based on the 3month evaluation . table 3 presents secondary objective outcomes based on binomial comparisons of preoperative to postoperative changes for cnc words and azbio sentences for the implanted ear at the 6month endpoint . the secondary endpoint objectives were met : over 75% of the subjects demonstrated equal or improved performance on cnc words , phonemes , and azbio sentences with the hybrid implant relative to performance with a hearing aid . specifically , 96% and 92% of subjects performed equal or better on cnc words and phonemes , respectively , and 90% on azbio sentences . furthermore , 82% and 86% showed improved performance on cnc words and phonemes , respectively , and 74% improved on sentences . the consistency of the primary endpoints for the treated ear was examined across subject subgroups defined by baseline characteristics : gender , age , duration of hearing loss , duration of severetoprofound highfrequency hearing loss , etiology , and baseline speech perception scores . results indicated that baseline characteristics gender , age , and duration of hearing loss were the main factors in terms of speech perception outcomes . this was not the case for duration of severetoprofound highfrequency hearing loss , etiology , and baseline speech scores . mean benefit scores ( i.e. , improvement ) for females were significantly greater than males for cnc words ( females : 48.8% ; males : 25.7% ) and azbio tests ( females : 42.6% ; males : 23.5% ) ( p = 0.002 and 0.02 , respectively . ) subjects under the median implantation age of 68 years showed significantly greater benefit for cncs ( < 68 years : 46.6% ; > 68 years : 27.8% ) ( p = 0.01 ) but not azbio sentences ( < 68 years : 41.0% ; > 68 years : 25.0% ) ( p = 0.05 ) , although the trend favored younger subjects . the mean benefit for subjects below the median hearing loss duration of 23.5 years was significantly better ( p = 0.01 ) than for hearing loss durations above 23.5 years for cncs ( < 23.5 years : 46.2% ; > 23.5 years : 27.5% ) but not azbio sentences ( < 23.5 years : 40.7% ; > 23.5 years : 24.7% ) ( p = 0.05 ) , although the trend favored shorter durations . mean differences for cnc words and azbio sentences in noise at 6months postactivation , using the implant and contralateral hearing aid , were preoperatively compared to bilateral amplification . for cncs , subjects ( n = 49 ) showed significant ( p < 0.001 ) improvement of 34.7 percentage points ( sd = 17.4 ) compared to bilateral amplification . for azbio sentences , subjects ( n = 49 ) showed significant ( p < 0.001 ) improvement of 33.0 percentage points ( sd = 23.5 ) compared to bilateral amplification . no subject showed a significant decrement preoperatively to postoperatively on either measure . at the 6month endpoint , all subjects performed equal or better than preoperatively with bilateral amplification with hearing aids . fortyeight subjects completed the ssq preoperatively using hearing aids and after 6 months using the hybrid system in the everyday listening condition . for the speech hearing scale , subjects improved significantly ( p < 0.001 ) , showing a mean change score of 2.2 ( sd = 1.8 ) . on the spatial hearing scale , there was a significant ( p < 0.003 ) mean change score of .9 ( sd = 2.0 ) ; on the sound quality scale , subjects experienced significantly ( p < 0.001 ) improved mean change of 1.3 ( sd = 2.0 ) . of the 48 subjects who completed the device use survey , four ( 8% ) were the type and frequency of events were consistent with those reported in cochlear implantation ( e.g. , electrode open or short circuits , postoperative dizziness , changes in tinnitus ) or other mastoid operations ; no unanticipated adverse events were reported . fifty events were medical / surgical in nature and included instances of increased tinnitus , vertigo , and other symptoms associated with a mastoidectomy with facial recess approach used in cochlear implantation . it should be noted that the nine adverse events reporting of dizziness , imbalance , and vertigo were likely reported by a few patients and not nine separate patients ; one could have symptoms of dizziness , imbalance , and vertigo . this trial specified implanting subjects with functional lowfrequency acoustic hearing . unlike prior ci trials , this was the first to quantify changes in residual hearing ; any changes in preoperative to postoperative hearing sensitivity were measured throughout the study period . changes resulting in profound ( > 90 db hl ) hearing loss were reported as anticipated adverse events . at 6months postactivation , 66% of subjects ( 33 of 50 ) retained functional acoustic sensitivity determined by a 5frequency pure tone average ( 125 , 250 , 500 , 750 , 1000 hz ) of a severe degree or better ( 90 db hl ) . the degree of hearing loss and the number of subjects in each hearing loss category and their postintervention outcomes are depicted in figure 2a and b. in addition , the amount of residual hearing and the number of subjects in each category and their postintervention outcomes are depicted in figure 3a and 3b . subjects with aidable , residual hearing performed better that those without aidable , residual hearing . however , even if subjects had no residual , aidable hearing , they performed better in the ci electriconly condition than preoperatively with hearing aids . regarding 17 subjects who did not maintain functional acoustic hearing , five chose to have the hybrid implant explanted and replaced with a standard ci . improved speech perception of varying degrees was observed compared to that obtained preoperatively with a hearing aid and at the most recent hybrid evaluation prior to revision surgery . ( a ) cnc word scores for subjects with < 10 , 1020 , 2030 , and > 30 db of hearing loss at 6 months postcochelar implant activation . the number of subjects in each category of hearing loss is shown . ( b ) azbio + 5 db signaltonoise ratio scores for subjects with < 10 , 1020 , 2030 , and > 30 db of hearing loss at 6 months postcochlear implant activation . the number of subjects in each category of hearing loss is shown . [ color figure can be viewed in the online issue , which is available at www.laryngoscope.com . ] ( a ) the cnc word scores for subjects in each category of lowfrequency hearing loss . ( b ) the azbio + 5 db signal to noise ratio scores for subjects in each category of lowfrequency hearing loss . [ color figure can be viewed in the online issue , which is available at www.laryngoscope.com . apart from cases of profound hearing loss , all but two events ( one sound quality issue and one decreased performance ) were resolved as of database closure . association of baseline characteristics with adverse events , including profound hearing loss , was examined by univariate cox proportional hazards regression models . baseline characteristics evaluated included age at implantation , hearing loss duration , severetoprofound hearing loss duration , etiology , and preoperative speech perception . none were found to be significantly associated with either outcome of an adverse event or profound hearing loss . results from this study support the conclusion that the nucleus hybrid system ( cochlear ) delivers significantly improved speech understanding in quiet and noise compared to a hearing aid for individuals with bilateral , severe highfrequency hearing loss . ninety percent of subjects achieved the same or better performance on both speech perception measures when listening with the hybrid system . when using both ears , all subjects performed equal or better than preoperatively on both measures . the ssq selfassessment supported speech intelligibility results , with significant improvement on all scales and with greatest improvement on the hearing speech scale . on overall listening satisfaction , the number of individuals satisfied increased from 8% preoperatively with amplification to 79% with the hybrid system . this system delivers important highfrequency information through electrical stimulation and the opportunity to combine it with beneficial lowfrequency residual hearing in one or both ears . outcomes for five subjects undergoing revision surgery suggest that a standard ci remains a viable treatment when hybrid implantation does not meet expectations . current hearing aid technology often can not provide audible , clear highfrequency sound for individuals with this type of hearing loss . individuals with substantial highfrequency losses frequently have nonfunctional inner and outer hair cells ; therefore , amplification can not be effective . individuals with precipitously sloping losses predictably are frustrated due to significant communication struggles ; they regularly reject amplification , leaving them with no alternative treatments prior to availability of the hybrid system . limitations to the study include the nonrandomized design , limited sample size , and duration of followup . using subjects as their own control enables clinically meaningful comparisons that account for patient heterogeneity , and use of standardized objective measures of hearing helps ensure validity . the effect and sample size were large enough to produce statistically significant improvements after 6 months followup ; additional longer term followup for safety and study of the device in larger and diverse subgroups is important . typically , this is not accessible through amplification for individuals with bilateral severe highfrequency hearing loss and beneficial , aidable lowfrequency hearing . this system is a new and effective treatment that provides clinically significant improvements in speech understanding through integrated electric and acoustic stimulation in the implanted ear , with additional benefit when listening using both ears thus fulfilling a need in individuals who to date have had no other treatment options . canine visceral leishmaniasis ( cvl ) is an infectious disease transmitted by sand flies and caused by leishmania infantum ( 1 ) . it is a zoonotic endemic disease in most of the mediterranean area , asia , and latin america ( 2 , 3 ) . in iran the prevalence of cvl in various parts of iran is different and it is correlated by weather condition and humidity ( 4 ) . it is endemic in ardebil , east azerbaijan , fars and bushehr provinces ( 5 ) . domestic dogs ( canis familiaris ) are the main reservoir hosts of human visceral leishmaniasis in iran ( 6 ) and the prevalence of vl in human , in distinct area , is associated with amount of cvl in dogs ( 4 ) . in foci of canine leishmaniasis , symptomatic disease is low and due to the systemic nature of the disease , clinical manifestations are variable ( 68 ) . it seemed among different control strategies of the disease vaccination , if an efficient vaccine being accessible , is the best possible way for eradication of this disease in dogs ( 6 ) . immunization with naked dna is the latest method , which promote both cd4- and cd8-mediated responses ( 9 , 10 ) . in the past few years , studies focused on some antigens such as gp63 , cp , tsa , gp64 , lmsti1 , leif and p8 , p4 and lack ( 11 ) . among of these antigens , lack gene is one of the best candidates in other strains of this pathogen ( 12 ) lack ( leishmania homologue of receptors for activated c kinase ) is a 36 kda protein localized in cytosol and external surface of the membrane ( 13 ) . it is expressed in both promastigote and amastigote forms of the parasite ( 14 ) . the protective effect of the lack vaccine was mediated by il-12-dependent ifn - g production ( 15 ) . the objective of this study was to investigate of the quality of lack protein expression of iran strain of l. infantum . leishmania infantum iran strain [ mcan / ir/07/moheb - gh ] was provided by the school of public health tehran university of medical sciences . promastigotes were grown in rpmi 1640 medium ( gibco , germany ) supplemented with 5% fetal calf serum and 200iu /ml penicillin g [ crystalizeh ] . dna extracted by dna extraction kit - mbst - iran and electrophoresed on 1% agarose gel . the extracted dna was used as a template to amplify the lack gene by pcr . the reaction was performed in 100l of the solution containing : 1l of template dna , 2l of dntp ( 100 mol ) , 0.5l of taq dna polymerase ( 5u/l ) , 10l of 10x pcr buffer , 2 l of mgcl2 ( 50 mmol ) , 79.5l of distilled water and 2l of each of primers ( 10 pmol/l).we designed a pair of primer based on lack gene sequence ( accession number u49695 ) with bamhi and hindii restriction enzymes on 5 forward and reverse primer lack f 5- ggatcc a tga act acg ag - g gtc acc -3 reverse primer : introduced hindiii . pcr product was purified ( purification kit mbst - iran ) , cloned into ptz57r / t vector ( fermentase t / a vector cat . / t - lack was digeted by bamhi and released lack gene purified and sub cloned into pcdna3.1 shuttel vector . briefly , the reaction was performed in 10 l of the solution containing : 5 l of the purified lack gene , 2 l of 5x buffer , 0.5 l of t4 dna ligase ( 5u/l ) ( fermentase ) , 1.5 l of d.w ( distilled - water ) in 4c for overnight and transformed in to competent cell as previously described ( 16 ) . analysis of recombinant colony was done by three methods : colony pcr reaction , restriction analysis and sequencing . the universal primers are used for detection are t7promoter , taa tac gac tca cta tag gc and bgh - rev , cta gaa ggc aca gtc gag gc . a single colony of pc - lack was cultured , when its od reach to 0.6 was induced by 0.1 molar iptg . electrophoresed proteins were transferred to nitrocellulose membrane and western blot analysis was done and specific protein was detected by leishmania antibody - positive dog serum and anti - dog conjugate ( anti - igg dog sigma ) . for dilution of serum in western blot , the best antigen - antibody reaction was found by dot blot . leishmania infantum iran strain [ mcan / ir/07/moheb - gh ] was provided by the school of public health tehran university of medical sciences . promastigotes were grown in rpmi 1640 medium ( gibco , germany ) supplemented with 5% fetal calf serum and 200iu /ml penicillin g [ crystalizeh ] . dna extracted by dna extraction kit - mbst - iran and electrophoresed on 1% agarose gel . the extracted dna was used as a template to amplify the lack gene by pcr . the reaction was performed in 100l of the solution containing : 1l of template dna , 2l of dntp ( 100 mol ) , 0.5l of taq dna polymerase ( 5u/l ) , 10l of 10x pcr buffer , 2 l of mgcl2 ( 50 mmol ) , 79.5l of distilled water and 2l of each of primers ( 10 pmol/l).we designed a pair of primer based on lack gene sequence ( accession number u49695 ) with bamhi and hindii restriction enzymes on 5 forward and reverse primer lack f 5- ggatcc a tga act acg ag - g gtc acc -3 reverse primer : introduced hindiii . pcr product was purified ( purification kit mbst - iran ) , cloned into ptz57r / t vector ( fermentase t / a vector cat . recombinant ptz57r / t - lack was digeted by bamhi and released lack gene purified and sub cloned into pcdna3.1 shuttel vector . briefly , the reaction was performed in 10 l of the solution containing : 5 l of the purified lack gene , 2 l of 5x buffer , 0.5 l of t4 dna ligase ( 5u/l ) ( fermentase ) , 1.5 l of d.w ( distilled - water ) in 4c for overnight and transformed in to competent cell as previously described ( 16 ) . analysis of recombinant colony was done by three methods : colony pcr reaction , restriction analysis and sequencing . the universal primers are used for detection are t7promoter , taa tac gac tca cta tag gc and bgh - rev , cta gaa ggc aca gtc gag gc . a single colony of pc - lack was cultured , when its od reach to 0.6 was induced by 0.1 molar iptg . electrophoresed proteins were transferred to nitrocellulose membrane and western blot analysis was done and specific protein was detected by leishmania antibody - positive dog serum and anti - dog conjugate ( anti - igg dog sigma ) . specific binding was revealed with diaminobenzidine ( dab ) ( dako , denmark ) . for dilution of serum in western blot , leishmania infantum genomic dna was extracted and lack gene amplified by pcr reaction . then gene was cloned into ptz57r / t vector and after confirmation plasmid by colony pcr and restriction analysis , recombinant plasmid was digested by bamhi and sub cloned into pcdna3.1 expressing vector ( fig . 3 ) . electrophoresis of t / a - lack and pc- lack recombinant plasmids and pcdna3 plasmid were loaded on a 1% agarose gel./ column1 : t / a - lack recombinant plasmid / column2 : pc- lack recombinant plasmid / column3 : the band of pcdna3 electrophoresis of pcr , amplification of lack gene / with pc - lack./ column 1 : dna ladder / column 27 : pcr amplification of lack gene with pc - lack / column 8 : control negative electrophoresis of extracted pc - lack after digestion by enzyme/ column 1 : dna ladder / column 2 : pclack digested by bamh1 recombinant pcdna3.1 plasmid was purified and sequenced by dideoxy chain termination method and compared with lack gene ( accession no u49695.1 ) by www.ncbi.nlm.nih.gov/blast-showed high homology of 98 % ( fig . 4 ) . sequencing of lack gene with universal primers of pc - dna3.1 a 36 kd band was recognized by leishmania antibody - positive [ polyclonal ] dog sera in protein extracts of the cells transfected with pc - lack . in western blot lack protein ( fig . 5 , 6 ) sds - page analysis of expressed gene product column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg western blot analysis of expressed gene product column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg leishmania infantum genomic dna was extracted and lack gene amplified by pcr reaction . then gene was cloned into ptz57r / t vector and after confirmation plasmid by colony pcr and restriction analysis , recombinant plasmid was digested by bamhi and sub cloned into pcdna3.1 expressing vector ( fig . 3 ) . electrophoresis of t / a - lack and pc- lack recombinant plasmids and pcdna3 plasmid were loaded on a 1% agarose gel./ column1 : t / a - lack recombinant plasmid / column2 : pc- lack recombinant plasmid / column3 : the band of pcdna3 electrophoresis of pcr , amplification of lack gene / with pc - lack./ column 1 : dna ladder / column 27 : pcr amplification of lack gene with pc - lack / column 8 : control negative electrophoresis of extracted pc - lack after digestion by enzyme/ column 1 : dna ladder / column 2 : pclack digested by bamh1 recombinant pcdna3.1 plasmid was purified and sequenced by dideoxy chain termination method and compared with lack gene ( accession no u49695.1 ) by www.ncbi.nlm.nih.gov/blast-showed high homology of 98 % ( fig . a 36 kd band was recognized by leishmania antibody - positive [ polyclonal ] dog sera in protein extracts of the cells transfected with pc - lack . in western blot lack protein ( fig . 5 , 6 ) sds - page analysis of expressed gene product column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg western blot analysis of expressed gene product column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg canine s family , particularly domestic dogs are considered the main reservoir of zoonotic transmission ( 17 ) . dogs usually develop the systemic form of infection , with a highly variable clinical appearance that may involve any organ , tissue , or body fluid and is manifested by non - specific clinical signs . both symptomatic and asymptomatic leishmania - infected dogs act as a source of parasites for vl transmission ( 17 ) . although anti leishmania drugs successfully used for human , vl therapy show low efficacy in canines ( 9 , 18 ) . thus , recent studies have tended to cvl control instead of treating . control methods are variable , such as reservoir control , vector control , insecticide - impregnated materials and culling . but none of them were useful ( 8 , 9 , 19 ) . due to the simple nature of the parasite and this fact that recovery and resistance are the results of reinfection in leishmaniasis , vaccination against vl is feasible ( 13 , 20 ) . studies on a protective vaccine candidate have advanced in recent years and several vaccination methods and several antigens were tested . immunization with naked dna ( dna vaccination ) is a new approach that promote both cd4 and cd8 mediated responses and helped to inducing a protective response against infection ( 10 , 15 , 21 , 22 ) . . found that lack is required for parasite viability and it can be a potential drug target for leishmaniasis ( 20 ) . lack might bind and enhance plasminogen activation in vivo promoting the formation of plasmin and might contribute to the invasiveness of the parasite ( 13 ) . in some studies , for example , the study on the combination of dna vectors expressing il-12 + il-18 and a booster with a vaccinia virus recombinant expressing lack in mice revealed that this combined prime / booster immunization regime is an efficient approach to protect against leishmaniasis ( 14 ) . heterologous prime - boost regime using dna and recombinant vaccinia virus vectors expressing lack was tested in some studies and relative protection was achieved ( 2225 ) . lack dna vaccine induced a robust parasite - specific th1 immune response ( ifn- ) but it is not protective against cutaneous or systemic l. donovani challenge ( 15 ) . cloning and expression of the lack gene of l. major iranian strain for immunology study and production of recombinant vaccine was done before ( 26 , 27 ) . according to prevalence and importance of visceral leishmaniasis in iran and necessity of production an efficient vaccine , we did this study . the results of the blast in gene bank and western blot analyze showed that the gene correctly cloned in vector and it is active in immunological aspect . therefore , this study is a start of design a recombinant vaccine against canine visceral leishmaniasis in the future .

objectives / hypothesisto evaluate the safety and efficacy of acoustic and electric sound processing for individuals with significant residual lowfrequency hearing and severetoprofound highfrequency sensorineural hearing loss.study designprospective , singlearm repeated measures , singlesubject design.methodsfifty individuals , 18 years old , with lowfrequency hearing and severe highfrequency loss were implanted with the cochlear nucleus hybrid l24 implant at 10 investigational sites . preoperatively , subjects demonstrated consonantnucleusconsonant word scores of 10% through 60% in the ear to be implanted . subjects were assessed prospectively , preoperatively , and postoperatively on coprimary endpoints of consonantnucleusconsonant words , azbio sentences in noise , and selfassessment measures.resultssignificant mean improvements were observed for coprimary endpoints : consonantnucleusconsonant words ( 35.8 percentage points ) and azbio sentences in noise ( 32.0 percentage points ) , both at p < 0.001 . ninetysix percent of subjects performed equal or better on speech in quiet and 90% in noise . eightytwo percent of subjects showed improved performance on speech in quiet and 74% in noise . selfassessments were positive , corroborating speech perception results.conclusionthe nucleus hybrid system provides significant improvements in speech intelligibility in quiet and noise for individuals with severe highfrequency loss and some lowfrequency hearing . this device expands indications to hearingimpaired individuals who perform poorly with amplification due to bilateral highfrequency hearing loss and who previously were not implant candidates.level of evidence2b . laryngoscope , 126:175181 , 2016 background : there are several methods , such as vaccination , to control visceral leishmaniasis . although there is no efficient vaccine , it seem dna vaccination with stimulates both cellular and humoral immunity apparently is the best way . the aim of this study was cloning and expression of lack gene , a 36kd protein , as a candidate protein for vaccination against iranian l. infantum.methods:iranian strain of l. infantum [ mcan / ir/07/moheb - gh ] was used as a template for pcr to amplify lack gene . the lack gene was cloned in ptz57r / t vector and after confirmation it was digested by restriction enzymes ( bamh1 ) and cloned in pcdna3.1 expression vector . recombinant plasmid was extracted and analyzed by sequencing , restriction digestion analysis and pcr reaction . the pc- lack recombinant plasmid was purified from transformed e.coli ( dh5 ) and its expression was analyzed by sds - page and western blot.results:the results of sequencing , restriction digestion analysis and pcr reaction revealed that lack gene was cloned correctly in pcdna3.1 vector and the results of sds page and western blot emphasized that lack protein of iranian l. infantum is a well - expressed protein.conclusion:we amplified , cloned and expressed iranian l. infantum lack gene successfully .

arterial stiffness is increased in patients with type 2 diabetes mellitus ( t2 dm ) , which can potentially increase the risk of morbidity and mortality associated with cardiovascular diseases ( cvd ) . the brachial - ankle pulse wave velocity ( bapwv ) is a non - invasive technique often used clinically for assessment of arterial wall stiffness and evaluation of the state of atherosclerosis and prediction of cvd in t2 dm patients [ 2 , 3 ] . previous studies demonstrated that conventional risk factors for cvd , such as age , gender , body mass index ( bmi ) , duration of t2 dm , glycemic control , dyslipidemia , systolic blood pressure ( sbp ) , estimated glomerular filtration rate ( egfr ) , uric acid and albuminuria , are associated with increased arterial stiffness in patients with t2 dm [ 1 , 4 - 6 ] . however , life style - related problems were not fully taken into consideration in these studies , although the risk of cvd in patients with t2 dm correlates with numerous lifestyle problems [ 7 , 8 ] . in fact , we recently demonstrated that poor sleep quality in patients with t2 dm correlates with increased arterial stiffness ; however , in that study , we did not take conventional cardiovascular risk factors into full consideration . another study showed that low physical activity was associated with increased arterial stiffness in newly diagnosed patients with t2 dm . on the other hand , the association of other lifestyle habits , such as energy intake , morningness - eveningness , sleep duration and depressive state , with bapwv remains largely unknown in patients with t2 dm . the aim of the present study was to identify lifestyle habits that are associated with bapwv in t2 dm patients free of apparent cvd , using a model adjusted for numerous conventional cardiovascular risk factors and lifestyle habits . the subjects of this cohort study were recruited from three institutions : the diabetes outpatient clinic of juntendo university ( tokyo , japan ) , naka memorial clinic ( naka , japan ) , and secomedic hospital ( funabashi , japan ) [ 7 , 9 , 10 ] . the inclusion criteria were as follows : 1 ) male and female t2 dm patients , 2 ) age 25 and < 70 years , and 3 ) signing consent form for participation in the study . the following exclusion criteria were also applied : 1 ) type 1 or secondary diabetes , 2 ) presence of severe infection , recent surgery , or severe trauma , 3 ) history of myocardial infarction , angina pectoris , or stroke , 4 ) chronic renal failure requiring hemodialysis , 5 ) liver cirrhosis , 6 ) moderate or severe heart failure ( nyha / new york heart association stage iii or higher ) , 7 ) active malignancy , 8) pregnancy , lactation , or possible pregnancy , and 9 ) patients judged as ineligible by the clinical investigators . a total of 1,032 consecutive subjects were screened . between june 2013 and january 2014 , 906 of 1,032 patients who met the above eligibility criteria were enrolled in this study . among the 906 patients , 736 patients agreed to participate in the study . the self - administered questionnaire survey to evaluate sleep quality used in the present study was the pittsburgh sleep quality index ( psqi ) [ 9 , 11 ] . based on the total psqi score , patients were divided into three groups : the good sleep quality group with psqi score of 5 , average sleep quality group with psqi 6 - 8 , and poor sleep quality group with psqi 9 . we also used the morning - evening questionnaire ( meq ) to evaluate morningness and eveningness in individuals . the remaining five questions allowed for choice of time scales and scored from 1 to 5 . the sum of all scores was converted into three - point meq scales as follows : scores 16 - 52 represented the evening type , scores 53 - 64 represented the neither type , and scores 65 - 86 was considered the morning type , as reported previously . the participating patients also completed the beck depression inventory ( bdi)-ii , which is a 21-item questionnaire that assesses hopelessness , irritability , cognition , guilt , fatigue , weight loss , and sexual interest , representing depression - related symptoms . dietary habits during the preceding month were also assessed by the self - administered diet history questionnaire ( bdhq ) . briefly , the four - page structured bdhq includes questions on selected foods and is designed to estimate the dietary consumption of 56 food and beverage items . physical activity level was assessed with the four - question international physical activity questionnaire ( ipaq ) , and the results were expressed as metabolic equivalent scores ( metsh / week ) . in the above questionnaires , the subjects were also divided into non - smokers , former smokers and current smokers . blood samples were collected at the outpatient clinic after overnight fast and liver and renal function tests , lipid profile and hba1c ( national glycohemoglobin standardization program ) were measured with standard techniques . urinary albumin excretion ( uae ) was measured by the latex agglutination assay using a spot urine sample . the egfr was calculated by the formula : egfr ( ml / min/1.73 m ) = 194 age serum creatinine ( 0.739 for females ) . bapwv was measured using an automatic waveform analyzer ( bp-203rpe ; colin medical technology , komaki , japan ) , as described previously [ 7 , 9 , 10 ] . briefly , recording was performed with the patients in the supine position after 5-min rest . occlusion and monitoring cuffs were placed snugly around both areas in the upper and lower extremities . the pressure waveforms were then recorded simultaneously from the brachial arteries by the oscillometric method . all scans were automatically conducted by well - trained investigators who were blinded to the clinical information . a resting ankle - brachial index of 0.90 was considered to reflect the presence of peripheral artery disease . the diagnosis of peripheral artery disease based on the ankle - brachial index was further confirmed by computed tomographic angiography , magnetic resonance angiography or catheter angiography . based on the data obtained from the above procedures , six patients with arterial lumen narrowing of 50% were excluded from analysis . results were presented as mean sd or median ( interquartile range : 25 - 75% ) for continuous variables or number ( proportion ) of patients for categorical variables . multiple linear regression analysis was performed to investigate whether possible cardiovascular risk factors evaluated by clinical , biochemical , metabolic tests and lifestyle habits were associated with bapwv in patients with t2 dm free of apparent cvd . conventional atherosclerotic risk factors based on clinical judgment and certain life styles were included in the model . all analyses were performed using the sas software version 9.3 ( sas institute , cary , nc ) . the subjects of this cohort study were recruited from three institutions : the diabetes outpatient clinic of juntendo university ( tokyo , japan ) , naka memorial clinic ( naka , japan ) , and secomedic hospital ( funabashi , japan ) [ 7 , 9 , 10 ] . the inclusion criteria were as follows : 1 ) male and female t2 dm patients , 2 ) age 25 and < 70 years , and 3 ) signing consent form for participation in the study . the following exclusion criteria were also applied : 1 ) type 1 or secondary diabetes , 2 ) presence of severe infection , recent surgery , or severe trauma , 3 ) history of myocardial infarction , angina pectoris , or stroke , 4 ) chronic renal failure requiring hemodialysis , 5 ) liver cirrhosis , 6 ) moderate or severe heart failure ( nyha / new york heart association stage iii or higher ) , 7 ) active malignancy , 8) pregnancy , lactation , or possible pregnancy , and 9 ) patients judged as ineligible by the clinical investigators . a total of 1,032 consecutive subjects were screened . between june 2013 and january 2014 , 906 of 1,032 patients who met the above eligibility criteria were enrolled in this study . among the 906 patients , 736 patients agreed to participate in the study the self - administered questionnaire survey to evaluate sleep quality used in the present study was the pittsburgh sleep quality index ( psqi ) [ 9 , 11 ] . based on the total psqi score , patients were divided into three groups : the good sleep quality group with psqi score of 5 , average sleep quality group with psqi 6 - 8 , and poor sleep quality group with psqi 9 . we also used the morning - evening questionnaire ( meq ) to evaluate morningness and eveningness in individuals . briefly , 11 questions allowed for choice and scored from 1 to 4 . two questions allowed for choice and scored 0 , 2 , 4 and 6 . the remaining five questions allowed for choice of time scales and scored from 1 to 5 . the sum of all scores was converted into three - point meq scales as follows : scores 16 - 52 represented the evening type , scores 53 - 64 represented the neither type , and scores 65 - 86 was considered the morning type , as reported previously . the participating patients also completed the beck depression inventory ( bdi)-ii , which is a 21-item questionnaire that assesses hopelessness , irritability , cognition , guilt , fatigue , weight loss , and sexual interest , representing depression - related symptoms . dietary habits during the preceding month were also assessed by the self - administered diet history questionnaire ( bdhq ) . briefly , the four - page structured bdhq includes questions on selected foods and is designed to estimate the dietary consumption of 56 food and beverage items . physical activity level was assessed with the four - question international physical activity questionnaire ( ipaq ) , and the results were expressed as metabolic equivalent scores ( metsh / week ) . in the above questionnaires , the subjects were also divided into non - smokers , former smokers and current smokers . blood samples were collected at the outpatient clinic after overnight fast and liver and renal function tests , lipid profile and hba1c ( national glycohemoglobin standardization program ) were measured with standard techniques . urinary albumin excretion ( uae ) was measured by the latex agglutination assay using a spot urine sample . the egfr was calculated by the formula : egfr ( ml / min/1.73 m ) = 194 age serum creatinine ( 0.739 for females ) . bapwv was measured using an automatic waveform analyzer ( bp-203rpe ; colin medical technology , komaki , japan ) , as described previously [ 7 , 9 , 10 ] . briefly , recording was performed with the patients in the supine position after 5-min rest . occlusion and monitoring cuffs were placed snugly around both areas in the upper and lower extremities . the pressure waveforms were then recorded simultaneously from the brachial arteries by the oscillometric method . all scans were automatically conducted by well - trained investigators who were blinded to the clinical information . a resting ankle - brachial index of 0.90 was considered to reflect the presence of peripheral artery disease . the diagnosis of peripheral artery disease based on the ankle - brachial index was further confirmed by computed tomographic angiography , magnetic resonance angiography or catheter angiography . based on the data obtained from the above procedures , six patients with arterial lumen narrowing of 50% were excluded from analysis . results were presented as mean sd or median ( interquartile range : 25 - 75% ) for continuous variables or number ( proportion ) of patients for categorical variables . multiple linear regression analysis was performed to investigate whether possible cardiovascular risk factors evaluated by clinical , biochemical , metabolic tests and lifestyle habits were associated with bapwv in patients with t2 dm free of apparent cvd . conventional atherosclerotic risk factors based on clinical judgment and certain life styles were included in the model . all analyses were performed using the sas software version 9.3 ( sas institute , cary , nc ) . among the 736 participating patients , 12 did not complete the questionnaires and/or bapwv measurement and/or had an ankle - brachial index of 0.90 , and accordingly were excluded from analysis . table 1 summarizes the characteristics of the remaining 724 japanese patients with t2 dm . the mean age was 57.8 8.6 years , and 62.8% of the subjects were males . the mean hba1c was 7.01.0% , and the estimated duration of t2 dm was 9.9 7.2 years . most subjects had attended educational programs in the past about diet and exercise therapy and received appropriate medical treatments . alt : alanine aminotransferase ; ast : aspartate aminotransferase ; bapwv : brachial - ankle pulse wave velocity ; egfr : estimated glomerular filtration rate ; hdl - c : high - density lipoprotein - cholesterol ; uae : urinary albumin excretion ; -gtp : -glutamyl transpeptidase . the results of multivariable linear regression analysis are shown in table 2 . the regression analysis that included age and gender , which are major confounding factors for atherosclerosis , demonstrated that age and male sex were positively associated with bapwv ( model 1 ) . in model 2 adjusted for conventional cardiovascular risk factors ( + model 1 ) , age , duration of t2 dm , hba1c , sbp , uric acid and uae were positively associated with bapwv while bmi was negatively associated with bapwv . almost similar findings were observed in model 3 adjusted for alcohol consumption , smoking , and background therapies for cvd risk factors ( + model 2 ) . in model 4 adjusted for lifestyle habits ( + model 3 ) , age , duration of t2 dm , sbp , uric acid , uae and poor sleep quality were positively associated with bapwv while bmi was negatively associated with bapwv . multiple linear regression analysis included age and gender ( model 1 ) , model 1 plus body mass index , estimated duration of diabetes , hba1c , systolic blood pressure , diastolic blood pressure , total cholesterol , high density lipoprotein - cholesterol , triglyceride , ast , alt , -gtp , serum uric acid , egfr and uae ( model 2 ) , model 2 plus current smoker , alcohol , diabetic retinopathy , insulin therapy , hypertension medication , hyperlipidemia medication and anti - platelet agents ( model 3 ) , model 3 plus morningness - eveningness questionnaire , pittsburgh sleep quality index , beck depression inventory - ii , energy intake ( kcal / day ) , physical activity ( kcal / day ) and sleep time ( model 4 ) . est : standardized estimate of regression parameter ; alt : alanine aminotransferase ; ast : aspartate aminotransferase ; bapwv : brachial - ankle pulse wave velocity ; egfr : estimated glomerular filtration rate ; hdl - c : high - density lipoprotein - cholesterol ; uae : urinary albumin excretion ; -gtp : -glutamyl transpeptidase . in agreement with previous studies [ 1 , 4 - 6 , 20 ] , our data demonstrated that conventional cardiovascular risk factors , such as age , duration of t2 dm , sbp , uric acid and uae , were associated with increased arterial stiffness in patients with t2 dm even after adjustment for some lifestyle habits . furthermore , we found that poor sleep quality was associated with arterial stiffness even after adjustment for several conventional cardiovascular risk factors and other lifestyle habits . in this regard , a previous report demonstrated that high levels of catecholamines caused by poor sleep quality may be associated with progression of arterial stiffness in healthy middle - aged adults . high catecholamine levels could promote smooth muscle cell proliferation and fibrosis , leading to structural changes in the arterial wall . although the study design does not allow evaluation of causal relations , the above data suggest that poor sleep quality may be an important target of interventions that can prevent arterial stiffness in patients with t2 dm . in contrast , our data failed to show close association of other lifestyle problems , such as energy intake , physical activity , morningness - eveningness , sleep duration and depressive state , with arterial stiffness in patients with t2 dm . a recent clinical trial demonstrated that life style interventions , with a special focus on reduced calorie intake and increased physical activity , did not affect the rate of cvd in obese t2 dm patients . it is possible that not only caloric intake , but rather dietary composition , may need to be modified to prevent or delay the development of cvd in patients with t2 dm . while previous reports demonstrated that bmi was positively associated with arterial stiffness [ 5 , 20 , 24 , 25 ] , bmi was inversely associated with arterial stiffness in this study , consistent with a previous report in non - t2 dm patients . the exact explanation for the conflicting results is unknown ; however , a previous study showed higher bapwv levels in t2 dm patients with normal bmi and increased visceral adiposity compared to those with higher bmi and normal visceral adiposity . generally , increased visceral adiposity is associated with insulin resistance , which increases the production of inflammatory cytokines , leading to arterial stiffness . since asians tend to have insulin resistance even with normal bmi , evaluation of visceral adiposity may be helpful in the assessment of effect of obesity on arterial stiffness in these subjects . a close association between serum uric acid and arterial stiffness in male patients with newly diagnosed t2 dm was reported previously , even after adjustment for several risk factors for atherosclerosis . in the present study , we demonstrated a significant association between serum uric acid and arterial stiffness in a wider range of t2 dm population than the above study , even after adjustment for lifestyle habits in addition to numerous cardiovascular risk factors . however , whether serum uric acid plays an important role or is a potentially useful sensitive marker for identifying patients at high risk for cvd is not clear in this cross - sectional study . indeed , the deleterious effect of hyperuricemia on arterial stiffness may be mediated through other interactive cardiovascular risk factors and/or the effect of hyperuricemia may depend on the stage of atherosclerosis [ 6 , 29 ] . first , the cross - sectional design does not allow evaluation of the causal relationship between risk factors for atherosclerosis and arterial stiffness . third , we can not rule out the possible effects of other lifestyle habits that we did not assessed in this study on arterial stiffness . in conclusion , we found that several conventional cardiovascular risk factors , such as age , duration of t2 dm , sbp , serum uric acid , uae , lower bmi and poor sleep quality were associated with bapwv even after adjustment for multiple traditional cardiovascular risk factors and lifestyle habits . in conclusion , we found that several conventional cardiovascular risk factors , such as age , duration of t2 dm , sbp , serum uric acid , uae , lower bmi and poor sleep quality were associated with bapwv even after adjustment for multiple traditional cardiovascular risk factors and lifestyle habits . the study was approved by the institutional review board of our hospital and conducted in accordance with the principles described in the declaration of helsinki . all patients provided written informed consent before participation and registered on the university hospital medical information network clinical trials registry ( umin000010932 ) . hm is an employee of takeda pharmaceutical co. hw has received lecture fees from novo nordisk , inc . , eli lilly and company , sanofi , dainippon sumitomo pharma co. , fujifilm , bayer health care , kissei pharmaceutical company , mochida pharmaceutical company , msd , takeda pharmaceutical company , boehringer ingelheim pharmaceuticals , inc . , daiichi - sankyo , ono pharmaceutical co. , ltd , novartis pharmaceuticals corporation , mitsubishi tanabe pharma corporation , astrazeneca lp , kyowa hakko kirin company , ltd , sanwa kagaku kenkyusyo company , ltd , kowa company ltd , astellas pharma , inc . , advisory fees from novo nordisk , inc . , mochida pharma company , astrazeneca lp , kowa company , astellas pharma , inc . , msd , mitsubishi tanabe pharma corporation , novartis pharmaceuticals corporation , dainippon sumitomo pharma co. , takeda pharmaceutical company , ono pharmaceutical co. , pfizer , inc . , kowa company and research funds from boehringer ingelheim , pfizer , mochida pharmaceutical co. , sanofi - aventis , novo nordisk pharma , novartis pharmaceuticals , sanwakagaku kenkyusho , terumo corp . , takeda pharmaceutical co. , msd , shionogi , pharma , dainippon sumitomo pharma , kissei pharma , and astrazeneca . all authors contributed to the study design and were involved at all stages of manuscript development . all authors were also involved in the collection , analysis and interpretation of data , reviewed / edited the manuscript and approved the final manuscript . hw is the principal guarantors of this work and has full access to all study data and takes responsibility for the integrity of the data and accuracy of data analysis . this study was conducted through a research grant from the manpei suzuki diabetes foundation ( to tm ) . the funding agency had no role in the study design , data collection or analysis , decision to publish , or preparation of the manuscript . alanine aminotransferase brachial - ankle pulse wave velocity brief , self - administered diet history questionnaire beck depression inventory systolic blood pressure cardiovascular disease estimated glomerular filtration rate -glutamyl transpeptidase high - density lipoprotein - cholesterol international physical activity questionnaire morningness - eveningness questionnaire pittsburgh sleep quality index type 2 diabetes mellitus urinary albumin excretion we began our investigation of what high reliability might mean for health care by analyzing what is known about how highly reliable organizations function . weick and sutcliffe provide the most compelling depiction of how high - reliability organizations ( hros ) stay safe . they describe an environment of collective mindfulness in which all workers look for , and report , small problems or unsafe conditions before they pose a substantial risk to the organization and when they are easy to fix ( weick and sutcliffe 2007 ) . they prize the identification of errors and close calls for the lessons they can extract from a careful analysis of what occurred before these events . these lessons point to specific weaknesses in safety protocols or procedures that can be remedied to reduce the risk of future failures . the five high - reliability principles that weick and sutcliffe spell out further elucidate the capability of high - reliability organizations to achieve and maintain exemplary levels of safety . hros are preoccupied with failure , never satisfied that they have not had an accident for many months or many years , and they are always alert to the smallest signal that a new threat to safety may be developing . people who work in hros also resist the temptation to simplify their observations and their experiences of their environment . threats to safety can be complex and present themselves in many different forms . accordingly , being able to identify the often subtle differences among threats may make the difference between early and late recognition between finding an unsafe condition when it is easy to correct and failing to recognize a problem until it is getting out of control . hros recognize that the earliest indicators of threats to organizational performance typically appear in small changes in the organization 's operations . they thus take great pains to ensure that all those workers who are most intimately involved in operations always report any deviations from expected performance . in addition , hros make sure that everyone not only feels free to speak up with any concerns but also recognizes an obligation to do so because of how highly the organization values the information as a vital component of its ability to achieve its highest priority : near - perfect safety . hros recognize that despite all their best efforts and past safety successes , errors will occur and safety will be threatened . the hallmark of an hro is not that it is error - free but that errors do n't disable it resilience refers to an organization 's capability to recognize errors quickly and contain them , thereby preventing the harm that results when small errors propagate , are compounded , and mushroom into major problems . when confronted by a new threat , hros have mechanisms in place to identify the individuals with the greatest expertise relevant to managing the new situation and to place decision - making authority in the hands of that person or group . they do not invoke organizational hierarchy or expect that the person with the most seniority or highest rank will be the most effective at dealing with the problem . how close or far away is the typical hospital today from this state of high reliability ? , we rarely observe the five principles of high reliability guiding the actions of organizations , their leaders , and caregivers . as opposed to a preoccupation with avoiding failure , hospitals and other health care organizations behave as if they accept failure as an inevitable feature of their daily work . how else could we explain the estimates that 99,000 americans die in hospitals each year from health care associated infections while hand hygiene compliance routinely registers in the 40 percent range among many other examples ? fortunately , these events happen only rarely from the perspective of an individual surgeon or hospital but we are not close to eliminating them entirely from american health care . in health care , the rarity of adverse events like these tends to reinforce organizations beliefs that they will never experience them and leads to a misplaced confidence that their safety systems are adequate . this complacency blunts the alertness of surgical teams to the small signs of a risk of a surgical fire or wrong - site surgery . hros recognize that complacency itself is a threat to safety and so take great pains to not let it take root . failing to resist for example , we often approach a quality problem with a simple , one - size - fits - all best practice solution . the joint commission 's universal protocol , developed to eliminate wrong - site surgery , is one such example . it consists of three simple steps : ( 1 ) verify the identity of the patient and the intended procedure , ( 2 ) mark the surgical site , and ( 3 ) conduct a time - out in the operating room just before the surgery begins in order to verify again that the patient , the procedure , and the operative site are correctly identified . but this overly simple approach has not eliminated the problem , in large part because it fails to account for the complexities of the surgical process and all the different ways in which risks of a wrong - site procedure may be introduced into it . for example , such risks may arise while scheduling the surgical procedure , a set of problems that the universal protocol does not address . one of the most pervasive safety problems in hospitals relates to their failure to be sensitive to operations . health care workers at all levels routinely observe unsafe conditions , behaviors , and practices , but they very often fail to bring those problems to the attention of managers who are placed appropriately in the daily work flow to address the problems quickly . transitions from one care setting to another ( so - called handoffs ) are fraught with the risk of error due to the incomplete or inaccurate communication of crucial patient information . when caregivers come to expect poor communication , they become desensitized to its hazards . in one analysis , such a culture of low expectations explained a substantial number of the errors that led to a patient 's undergoing an invasive procedure that was intended for someone else ( chassin and becher 2002 ) . thus , the lack of recognition of unsafe conditions or practices is one important reason they are not reported . in addition , health care workers of all kinds are exposed to an inordinate amount of intimidating behavior that suppresses their reporting of safety problems . physicians are often seen as the initiators of intimidating or disrespectful behavior , and nurses are commonly seen as its targets ( leape et al . 2012 ; saxton , hines , and enriquez 2009 ) . but caregivers of all kinds are involved in these unsafe situations . in 2004 , the institute for safe medication practices published the results of its workplace intimidation survey , which focused on the process of receiving , interpreting , and acting on medication orders ( institute for safe medication practices 2004 ) . more than two thousand respondents , mainly nurses and pharmacists , reported a variety of these behaviors that they had personally experienced in the preceding twelve months . the most common behaviors perceived as intimidating were not the flagrantly abusive practices of throwing objects or using loud or profane language . rather , the failure to return phone calls or pages , the use of condescending language , and impatience with questions topped the list . between 60 and 67 percent of the respondents said they had personally experienced such behaviors initiated by physicians three or more times in the preceding year , and 20 to 28 percent said that they had experienced those behaviors more than ten times . the caregivers who experienced these behaviors employed a variety of strategies all suboptimal and risky to deal with them , including asking someone else to talk to an intimidating prescriber about a safety concern regarding a medication order ( 39% ) , refraining from contacting the prescriber while attempting to clarify the safety of a drug order on their own ( 67% ) , or asking colleagues to help interpret an order to avoid having to interact with a particular prescriber ( 75% ) . hros do not tolerate the existence of intimidating behaviors that suppress the reporting of safety concerns and perpetuate the existence of unsafe conditions . a specific example helps illuminate the complexities of the barriers that hospitals face in trying to be sensitive to these safety signals . many medical devices employed in routine hospital care come equipped with alarms that make various sounds when preset parameters are exceeded . intravenous infusion pumps , cardiac rate and rhythm monitors , mechanical ventilators , and blood oxygen monitors are some of the more common ones . caregivers are bombarded hourly by these alarms , especially those working in intensive care areas housing the sickest patients with the greatest number of devices per patient . the number of alarms that sound per patient per day can total several hundred . for a variety of reasons , the vast majority ( perhaps as many as 85% to 99% ) of these alarm sounds do not signify clinical situations of danger . these reasons include poor integration of devices with one another , equipment malfunction , inappropriate alarm settings , and gaps in staff training . the result is that caregivers experience alarm fatigue and may take a variety of unsafe actions , such as turning off the alarms entirely , turning down the sound volume to the point of inaudibility , resetting the alarm to unsafe levels , or ignoring the alarm sounds altogether ( joint commission 2013 ) . the joint commission 's voluntary adverse event reporting program recorded ninety - eight alarm - related events between 2009 and june 2012 , with eighty of them resulting in death . the ecri institute has cited this problem as one of the top ten health technology hazards each year since 2007 ( ecri institute 2012 ) . a comprehensive solution to this problem would require many stakeholders to work together , including device manufacturers , information technology experts , physicians , medical informatics professionals , nurses , clinical engineers , and hospital administrators . imagine the risks to safety if a nuclear power plant had alarm systems that functioned in this fashion . hospitals and health care organizations do not exhibit the features of resilience that characterize hros . in a high - reliability environment , errors and unsafe conditions are recognized early and prevented by rapid remediation from causing harm . but in health care , uncoordinated and poorly designed and maintained mechanical systems ( like medical device alarms ) are tolerated , even though they are not safe . intimidating behaviors suppress reporting and lead to additional unsafe behaviors as caregivers create workarounds to avoid repetitive exposure to intimidators . errors are not seen as valuable information , essential to a hospital 's ability to improve patient safety . in its 2012 report of the results of its annual patient safety culture survey , the federal agency for healthcare research and quality stated that on average , 65 percent of respondents from 1,128 hospitals worried that mistakes they had made were kept in their personnel files , and 50 percent agreed that staff felt that their mistakes were held against them ( agency for healthcare research and quality 2012 ) . finally , in attempting to solve safety and quality problems , hospitals do not regularly permit the most expert individual to implement solutions . pharmacists often have a difficult time bringing their considerable expertise to bear to avoid medication errors . too often , health care teams are multidisciplinary in name only , with physicians or senior administrators dominating the scene . westrum coined this term during a sociological analysis of why pediatricians failed to identify child abuse until the 1960s . he suggested that one of the important underlying phenomena was pediatricians ingrained belief that they were if something as crucial to a child 's health as physical abuse by a parent were going on , surely pediatricians would know about it and bring it to the attention of other pediatricians . but they did n't know about it , and therefore it was n't happening ( westrum 1982 ) . in health care , the risk of an individual 's falling prey to the fallacy of centrality would seem to increase with seniority . this mind - set is particularly risky for organizational leaders because it encourages the risky belief that no news is good news . in hospitals , no news most often means that intimidated caregivers are not recognizing or reporting the unsafe conditions that will , soon enough , harm patients . thus , available data and considerable experience suggest strongly that the five principles of high reliability would be unrecognizable in an average hospital 's daily work . to the contrary , in several instances , particularly those involving the rapid identification and management of errors and unsafe conditions , it appears that today 's hospitals often exhibit the very opposite of high reliability . there is an important corollary to the observation that hospitals are currently characterized by low reliability . this fact implies strongly that hospitals can not solve these problems by simply and directly adopting high - reliability principles and practices all at once . imagine what might happen if all the workers in a hospital suddenly acquired a keen sense of collective mindfulness and began to recognize and report all the unsafe conditions and errors they encountered from the moment they arrived at the hospital . the organization would soon be so deluged with such reports that its capacity to fix the problems uncovered by the reports would be overwhelmed , and many unsafe conditions would necessarily remain unaddressed . of course , such a transformation of an organization 's culture can not take place over night . we must take careful note of how hospitals function today in all the key arenas that must change if high reliability is to become possible for them . this possibility will become more real if we can accurately describe hospitals current state and chart a plausible and feasible pathway toward high reliability , one that defines specific milestones representing incremental progress . is there any guidance in the high - reliability literature on how to chart such a pathway ? not much . weick and sutcliffe offer a series of audits or rating scales that assess the extent to which an organization is behaving like an hro and give some general advice about how to improve ( weick and sutcliffe 2007 , chaps . 5 and 6 ) . but reason offers a similar assessment tool , his checklist for assessing institutional resilience ( cair ) , and has adapted it for health care ( carthey , de leval , and reason 2001 ) . these thoughtful contributions help focus us on what hospitals should be doing to become highly reliable . but they do not give us much insight into precisely how these goals can be accomplished . in brief , we know of no well - documented blueprints for elevating a low - reliability organization or industry into a highly reliable one and sustaining that achievement over time . as noted earlier , we described elsewhere a broad conceptual framework for adapting high - reliability science to health care organizations ( chassin and loeb 2011 ) . this framework was derived from the integration of high - reliability science , our considerable experience working with the thousands of health care organizations that the joint commission accredits or certifies , and some studies explaining how some hospitals have started to adapt high - reliability principles to their operations ( dixon and shofer 2006 ; fei and vlasses 2008 ; frankel , leonard , and denham 2006 ; may 2013 ; wolterman and shabot 2012 ) . we explored three major changes that health care organizations would have to undertake in order to make substantial progress toward high reliability : ( 1 ) the leadership 's commitment to the ultimate goal of zero patient harm , ( 2 ) the incorporation of all the principles and practices of a safety culture throughout the organization , and ( 3 ) the widespread adoption and deployment of the most effective process improvement tools and methods . we elaborate here these three changes with specific respect to hospitals and health systems . by leadership commitment , we mean the aligned agreement of the governing body , typically a board of trustees or directors , senior management , and physician and nurse leaders . all the constituencies of leadership , both formal and informal , must share the same singular vision of eventually eliminating harms to patients . this is an essential initial requirement , because the success of all the other changes depends on it . the goal of zero also is important because one of the most salient characteristics of high - reliability organizations is that they are not satisfied with whatever their current level of safety might be . commercial aviation averaged 129 deaths per year from accidents and logged an average of 9.3 million flights per year , translating into a death rate of 13.9 deaths per million flights . in the next decade , however , from 2002 to 2011 , that death rate plummeted by a remarkable 88 percent to 1.6 deaths per million flights . even though the average annual number of flights increased to 10.4 million per year , the number of deaths dropped to an average of 16.6 per year ( u.s . department of transportation 2012 ) . for the past thirty years , commercial aviation has invested heavily in radically changing flight crews culture in order to advance airline safety . this work began following research conducted by the national aeronautics and space administration in the 1970s demonstrating that the majority of airplane crashes were caused not by catastrophic mechanical failures but by failures of communication among pilots and crew . the development and worldwide deployment of focused and highly effective training programs to establish a safety culture on aircraft flight decks followed . these programs , known as crew resource management , are widely credited with playing the most important role in the dramatic safety improvements the industry witnessed over this time period ( helmreich , merritt , and wilhelm 1999 ) . one of the original developers of crew resource management for the airline industry has since turned his attention to health care . he and his colleagues found that the professional culture in operating rooms and the communication errors related to it were quite similar to those found among aircraft crews ( helmreich 2000 ) . this work has led to a series of efforts to apply the principles and methods of crew resource management to health care ( gordon , mendenhall , and o'connor 2013 ) . since 2009 , the joint commission has required the leadership of all health care organizations that it accredits to create and maintain a culture of safety consequently , many hospitals now conduct staff surveys to assess their safety culture ( agency for healthcare research and quality 2012 ; sexton et al . few , however , have moved beyond tabulating survey results to taking effective actions that have resulted in creating the kind of safety culture that supports high reliability . we have few proven tools or methods that can guide hospital leaders to achieve a fully functional safety culture . the model we describe in our practical framework is derived from the work of reason and hobbs ( reason and hobbs 2003 ) . the organizational culture they depict is based on reason 's years of studying complex organizations and how they prevent or fail to prevent accidents that cause harm . we believe that this model is the one most adaptable and appropriate to health care . the third of the major changes relates to how hospitals carry out efforts to improve the performance of their care processes . it is in this domain that high - reliability science provides the least guidance to health care . hros do not have safety processes that fail 40 to 60 percent of the time , which is the case in health care ( e.g. , hand hygiene and handoff communication ) ( bodenheimer 2008 ; erasmus et al . the specific tools and methods that hros use to maintain their nearly perfect safety procedures are not directly relevant in a setting with reliability as low as that of health care . we believe that three sets of process improvement tools lean , six sigma , and change management constitute the most effective way for health care to dramatically enhance its capacity to create nearly perfect safety processes ( dellifraine , langabeer , and nembhard 2010 ; dupree et al . we call the three robust process improvement , or rpi ( joint commission center for transforming healthcare 2013 ) . they represent the next generation of process improvement methods that were developed in industry and imported into health care . they are proving to be far more effective in addressing complex clinical quality and safety problems than pdca ( plan , do , check , act ) or their more immediate predecessors ( continuous quality improvement and total quality management ) ( goldberg 2000 ) . one of the most important distinguishing features of these newer improvement methods is their systematic attention to uncovering all the very specific causes of the failures of safety processes ( e.g. , hand hygiene ) . by pinpointing specific causes ( e.g. , improper use of gloves or faulty maintenance procedures that do not keep hand gel dispensers full ) and by measuring which ones are most prevalent in a particular area of a hospital , these tools direct improvement efforts to eliminate the causes of the majority of failures . by their careful attention to unraveling the complexities of health care quality and safety problems , the tools of robust process improvement offer health care the means to implement the reluctance to simplify principle of high reliability . the joint commission has adopted rpi as its internal process improvement methodology and , in the first five years of the program , which began in 2008 , trained 35 percent of its workforce in using these tools ( adrian 2009 ) . since 2009 , the joint commission 's center for transforming healthcare has been applying these rpi tools together with teams from hospitals around the country that also have mastered their use to address a number of health care 's most persistent quality and safety problems . table 1 shows the rates of improvement demonstrated in the center 's first four projects . the joint commission 's experience is consistent with that of companies such as ge that have employed the same tools for many years with great benefit ( bartlett and wozny 2005 ; rao 2011 ) . improvements seen in four projects using robust process improvement notes : robust process improvement is a combination of three complementary process improvement methods : lean , six sigma , and change management . percentage of times that caregivers cleaned their hands before walking into or out of a patient 's room . having established these three major domains of change , we then considered how hospitals and health systems operate today and how they might evolve ( slowly or rapidly ) toward high reliability in each of these three areas . clearly , the industry contains much heterogeneity , but observing those differences helps better characterize the current state and directions for that evolution . in devising this framework , we identified several specific components of each of the three domains of change ( fourteen components in all ) and four stages of maturity for each of them that would define progress toward high reliability . we observed hospitals or health systems in which a few or several of these components currently reside in each of these four stages of high reliability . but we intentionally did not attempt to add a fifth stage ( perhaps to be labeled arriving in the future ) that would describe a high - reliability hospital , because we know of no hospitals that have achieved high reliability across all their activities and , therefore , have no firsthand observations to use for such a description . we created this framework over a two - year period , employing a variety of methods and sources . a team at the joint commission has been engaged with high - reliability experts from academia and industry since 2010 to assimilate what is known about hros with the institutional knowledge we have gained from our work in health care quality and safety . these experts include widely published authors and officials and executives from hros in commercial aviation , the chemical and petroleum industries , nuclear power , and the military . among other activities , the joint commission hosted the fifth international high - reliability conference in may 2012 , at which health care executives interacted with representatives from academia and hros from ten different industries ( joint commission 2012 ) . to produce the first draft of the framework , we combined the information gleaned from these experiences with the empirical literature on the characteristics of hospitals associated with improved safety and quality . we then conducted two rounds of pilot testing with health care leaders . in the first round , a small group of five individuals with hospital leadership roles as chief quality officers , chief medical officers , or chief executive officers examined the framework and provided qualitative assessments of its face validity , including whether all appropriate elements were included in the framework and whether any should be eliminated or defined differently . separately , we produced for their review a self - assessment questionnaire designed to elicit information from hospital leaders that would permit us to assign each of the fourteen components of high reliability to one of the four stages of maturity . based on this first round of reviews , we made appropriate changes in the framework and the questionnaire . in the second round of testing each team engaged in this process four to six leaders from a variety of different leadership perspectives , which included chief executive officers , chief nursing officers , chief medical officers , chief quality officers , chief information officers , and others with similar responsibilities . we compiled the data from this round of testing and convened a face - to - face meeting with the teams leaders , typically the chief executive officer , of each of the seven hospitals to discuss the experiences of their teams . the results of this round of testing were incorporated into the framework and questionnaire , which were finalized for further field - testing . table 2 depicts the six components of leadership and each one 's characteristics in the four stages of maturity . the six components are the board of trustees , the chief executive officer and all senior management ( including nursing leaders ) , the engagement of physicians , the hospital 's quality strategy , its use and dissemination of data on measures of quality , and the use of information technology to support quality and safety improvement . the identification of these specific components is supported by published literature linking them to better quality performance ( goeschel , wachter , and pronovost 2010 ; jha and epstein 2010 ; weiner , shortell , and alexander 1997 ) . the hospital leaders commitment to high reliability must include a prominent role for the board of trustees or directors . the board must be part of the leadership 's commitment to eventually achieve zero patient harm and to elevate quality and patient safety to the organization 's highest strategic goal . if the board is left out , management will find its efforts unsupported or misunderstood . today , hospital boards vary over a wide spectrum of involvement in the quality programs of the hospitals they oversee ( jha and epstein 2010 ) . leadership and high reliability : stages of organizational maturity in addition , physicians are essential to the success of any quality initiative in hospitals . table 2 identifies two vital components of physicians role : leadership and participation . in order to move effectively toward high reliability , physicians must routinely champion quality improvement initiatives throughout the hospital . both the formally appointed leaders ( chief medical officer , vice president for medical affairs ) and the informal leaders ( medical staff president , voluntary medical staff leaders ) need to be visible and active enthusiasts for quality , including physician leaders who are not employees of the hospital . it is difficult to imagine a hospital getting close to high reliability if quality is merely one of many competing priorities . memorial hermann health system , a twelve - hospital health care system based in houston , has explicitly committed to becoming highly reliable and has specified the importance of all the major ingredients in this framework to their efforts ( shabot et al . , the system 's ceo pointed out , ensuring patient safety is our core value , and it 's our only core value ( wolterman and shabot 2012 ) . to accelerate the progress toward zero harm , quality must be measured and data on those measures must be widely available both within the hospital and to the public . not only is such transparency valuable in its own right , but public reporting also is a powerful added force that drives improvement . the quality program and its measures should focus on meeting the needs and addressing the specific quality problems of the hospital 's patient population . other incentives , such as the judicious use of financial rewards and staff advancement opportunities based on performance against quality measures , are important accelerants as well . finally , leaders are obligated to employ health information technology ( it ) effectively in the service of quality improvement . it is particularly important to an hro , because it is frequently the vehicle by which nearly perfect processes sustain their performance . if a process has been so effectively redesigned as to be highly reliable , automating it is the most effective way to maintain it in that state . unfortunately , in health care , automation is often deployed unsafely , a phenomenon that increases rather than decreases the risk of harm ( ash et al . in addition , various types of health it are often not coordinated , thereby increasing risk . for example , if programmable infusion devices are not supported by the same decision support rules that govern pharmacy systems and physician order entry systems , the resulting confusion can be life threatening for patients . a hospital approaching high reliability adopts health it solutions in a coordinated and integrated manner following the principles of safe adoption ( joint commission 2008b ; karsh 2004 ) . table 3 shows the five components of safety culture and their manifestations in each of the four stages of maturity toward high reliability . a culture of safety that fully supports high reliability has three central attributes : trust , report , and improve ( reason and hobbs 2003 ) . workers exhibit enough trust in their peers and the organization 's management that they routinely recognize and report errors and unsafe conditions . this trust is established when the organization eliminates intimidating behavior that suppresses reporting , acts in a timely way to fix the problems reported by workers , and communicates these improvements consistently to the individuals who reported the problems in the first place . that communication in turn strengthens the trust that led to the reports and fosters further identification and reporting of problems even further upstream from harm . when all three of these components of a safety culture ( trust , report , and improve ) are working well , they reinforce one another and produce a stable organizational culture that sustains high reliability . safety culture and high reliability : stages of organizational maturity maintaining trust also requires the organization to hold employees accountable for adhering to safety protocols and procedures . hros establish clear , equitable , and transparent processes for recognizing and separating the small , blameless errors that all people make every day from unsafe or reckless actions that are blameworthy . understanding how and why blameless errors occur is part of the learning process that hros employ to maintain their exemplary safety records . recognizing and dealing appropriately with blameworthy acts is an equally important dimension of an hro 's safety culture because of its vital role in maintaining trust . unfortunately , health care organizations too often punish staff for blameless acts while failing to implement equitable disciplinary procedures for those who commit blameworthy acts . nor have hospital leaders succeeded in eradicating intimidating behaviors ( institute for safe medication practices 2004 ) . these failings explain the lack of trust among hospital staff noted earlier ( agency for healthcare research and quality 2012 ) . hospitals that move toward high reliability establish codes of behavior that are modeled by leaders ( including nurses and physicians ) who champion efforts to eliminate intimidation and encourage and reward the reporting of blameless errors and unsafe conditions . accountability for adhering to safe practices should be ingrained in all employees and is spurred by implementing standards for invoking disciplinary procedures that apply to all staff , regardless of seniority or professional credentials . for example , maimonides medical center in new york city has established such a program in its code of mutual respect , which commits all stakeholders ( including physicians , nurses , staff , students , vendors , consultants , and volunteers ) to working harmoniously together and to eliminate intimidating behaviors . the program includes progressive interventions , including disciplinary actions for individuals who repeatedly violate the code ( maimonides medical center 2009 ) . hros also proactively assess the strength and resilience of their safety systems and the organizational defenses that prevent errors from propagating and leading to harm . today 's hospitals function in primarily a reactive mode , investigating incidents in which patients have already been harmed , conducting root cause analyses , and instituting corrective action plans to prevent future occurrences . becoming much safer requires caregivers willingness and ability to recognize and report close calls and unsafe conditions , combined with an organizational capacity to act effectively on those reports to eliminate the risks they embody . furthermore , as opposed to today 's norm of focusing on single events , hospitals should compile the results of their investigations across many harm events and close calls to identify which of their safety systems or defenses are most in need of improvement . these evaluations should lead to the development of proactive assessments of key safety systems ( e.g. , those that relate to medication administration and infection prevention and control ) so that weaknesses can be identified and remedied before they pose any significant risk to patients . finally , progress toward establishing all these elements of a culture of safety should be measured . today , many hospitals regularly use one of several available staff surveys to assess their safety culture . few , however , analyze the meaning of the survey data , evaluate where each area of the hospital is falling short , and undertake specific , focused interventions to remedy those shortcomings . as hospitals make more progress toward high reliability , they will include safety culture metrics as part of their strategic planning programs , set goals for improving on those metrics , and report on those metrics to their boards , just as they report on metrics related to financial performance or patient satisfaction . hospitals need new process improvement tools and methods to break out of their current state of low reliability . we have argued that robust process improvement ( rpi)a combination of lean , six sigma , and change management is a much more potent set of tools than health care currently uses to address safety and quality problems . briefly , and oversimplifying somewhat , lean is a set of tools and a philosophy of employee - empowered improvement that identifies and removes wasted effort from processes without compromising the quality of the outcome . six sigma tools focus on improving the outcomes of a process by radically reducing the frequency with which defective products or outcomes occur . change management is a systematic approach , used alongside lean and six sigma , that prepares an organization to accept , implement , and sustain the improved processes that result from the application of lean and six sigma tools . these three sets of tools are complementary , and together they provide the best available methods for hospitals to achieve major improvements in faulty processes . table 4 shows the three components of rpi and how each changes as a hospital comes closer to high reliability . like ge , best buy , and other companies that have benefited from rpi , we believe that getting the most benefit from them requires that they be employed as a common language throughout the entire organization ( bartlett and wozny 2005 ; rao 2011 ) . nearly all employees should be trained at levels appropriate to each one 's job . the tools should be used throughout the organization for all improvement work . finally , proficiency in the use of rpi should be a part of every employee 's performance appraisal and be required for career advancement within the organization . these elements provide vital support for spreading the use of these tools . for hros , quality and safety are the personal responsibility of every employee , and being armed with highly effective ways to solve complex problems gives employees some of what they need to exercise that responsibility . robust process improvement and high reliability : stages of organizational maturity today 's hospitals generally lag far behind this ideal state . some have used some of the elements of rpi , often starting with lean , but relatively few hospitals have adopted the full suite of rpi tools . rpi provides highly effective tools for obtaining the voice of the customer , and the perspective of patients on what constitutes a high - quality outcome for a particular care process is vital to its improvement . making substantial progress toward high reliability in safety and quality requires the application of tools like rpi that can generate extremely high rates of sustainable improvement when applied to the poorly performing safety processes that exist in most hospitals today ( see table 1 ) . we know of no other approach to process improvement available at present that is capable of generating and sustaining rates of improvement of this magnitude consistently over the widest array of areas from clinical quality to business processes ( chassin 1998 ; r. chassin 2008 ) . enabling hospitals to use this high - reliability health care framework requires additional work . to advance toward high reliability , hospitals must be able to assess their current state of maturity with respect to each of the framework 's fourteen components and then to access proven strategies , tools , and methods to advance to more mature levels . the joint commission is developing and testing an instrument that will permit hospital leaders to perform such an assessment . the utility of the assessment to hospitals in identifying their most pressing opportunities for making progress toward high reliability and the availability of specific tools to facilitate such progress is currently being field - tested . this initiative , the south carolina safe care commitment , is being led by the joint commission center for transforming healthcare and the south carolina hospital association ( may 2013 ) . those stakeholders with a vested interest in moving health care further and faster toward high reliability include state and federal government health agencies , consumer and patient advocacy groups , employers , public and private payers , health care professional organizations , and hospitals and health systems themselves . although a comprehensive assessment of these roles is beyond the scope of this article , several observations are nonetheless pertinent here . regulation had only a modest and supportive role in the dramatic quality and safety improvements in other industries ( e.g. , commercial aviation , car manufacturing , and consumer electronics ) . in health care , regulators should pay attention first and foremost to identifying and eliminating requirements that obstruct progress toward high reliability . in some instances , they impose unproductive work on regulated organizations that distracts them from dealing more effectively with their quality challenges . regulators can support the transformation to high reliability , for example , by well - crafted programs of publicly reporting reliable and valid measures of quality . other u.s . industries have undergone transformations in quality stimulated primarily by competitive pressures ( e.g. , from japanese automakers ) . a similar occurrence in health care is difficult to imagine because of the intensely local environment in which the large majority of hospitals and health systems operate ( becher and chassin 2001 ) . because the changes health care must undergo to become highly reliable are so thoroughgoing and profound , the primary drive for change must ultimately come from the health care organizations themselves . as the saying goes , it takes only one psychiatrist to change a lightbulb , but the lightbulb has to want to change . many health care leaders are reluctant to commit to the goal of high reliability because they regard it as unrealistic or unachievable or a distraction from their current serious fiscal and regulatory pressures . one of the important roles for policymakers and stakeholders is to encourage , persuade , and demand that health care organizations embark on this journey . even after they have committed to do so , how long it will take for health care organizations to reach high reliability is unknown , because none has arrived at that destination yet . cincinnati children 's hospital medical center has been working toward this goal for a more than a decade , and its current strategic plan calls for eliminating serious patient harm by 2015 ( cincinnati children 's hospital medical center 2013 ) . finally , hospitals and systems like memorial hermann and cincinnati children 's that have been trailblazers in striving for high - reliability health care have developed their own strategies and tools , largely through trial and error . for this movement to broaden and deepen , the next wave of hospitals and health systems will need proven tools and methods to speed their journey through higher levels of maturity . many stakeholders could contribute to the development and evaluation of such tools , and policymakers at several levels of government could facilitate this process by focused funding efforts . table 2 depicts the six components of leadership and each one 's characteristics in the four stages of maturity . the six components are the board of trustees , the chief executive officer and all senior management ( including nursing leaders ) , the engagement of physicians , the hospital 's quality strategy , its use and dissemination of data on measures of quality , and the use of information technology to support quality and safety improvement . the identification of these specific components is supported by published literature linking them to better quality performance ( goeschel , wachter , and pronovost 2010 ; jha and epstein 2010 ; weiner , shortell , and alexander 1997 ) . the hospital leaders commitment to high reliability must include a prominent role for the board of trustees or directors . the board must be part of the leadership 's commitment to eventually achieve zero patient harm and to elevate quality and patient safety to the organization 's highest strategic goal . if the board is left out , management will find its efforts unsupported or misunderstood . today , hospital boards vary over a wide spectrum of involvement in the quality programs of the hospitals they oversee ( jha and epstein 2010 ) . leadership and high reliability : stages of organizational maturity in addition , physicians are essential to the success of any quality initiative in hospitals . table 2 identifies two vital components of physicians role : leadership and participation . in order to move effectively toward high reliability both the formally appointed leaders ( chief medical officer , vice president for medical affairs ) and the informal leaders ( medical staff president , voluntary medical staff leaders ) need to be visible and active enthusiasts for quality , including physician leaders who are not employees of the hospital . it is difficult to imagine a hospital getting close to high reliability if quality is merely one of many competing priorities . memorial hermann health system , a twelve - hospital health care system based in houston , has explicitly committed to becoming highly reliable and has specified the importance of all the major ingredients in this framework to their efforts ( shabot et al . , the system 's ceo pointed out , ensuring patient safety is our core value , and it 's our only core value ( wolterman and shabot 2012 ) . to accelerate the progress toward zero harm , quality must be measured and data on those measures must be widely available both within the hospital and to the public . not only is such transparency valuable in its own right , but public reporting also is a powerful added force that drives improvement . the quality program and its measures should focus on meeting the needs and addressing the specific quality problems of the hospital 's patient population . other incentives , such as the judicious use of financial rewards and staff advancement opportunities based on performance against quality measures , are important accelerants as well . finally , leaders are obligated to employ health information technology ( it ) effectively in the service of quality improvement . it is particularly important to an hro , because it is frequently the vehicle by which nearly perfect processes sustain their performance . if a process has been so effectively redesigned as to be highly reliable , automating it is the most effective way to maintain it in that state . unfortunately , in health care , automation is often deployed unsafely , a phenomenon that increases rather than decreases the risk of harm ( ash et al . 2007 , 2009 ; joint commission 2008b ; koppel et al . 2005 ; sparnon and marella 2012 ) . in addition , various types of health it are often not coordinated , thereby increasing risk . for example , if programmable infusion devices are not supported by the same decision support rules that govern pharmacy systems and physician order entry systems , the resulting confusion can be life threatening for patients . a hospital approaching high reliability adopts health it solutions in a coordinated and integrated manner following the principles of safe adoption ( joint commission 2008b ; karsh 2004 ) . table 3 shows the five components of safety culture and their manifestations in each of the four stages of maturity toward high reliability . a culture of safety that fully supports high reliability has three central attributes : trust , report , and improve ( reason and hobbs 2003 ) . workers exhibit enough trust in their peers and the organization 's management that they routinely recognize and report errors and unsafe conditions . this trust is established when the organization eliminates intimidating behavior that suppresses reporting , acts in a timely way to fix the problems reported by workers , and communicates these improvements consistently to the individuals who reported the problems in the first place . that communication in turn strengthens the trust that led to the reports and fosters further identification and reporting of problems even further upstream from harm . when all three of these components of a safety culture ( trust , report , and improve ) are working well , they reinforce one another and produce a stable organizational culture that sustains high reliability . safety culture and high reliability : stages of organizational maturity maintaining trust also requires the organization to hold employees accountable for adhering to safety protocols and procedures . hros establish clear , equitable , and transparent processes for recognizing and separating the small , blameless errors that all people make every day from unsafe or reckless actions that are blameworthy . understanding how and why blameless errors occur is part of the learning process that hros employ to maintain their exemplary safety records . recognizing and dealing appropriately with blameworthy acts is an equally important dimension of an hro 's safety culture because of its vital role in maintaining trust . unfortunately , health care organizations too often punish staff for blameless acts while failing to implement equitable disciplinary procedures for those who commit blameworthy acts . nor have hospital leaders succeeded in eradicating intimidating behaviors ( institute for safe medication practices 2004 ) . these failings explain the lack of trust among hospital staff noted earlier ( agency for healthcare research and quality 2012 ) . hospitals that move toward high reliability establish codes of behavior that are modeled by leaders ( including nurses and physicians ) who champion efforts to eliminate intimidation and encourage and reward the reporting of blameless errors and unsafe conditions . accountability for adhering to safe practices should be ingrained in all employees and is spurred by implementing standards for invoking disciplinary procedures that apply to all staff , regardless of seniority or professional credentials . for example , maimonides medical center in new york city has established such a program in its code of mutual respect , which commits all stakeholders ( including physicians , nurses , staff , students , vendors , consultants , and volunteers ) to working harmoniously together and to eliminate intimidating behaviors . the program includes progressive interventions , including disciplinary actions for individuals who repeatedly violate the code ( maimonides medical center 2009 ) . hros also proactively assess the strength and resilience of their safety systems and the organizational defenses that prevent errors from propagating and leading to harm . today 's hospitals function in primarily a reactive mode , investigating incidents in which patients have already been harmed , conducting root cause analyses , and instituting corrective action plans to prevent future occurrences . becoming much safer requires caregivers willingness and ability to recognize and report close calls and unsafe conditions , combined with an organizational capacity to act effectively on those reports to eliminate the risks they embody . furthermore , as opposed to today 's norm of focusing on single events , hospitals should compile the results of their investigations across many harm events and close calls to identify which of their safety systems or defenses are most in need of improvement . these evaluations should lead to the development of proactive assessments of key safety systems ( e.g. , those that relate to medication administration and infection prevention and control ) so that weaknesses can be identified and remedied before they pose any significant risk to patients . finally , progress toward establishing all these elements of a culture of safety should be measured . today , many hospitals regularly use one of several available staff surveys to assess their safety culture . few , however , analyze the meaning of the survey data , evaluate where each area of the hospital is falling short , and undertake specific , focused interventions to remedy those shortcomings . as hospitals make more progress toward high reliability , they will include safety culture metrics as part of their strategic planning programs , set goals for improving on those metrics , and report on those metrics to their boards , just as they report on metrics related to financial performance or patient satisfaction . hospitals need new process improvement tools and methods to break out of their current state of low reliability . we have argued that robust process improvement ( rpi)a combination of lean , six sigma , and change management is a much more potent set of tools than health care currently uses to address safety and quality problems . briefly , and oversimplifying somewhat , lean is a set of tools and a philosophy of employee - empowered improvement that identifies and removes wasted effort from processes without compromising the quality of the outcome . six sigma tools focus on improving the outcomes of a process by radically reducing the frequency with which defective products or outcomes occur . change management is a systematic approach , used alongside lean and six sigma , that prepares an organization to accept , implement , and sustain the improved processes that result from the application of lean and six sigma tools . these three sets of tools are complementary , and together they provide the best available methods for hospitals to achieve major improvements in faulty processes . table 4 shows the three components of rpi and how each changes as a hospital comes closer to high reliability . like ge , best buy , and other companies that have benefited from rpi , we believe that getting the most benefit from them requires that they be employed as a common language throughout the entire organization ( bartlett and wozny 2005 ; rao 2011 ) . nearly all employees should be trained at levels appropriate to each one 's job . , proficiency in the use of rpi should be a part of every employee 's performance appraisal and be required for career advancement within the organization . these elements provide vital support for spreading the use of these tools . for hros , quality and safety are the personal responsibility of every employee , and being armed with highly effective ways to solve complex problems gives employees some of what they need to exercise that responsibility . robust process improvement and high reliability : stages of organizational maturity today 's hospitals generally lag far behind this ideal state . some have used some of the elements of rpi , often starting with lean , but relatively few hospitals have adopted the full suite of rpi tools . fewer still have engaged patients when using these powerful tools to redesign care processes . rpi provides highly effective tools for obtaining the voice of the customer , and the perspective of patients on what constitutes a high - quality outcome for a particular care process is vital to its improvement . making substantial progress toward high reliability in safety and quality requires the application of tools like rpi that can generate extremely high rates of sustainable improvement when applied to the poorly performing safety processes that exist in most hospitals today ( see table 1 ) . we know of no other approach to process improvement available at present that is capable of generating and sustaining rates of improvement of this magnitude consistently over the widest array of areas from clinical quality to business processes ( chassin 1998 ; r. chassin 2008 ) . enabling hospitals to use this high - reliability health care framework requires additional work . to advance toward high reliability , hospitals must be able to assess their current state of maturity with respect to each of the framework 's fourteen components and then to access proven strategies , tools , and methods to advance to more mature levels . the joint commission is developing and testing an instrument that will permit hospital leaders to perform such an assessment . the utility of the assessment to hospitals in identifying their most pressing opportunities for making progress toward high reliability and the availability of specific tools to facilitate such progress is currently being field - tested . this initiative , the south carolina safe care commitment , is being led by the joint commission center for transforming healthcare and the south carolina hospital association ( may 2013 ) . those stakeholders with a vested interest in moving health care further and faster toward high reliability include state and federal government health agencies , consumer and patient advocacy groups , employers , public and private payers , health care professional organizations , and hospitals and health systems themselves . although a comprehensive assessment of these roles is beyond the scope of this article , several observations are nonetheless pertinent here . regulation had only a modest and supportive role in the dramatic quality and safety improvements in other industries ( e.g. , commercial aviation , car manufacturing , and consumer electronics ) . in health care , regulators should pay attention first and foremost to identifying and eliminating requirements that obstruct progress toward high reliability . in some instances , they impose unproductive work on regulated organizations that distracts them from dealing more effectively with their quality challenges . regulators can support the transformation to high reliability , for example , by well - crafted programs of publicly reporting reliable and valid measures of quality . other u.s . industries have undergone transformations in quality stimulated primarily by competitive pressures ( e.g. , from japanese automakers ) . a similar occurrence in health care is difficult to imagine because of the intensely local environment in which the large majority of hospitals and health systems operate ( becher and chassin 2001 ) . because the changes health care must undergo to become highly reliable are so thoroughgoing and profound , the primary drive for change must ultimately come from the health care organizations themselves . as the saying goes , it takes only one psychiatrist to change a lightbulb , but the lightbulb has to want to change . many health care leaders are reluctant to commit to the goal of high reliability because they regard it as unrealistic or unachievable or a distraction from their current serious fiscal and regulatory pressures . one of the important roles for policymakers and stakeholders is to encourage , persuade , and demand that health care organizations embark on this journey . even after they have committed to do so , how long it will take for health care organizations to reach high reliability is unknown , because none has arrived at that destination yet . cincinnati children 's hospital medical center has been working toward this goal for a more than a decade , and its current strategic plan calls for eliminating serious patient harm by 2015 ( cincinnati children 's hospital medical center 2013 ) . finally , hospitals and systems like memorial hermann and cincinnati children 's that have been trailblazers in striving for high - reliability health care have developed their own strategies and tools , largely through trial and error . for this movement to broaden and deepen , the next wave of hospitals and health systems will need proven tools and methods to speed their journey through higher levels of maturity . many stakeholders could contribute to the development and evaluation of such tools , and policymakers at several levels of government could facilitate this process by focused funding efforts . achieving high reliability in health care will require hospitals to undergo substantial changes that can not take place rapidly . we have outlined a framework , with fourteen components , for the practical application of these changes to hospitals . the components are distributed in three major domains : leadership , safety culture , and robust process improvement . we described for each component four evolutionary stages of maturity on the road to high reliability . each stage provides hospitals with specific guidance on what actions they need to take in order to advance toward high reliability . further research and experience derived from the application of this practical framework will be required to assess its effectiveness in facilitating hospitals advancement toward high reliability . finally , policymakers and stakeholders in various positions should evaluate how they can support and accelerate this transformation .

backgroundwhile conventional cardiovascular risk factors and certain lifestyle habits are associated with arterial stiffness in patients with type 2 diabetes mellitus ( t2 dm ) , it is still unknown whether they are actually associated with arterial stiffness even after adjustment for conventional cardiovascular risk factors and lifestyle habits . the aim of this study was to identify variables that are associated with brachial - ankle pulse wave velocity ( bapwv).methodsthe study participants comprised 724 japanese t2 dm outpatients free of history of cardiovascular diseases . lifestyle habits were analyzed using self - reported questionnaires . the associations among conventional cardiovascular risk factors and lifestyle habits with bapwv were investigated by multivariable linear regression analysis.resultsthe mean age of the study subjects was 57.8 8.6 years , and 62.8% of those were males . the mean hba1c was 7.01.0% , and the estimated duration of t2 dm was 9.9 7.2 years . multiple linear regression analysis that included age and gender demonstrated that age and male sex were positively associated with bapwv . in a model adjusted for numerous conventional cardiovascular risk factors and lifestyle habits , age , duration of t2 dm , systolic blood pressure , serum uric acid , urinary albumin excretion and poor sleep quality were positively associated with bapwv , while body mass index was negatively associated with bapwv.conclusionsin japanese t2 dm , in addition to several conventional cardiovascular risk factors , poor sleep quality was associated with bapwv even after adjustment for numerous conventional cardiovascular risk factors and lifestyle habits . contextdespite serious and widespread efforts to improve the quality of health care , many patients still suffer preventable harm every day . hospitals find improvement difficult to sustain , and they suffer project fatigue because so many problems need attention . no hospitals or health systems have achieved consistent excellence throughout their institutions . high - reliability science is the study of organizations in industries like commercial aviation and nuclear power that operate under hazardous conditions while maintaining safety levels that are far better than those of health care . adapting and applying the lessons of this science to health care offer the promise of enabling hospitals to reach levels of quality and safety that are comparable to those of the best high - reliability organizations.methodswe combined the joint commission 's knowledge of health care organizations with knowledge from the published literature and from experts in high - reliability industries and leading safety scholars outside health care . we developed a conceptual and practical framework for assessing hospitals readiness for and progress toward high reliability . by iterative testing with hospital leaders , we refined the framework and , for each of its fourteen components , defined stages of maturity through which we believe hospitals must pass to reach high reliability.findingswe discovered that the ways that high - reliability organizations generate and maintain high levels of safety can not be directly applied to today 's hospitals . we defined a series of incremental changes that hospitals should undertake to progress toward high reliability . these changes involve the leadership 's commitment to achieving zero patient harm , a fully functional culture of safety throughout the organization , and the widespread deployment of highly effective process improvement tools.conclusionshospitals can make substantial progress toward high reliability by undertaking several specific organizational change initiatives . further research and practical experience will be necessary to determine the validity and effectiveness of this framework for high - reliability health care .

acute generalized exanthematous pustulosis ( agep ) is a rare acute reaction that is drug - induced in 90% of the cases , characterized by a widespread , sterile pustular rash . cefepime is a fourth generation cephalosporin antibiotic used to treat febrile neutropenia , severe infections related to the urinary tract , skin , nosocomial pneumonia , brain abscess , and intra - abdominal and septic lateral / cavernous sinus thrombosis . a 67-year - old man with renal failure who had been on dialysis during the last 2 years and with an 8-year history of cardiac insufficiency was admitted to the hospital complaining of 6 days of diarrhea . the patient was taken to the semi - intensive care unit and treated with ciprofloxacin . as a consequence , his long - term medications had not been changed and consisted of acetylsalicylic acid , furosemide , captopril , carvedilol and clonazepam . on the seventh day , the patient became dyspneic and his chest radiograph showed a left lower lobe opacity . treatment for nosocomial pneumonia was promptly initiated with cefepime ( 1 g / day ) . five days later , he presented with a pruritic , erythematous , maculopapular eruption affecting the abdomen , neck and skin folds . one day later , he developed disseminated pustular lesions ( fig . 1 ) and his temperature was 37c . laboratory exams evidenced c - reactive protein 136 mg / l , white blood cells 14,700 cells/l ( normal 3,50010,500 cells/l ) with 11,995 cells/l neutrophils ( normal 1,7008,000 cells/l ) . histology showed a toxic pustuloderma with spongiform subcorneal pustules , edema in the papillary dermis and perivascular inflammatory infiltrate consisting of neutrophils ( fig . after withdrawal of cefepime and introduction of imipenem , the disseminated skin nonfollicular pustules cleared within 4 days following a desquamation . the patient denied previous adverse reaction to other drugs and no personal or family history of psoriasis was evident . agep is a disease characterized by the rapid onset of many sterile , nonfollicular pustules usually arising on an edematous erythema and frequently accompanied by leukocytosis and fever . skin symptoms usually arise rapidly after an insult and resolve spontaneously ( within a few days ) . agep often starts predominantly in intertriginous areas or on the face , spreading rapidly to the trunk and lower limbs . the mean duration of the pustules is 9.7 days , and an annular desquamation typically follows for a few days . complications are rare [ 1 , 3 ] . the agep validation score of the euroscar study group has been used to establish the diagnosis . a score between 8 and 12 for agep is a definitive diagnosis ( table 1 ) . the case score was 11 , according to the validation score of the euroscar study group ( table 2 ) . the main differential diagnosis of agep is pustular psoriasis . because the pustules clinically and histologically resemble the lesions of pustular psoriasis and because in a number of reports patients had a history of plaque psoriasis , some authors assume that agep is nothing more than an acute exacerbation of psoriasis caused by a variety of exogenous triggers however , many studies strongly suggest that agep is not associated with psoriasis [ 1 , 5 ] . up to now agep has been attributed to a variety of causes such as viral infections , chlamydia pneumoniae infection or hypersensitivity to mercury , but the skin reaction is primarily an adverse response to drugs . antibiotics , other than cefepime , have been implicated as the causative agents in 80% of individuals . in this group , the present case of agep has well defined criteria , and because correct diagnosis generally leads to spontaneous resolution once the causative drug is withdrawn , clinicians should keep the possibility of this cutaneous drug reaction in mind . paediatric age at the time of diagnosis represents a negative prognostic factor for keratoconus progression , with increased probability of corneal transplant . particularly younger patients represent a population at high risk for more rapid progression of the disease [ 1 , 2 ] . the long - term results reported in literature [ 35 ] have demonstrated the ability of cross - linking to slow the progression of keratoconus by a photo - polymerization reaction of stromal collagen fibres . cross - linking photodynamic reaction is induced by the combined action of a photosensitizing substance ( riboflavina o vitamin b2 ) and ultraviolet ( uv ) a light , allowing a corneal stiffening by increasing the number of intrafibrillar , interfibrillar covalent bonds , and corneal collagen resistance against enzymatic degradation [ 68 ] . the long - term effects of the technique are related also to a process of collagen neosynthesis with a different structure and higher molecular weight [ 912 ] which confers to the corneal stroma an increased resistance and lamellar compaction responsible of the variable functional modifications recorded after the treatment [ 1114 ] . according to international results [ 35 ] , cross - linking should be the primary choice in young patient with progressive keratoconus . to report a comparative prospective long - term functional analysis after cross - linking in three different age groups ( 18 years , between 1926 years , and 27 years ) of patients affected by progressive keratoconus . since 2004 to date more than 610 patients were treated by combined riboflavin uv a corneal collagen cross - linking . present prospective nonrandomized open study comprised 516 eyes of 413 patients aged between 10 and 40 years , affected by progressive keratoconus . the comparative functional analysis comprised the following : paediatric group ( 18 years and under ) included 152 eyes of 105 patients ( 29.5% ) , intermediate group ( 1926 years ) included 286 eyes of 243 patients ( 55.4% ) , adult group ( 27 years ) included 78 eyes of 65 patients ( 15.1% ) . all patients included in the treatment protocol were affected by progressive keratoconus with a documented clinical and instrumental worsening at least in the last three months of observation . the parameters we considered to establish keratoconus progression were worsening of ucva / bscva > 0.50 snellen lines , increase of sph / cyl > 0.50 d , increase of topographic symmetry index sai / si > 0.50 d , increase of maximum k reading > 0.50 d , reduction of the thinnest point at optical pachometry 10 m , clear cornea at biomicroscopic examination , absence of reticular dark striae at confocal laser microscopy in vivo . we considered significant for the inclusion in the study the variation of at least 3 of the parameters listed above ( one clinical plus two instrumental ) . statistical analysis was conducted by the mann - whitney u test for nonparametric data ( ucva and bscva ) and by the paired t test for parametric data ( maximum curvature power , symmetry indices and coma values ) . the surgical procedure of corneal cross - linking with riboflavina uva was performed in all patients according to the siena protocol using the vega cbm ( caporossi - baiocchi - mazzotta ) x linker ( cso , florence , italy ) developed in italy at the department of ophthalmology of siena university by the same authors , under intellectual property of siena university , italy . the treatment was conducted under topical anaesthesia ( 4% lidocaine drops ) . after applying the eyelid speculum , a 9 mm diameter marker was used to mark the corneal epithelium in a central circle , then epithelium was removed with a blunt metal spatula . after epithelial scraping , a disposable solution of riboflavin 0.1% and dextrane 20% ( ricrolin sooft , montegiorgio , italy ) was instilled for 10 minutes of corneal soaking before starting uv a irradiation . the riboflavin and dextrane solution was administered every 2.5 minutes for a total of 30 minutes of uva exposure ( 3 mw / cm ) . treated eyes were dressed with a therapeutic soft contact lens bandage for 4 days and medicated with antibiotics ( ofloxacin drops 4 times / day ) , nonsteroidal anti - inflammatory drugs ( diclofenac drops 4 times / day ) and lachrymal substitutes , until contact lens removal . after therapeutic corneal lens removal , fluorometholone 0.2% drops ( 3 times / day ) and lacrimal substitutes were administered for 6 to 8 weeks . ( 1 ) 152 eyes of 105 patients 18 years ( 91 eyes with followup of 12 months , 74 eyes at 24 months , 25 eyes at 36 months , 7 at 48 months ) . ( 2 ) 286 eyes of 243 patients between 19 and 26 years ( 108 eyes with followup 12 months , 83 eyes at 24 months , 56 eyes at 36 months , 11 at 48 months ) . ( 3 ) 78 eyes of 65 patients 27 ( 35 eyes with followup 12 months , 25 eyes at 24 months , 12 eyes at 36 months , 8 at 48 months ) . pre- and postoperative examination included uncorrected visual acuity ( ucva ) , best spectacle corrected visual acuity ( bscva ) , corneal topography and surface aberrometry ( cso eye top , florence , italy ) , optical pachometry ( visante oct , zeiss meditech , jena , germany ) , and in vivo confocal microscopy ( hrt ii , heidelberg , rostock cornea module , germany ) . according to epidemiology findings , we found a male / female ratio in the whole sample of 4 : 1 ; a male / female ratio in paediatric group of 6 : 1 , and a male / female ratio of 3 : 1 in patients between 19 and 40 years old . these results , even if in a small group , differ from epidemiological data reported in the literature were a male / female rate of 2 : 1 is reported . there was no statistical difference in the incidence of keratoconus between right and left eye in the whole population of 516 eyes and age - related groups . comparative ucva in patients 18 years showed a mean gain of + 0.14 ( p = 0.0037 ) , + 0.17 ( p = 0.0043 ) , + 0.16 ( p = 0.0051 ) and + 0.2 ( p = 0.006 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively . patients between 19 and 26 years showed a mean gain of + 0.13 ( p = 0.0034 ) , + 0.16 ( p = 0.0041 ) , + 0.12 ( p = 0.0032 ) , and + 0.14 ( p = 0.0073 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively . patients 27 years showed a mean gain of + 0.08 ( p = 0.0036 ) , + 0.09 ( p = 0.005 ) , + 0.12 ( p = 0.0047 ) and + 0.12 ( p = 0.0071 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively , ( figure 1 ) . comparative bscva in patients 18 years showed a mean gain of + 0.15 ( p = 0.0056 ) , + 0.19 ( p = 0.0031 ) , + 0.18 ( p = 0.0059 ) and + 0.21 ( p = 0.0079 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively . patients between 19 and 26 years showed a mean gain of + 0.10 ( p = 0.0052 ) , + 0.12 ( p = 0.0045 ) , + 0.13 ( p = 0.0056 ) , and + 0.2 ( p = 0.0075 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively . patients 27 years showed a mean gain of + 0.07 ( p = 0.0054 ) , + 0.06 ( p = 0.0067 ) , + 0.08 ( p = 0.0069 ) , and + 0.10 ( p = 0.0075 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively , ( figure 2 ) . k max in paediatric group varied by a mean of 0.7 d ( p = 0.006 ) , 0.8 d ( p = 0.0045 ) , 1.1 d ( p = 0.051 ) , and 0.9 d ( p = 0.071 ) ; in the intermediate group ( patients between 19 and 26 years ) varied by a mean of 0.6 d ( p = 0.0053 ) , 0.5 d ( p = 0.0051 ) , 0.3 d ( p = 0.0045 ) , and 0.6 d ( p = 0.0091 ) ; in adult group ( patients 27 years ) varied by a mean of 0.4 d ( p = 0.0065 ) , 0.6 d ( p = 0.0074 ) , 0.5 d ( p = 0.0095 ) , and 0.5 d ( p = 0.0091 ) , at 12 , 24 , 36 , and 48 months of follow - up , respectively for each group ( figure 3 ) . surface asymmetry index ( sai m ) in paediatric patients improved by a mean value of , 0.42 d ( p = 0.0054 ) , 0.18 d ( p = 0.0066 ) , 0.24 d ( p = 0.091 ) , and 0.10 d ( p = 0.096 ) ; in the intermediate group improved by a mean of , 1.05 d ( p = 0.0032 ) , 1.14 d ( p = 0.0021 ) , 0.84 d ( p = 0.0036 ) , and 0.65 d ( p = 0.076 ) ; in adult group improved by a mean of , 0.52 d ( p = 0.0067 ) , 1.0 d ( p = 0.0077 ) , 0.17 d ( p = 0.0081 ) , and 1.11 d ( p = 0.0094 ) , ( figure 4 ) . topographic superior - inferior symmetry index ( si m ) in paediatric patients varied by a mean of , + 0.3 d ( p = 0.0098),+ 0.6 d ( p = 0.011 ) , + 0.2 d ( p = 0.017 ) , and + 1.5 d ( p = 0.021 ) ; in the intermediate group changed by a mean of , 0.42 d ( p = 0.0086 ) , 0.55 d ( p = 0.0079 ) , + 0.90 d ( p = 0.091 ) , and + 0.40 d ( p = 0.099 ) ; in adult patients varied by a mean of , 0.26 d ( p = 0.0059 ) , 0.21 d ( p = 0.0048 ) , + 1.17 d ( p = 0.012 ) , and 0.21 d ( p = 0.0011 ) at 12 , 24 , 36 , and 48 months of followup , respectively , ( figure 5 ) . coma values in paediatric patients improved by a mean of 0.47 m ( p = 0.0034 ) , 0.52 m ( p = 0.0025 ) , 0.47 m ( p = 0.0022 ) , and 0.45 m ( p = 0.0054 ) ; in the intermediate group coma values decreased by a mean of 0.89 m ( p = 0.0034 ) , 0.96 m ( p = 0.0065 ) , 0.93 m ( p = 0.0074 ) , and 0.91 m ( p = 0.0081 ) ; in old patients we recorded a mean postoperative value of 0.2 m ( p = 0.0056 ) , 0.18 m ( p = 0.0045 ) , 0.21 m ( p = 0.0034 ) , and 0.19 m ( p = 0.0067 ) at 12 , 24 , 36 and 48 months of followup respectively , ( figure 6 ) . according to the literature in and our previous reports , keratoconus progression is more frequent and faster in younger patients under 18 years old at the time of diagnoses , with higher probability to undergo a corneal transplantation [ 1 , 3 ] . therefore paediatric patients represent the goal of photo - induced riboflavin uv a corneal collagen cross - linking . the italian pilot study siena cxl paediatrics , conducted on a large cohort of patients with a long - term follow up , demonstrated that there was a significant and fast functional improvement in younger patients after riboflavin uv a corneal cross - linking . as we recently published , it is however impossible to exactly predict the distribution of cross - links and the geometric redistribution of newly formed collagen [ 12 , 14 ] . long - term comparative analysis showed that functional results after riboflavin uv a corneal collagen cross - linking among paediatric patients were slightly better , but without statistically significant differences with the results recorded in the intermediate group patients . on the other hand , patients over 27 years showed a positive but poorer functional response compared with other age groups . the mean kmax variation and topographic surface asymmetry index results were statistically significant in the paediatric sample , particularly in the postoperative 24 months . after the 24th month , until the 48th month , the mean data results were statistically not significant , reasonably due to reduced number of patients in the longitudinal analysis . the comparative aberrometric data of coma values showed a significant improvement in all analyzed groups , justifying the rapid improvement of visual acuity in all treated patients . in the paediatric group of our cohort , there was a minority of patients ( about 5% ) that , despite the treatment , showed a worsening trend or at least an instability of keratoconus . in our opinion , this concept should be remarked because the disease in this age group is more aggressive and the possibility of progression higher than in the others age groups . the instability of certain cases should be explained by the different genetic patterns of keratoconus [ 1719 ] with relative biochemical modifications potentially occurring in corneal stroma associated with negative influences of some environmental factors ( allergy , atopy ) [ 2124 ] . every time we decide to treat a paediatric patient under 18 years affected by progressive keratoconus , parents and patient himself should be well informed about the possibility that the treatment in a minority of cases could not warrant a total and long - lasting stabilization of the disease , with the possibility to repeat the cross - linking or to undergo alternative treatments . pilot study demonstrated the effective ability of corneal cross - linking to retard keratoconus progression in all age groups with better functional response in patients under 26 years . treatment ensured a long - term keratoconus stabilization in over 90% of treated cases . the lower functional response observed in patients over 27 years plasticity in the adult age , as well demonstrated in the literature [ 25 , 26 ] . cross - linking treatment may result in less effectiveness with increased failure and complication rate particularly in adult patients over 35 years , as reported in the literature . according to our long - term comparative age - related analysis and results , the standard riboflavin uv a cross - linking with epithelium removal should be the first choice therapy of progressive keratoconus in paediatric and under-26-year old patients with corneal thickness at least of 400 m in the thinnest point .

acute generalized exanthematous pustulosis ( agep ) is a rare cutaneous rash characterized by widespread sterile nonfollicular pustules . cefepime is a fourth generation cephalosporin , used to treat severe infections . a 67-year - old man was admitted with acute gastroenterocolitis . on the seventh day , the patient developed a nosocomial pneumonia and cefepime was initiated . on the fourth day of cephalosporin treatment , he presented with a maculopapular , pruritic eruption affecting the face , neck , abdomen and limbs . one day later he developed disseminated pustular lesions and his temperature was 37c . laboratory analysis evidenced leukocytosis and skin biopsy showed subcorneal pustule , edema in the papillary dermis , perivascular inflammatory infiltrate consisting of neutrophils , leukocytoclasia and red cell extravasation in the epidermis . cefepime was suspended and within 4 days the non - follicular pustules cleared following a desquamation . agep is a disease attributed to a variety of causes , but in 90% of the cases it is due to an adverse drug reaction . antibiotics are implicated in 80% of these cases , mostly penicillins and macrolides . there are few cases associated with cephalosporins . it is very important to consider agep in cases of acute pustular rashes and drugs should be investigated as causative agents . purpose . to report a comparative prospective long - term functional analysis after riboflavin uv a corneal cross - linking ( cxl ) in three different age groups of patients affected by progressive keratoconus ( kc ) . methods . functional analysis comprised paediatric patients ( 18 years ) included 152 eyes ( 29.5% ) ; intermediate group ( 1926 years ) 286 eyes ( 55.4% ) , and adults ( 27 years ) 78 eyes ( 15.1% ) . cxl was performed according to the siena protocol by using the vega cbm ( caporossi - baiocchi - mazzotta ) x linker ( cso , florence , italy ) at siena university by the same authors . pre- and post - op examinations included ucva , bscva , corneal topography , and surface aberrometry ( cso eye top , florence , italy ) , at 48 months followup . results . at 48 months followup paediatrics , intermediate , and adult patients showed a mean gain in ucva of + 0.2 , + 0.14 and + 0.12 snellen lines . bscva gained by a mean of + 0.21 , + 0.2 , and + 0.1 snellen lines . kmax was reduced by a mean value of 0.9 d , 0.6 d , and 0.5 d , respectively . coma values improved by a mean of 0.45 m , 0.91 m , and 0.19 m , respectively . treatment ensured a long - term keratoconus stabilization in over 90% of treated patients . conclusion . according to our long - term comparative results , epithelium - off riboflavin uv a cross - linking should be the first choice therapy of progressive kc , particularly in paediatric age and patients under 26 years .

rufinamide ( ruf ) is a triazole derivative that is structurally unrelated to other antiepileptic drugs ( aed).1 it was approved by the united states , food and drug administration ( fda ) in 2004 for the treatment of lennox - gastaut syndrome ( lgs ) in patients aged 4 years and older.2 ruf was authorized for the same indication in europe in january 2007.2,3 the mechanism of action involves limiting the firing of excessive sodium - dependent action potentials.1 ruf has been reported to reduce the number of drop attacks and major motor seizure in about 60% of patients with lgs and has subsequently been regarded as an effective adjunctive therapeutic agent.2 in recent years , studies have been reported that ruf is also efficacious and well tolerated in the treatment of various epilepsy syndromes other than lgs , including cases of refractory epilepsy in pediatric patients.3 however , there are still limited data regarding the long term treatment results of ruf for pediatric refractory epilepsy . the aim of this study was to delineate the long term efficacy and tolerability of ruf in children and infants with intractable epilepsies . we performed a retrospective study for the patients who met the following inclusion criteria ; ( 1 ) who were followed up for more than one year at samsung medical center since the beginning of the ruf use , at the time of 31 aug 2011 , ( 2 ) who were less than 19 years of age at the time of the start of ruf treatment , ( 3 ) who were classified as intractable epilepsy , meaning presence of persistent seizures despite the use of more than three antiepileptic drugs , and ( 4 ) whose data on the clinical characteristics and seizure outcome were available . the data concerning demography , clinical characteristics , seizure - related characteristics , laboratory works including electroencephalography ( eeg ) and brain magnetic resonance imaging ( mri ) , and treatment outcome were collected . we conformed to the classification of the international league against epilepsy ( ilae ) in 1981 and 1989 in classifying the seizure and epilepsy of the patients.4,5 the efficacy of ruf was evaluated by the response rate , by comparing the seizure frequencies at the baseline and in the last three months . patients who showed seizure reduction of more than 50% in frequency was defined as responders . the retention rate was another index of efficacy and was defined by the portion of patients who continued with ruf treatment for one year . tolerability was evaluated by the presence of side effects . additionally , we investigated the influencing factors for the response rate or retention rate . chicago , il , usa ) was used in the analysis of nonparametric measures and statistical evaluation was performed by means of chi - square test . thirty seven patients ( 27 male , 10 female ) were included in this study . the etiology of epilepsy , epilepsy syndrome classification , type of seizures , number of prior and concurrent aeds and other treatment are presented on table 1 and 2 . the patients were taking many concurrent aeds ( mean=3.9 , range : 18 ) ; valproic acid ( n=26 ) , clobazam ( n=19 ) , topiramate ( n=15 ) , levetiracetam ( n=13 ) , lamotrigine ( n=13 ) , etc . initial starting dose of ruf was mean 7.8 ( 2.631.5 ) mg / kg / day , and the final maintenance dose was mean 31.4 ( 6.165.6 ) mg / kg / day . the mean duration of ruf therapy was 10.52.73 months and ruf was discontinued in 17 of 37 patients after a mean period of 5.6 ( 0.615.7 ) months . thirty seven patients ( 27 male , 10 female ) were included in this study . the etiology of epilepsy , epilepsy syndrome classification , type of seizures , number of prior and concurrent aeds and other treatment are presented on table 1 and 2 . the patients were taking many concurrent aeds ( mean=3.9 , range : 18 ) ; valproic acid ( n=26 ) , clobazam ( n=19 ) , topiramate ( n=15 ) , levetiracetam ( n=13 ) , lamotrigine ( n=13 ) , etc . initial starting dose of ruf was mean 7.8 ( 2.631.5 ) mg / kg / day , and the final maintenance dose was mean 31.4 ( 6.165.6 ) mg / kg / day . the mean duration of ruf therapy was 10.52.73 months and ruf was discontinued in 17 of 37 patients after a mean period of 5.6 ( 0.615.7 ) months . the overall response rate of ruf was 21.6% . the change of seizure frequency according to etiology response rates in patients with the atonic seizure type were higher than other seizure types but there were no statistical difference ( table 3 ) . the overall retention rate of ruf at one year was 54% ( 20/37 patients ) . the reasons of discontinuation were ineffectiveness ( n=11 , 64.7% ) , adverse effects ( n=3 , 17.6% ) , both ineffectiveness and adverse effects ( n=1 , 9.8% ) , and increased seizure frequency ( n=2 , 11.7% ) . the reasons for retention were as follows : responders ( n=8 , including two seizure free patients , including additive effect with other aeds < n=2 > ) , good initial response ( n=7 ) , improved cognitive function by parent s record ( n=2 ) , ruf being more effective than other previous aeds ( n=3 ) . of non - responders ( n=12 , 60% ) , seven patients showed a good initial response to ruf and two patients improved their cognitive abilities and increased their appropriateness by parent s record . the rest of three non - responders were able to decrease other aeds due to adjunctive treatment with ruf . two patients , one with seizure free outcome and the other with 50% in seizure reduction , were presumed to have additive effect with other aeds . furthermore , five patients showed great improvement in specific types of seizures ( atonic seizure type < n=2 > , generalized tonic seizure type < n=1 > , both < n=2 > ) . adverse effects were reported in ten patients ( 27% ) : insomnia ( n=3 ) , loss of appetite ( n=3 ) , somnolence ( n=2 ) , irritability ( n=2 ) , vomiting ( n=1 ) , and dizziness ( n=1 ) . four patients discontinued ruf due to adverse effects ( vomiting , insomnia , dizziness , loss of appetite , respectively ) and six patients continued with ruf . the overall response rate of ruf was 21.6% . the change of seizure frequency according to etiology response rates in patients with the atonic seizure type were higher than other seizure types but there were no statistical difference ( table 3 ) . the overall retention rate of ruf at one year was 54% ( 20/37 patients ) . the reasons of discontinuation were ineffectiveness ( n=11 , 64.7% ) , adverse effects ( n=3 , 17.6% ) , both ineffectiveness and adverse effects ( n=1 , 9.8% ) , and increased seizure frequency ( n=2 , 11.7% ) . the reasons for retention were as follows : responders ( n=8 , including two seizure free patients , including additive effect with other aeds < n=2 > ) , good initial response ( n=7 ) , improved cognitive function by parent s record ( n=2 ) , ruf being more effective than other previous aeds ( n=3 ) . of non - responders ( n=12 , 60% ) , seven patients showed a good initial response to ruf and two patients improved their cognitive abilities and increased their appropriateness by parent s record . the rest of three non - responders were able to decrease other aeds due to adjunctive treatment with ruf . two patients , one with seizure free outcome and the other with 50% in seizure reduction , were presumed to have additive effect with other aeds . furthermore , five patients showed great improvement in specific types of seizures ( atonic seizure type < n=2 > , generalized tonic seizure type < n=1 > , both < n=2 > ) . adverse effects were reported in ten patients ( 27% ) : insomnia ( n=3 ) , loss of appetite ( n=3 ) , somnolence ( n=2 ) , irritability ( n=2 ) , vomiting ( n=1 ) , and dizziness ( n=1 ) . four patients discontinued ruf due to adverse effects ( vomiting , insomnia , dizziness , loss of appetite , respectively ) and six patients continued with ruf . ruf has established its efficacy in lgs and has been approved as an adjunctive treatment for patients older than four years of age with lgs.6 in previous studies , ruf was also efficacious and well tolerated in treatment of various epilepsy syndromes other than lgs , including cases of refractory epilepsy in pediatric patients.3 however , the data about the efficacy of ruf in epilepsies other than lgs are limited.3,7 in this study , ruf showed a response rate of 21.6% in patients with lgs , intractable ge or lre . the retention rate at one year was 54% and a fourth of the patients showed various adverse effects . the response rate of ruf in patients with refractory epilepsies including lgs was reported to be 26.746.7%.1,3,7,8 those studies included both child and adult patients and the mean age ranged from 1 to 50 years . according to the study of kluger et al,7 which was conducted in 60 patients ( 45 children and 15 adults ) treated with ruf over the mean period of 14.5 months with inadequately controlled epilepsy syndromes ( range : 318 months ) , the overall response rate was 26.7% . in another study , the patients with refractory epilepsies showed the response rate of 46% at 21 weeks of ruf therapy.8 this study included pediatric patients with lgs or other intractable epilepsy and the mean age of the patients was 9.5 years in the range of 120 years . the response rate of the present study was 21.6% and it is slightly lower than previous studies mentioned above . this difference may result from the intractability of study population including the patients with lgs and highly intractable generalized epilepsy and localization related epilepsy . at the time of introduction of ruf , the patients had taken a mean 7.2 ( range : 413 ) aeds , which was much higher than the studies of kluger et al . and joseph et al.7,8 in our study , patients with lgs showed a slightly higher response rate of 30% than patients with other intractable ge or lre . in one prospective study with 43 patients with lgs , the overall response rate of ruf at 12 months was 60.5% without worsening of seizures.2 in another study involving children and young adults with lgs , the response rate was 54.8%.3 in those studies , patients with lgs took a large part of the total number of patients compared to our study group . due to such reason , response rate in other studies were higher than the present study . however , ruf was found to be more efficacious in patients with lgs than those with intractable ge or lre . as for seizure types , the highest response rate was shown in atonic seizure ( 27.8% ) . according to previous data , atonic seizure and tonic seizure were the most improved types of seizure by ruf as adjunctive therapy ( 63.6% and 48.6% , respectively).1,9 also , in a long - term , open - label extension study , 124 patients with lgs ( aged 437 years ) were treated with ruf for a median of 432 days . during the treatment , 47.9% of patients had 50% reduction in the frequency of tonic - atonic seizures.6 based on previous studies , ruf appeared to be an effective adjunctive therapy for intractable atonic seizures . in our study , partial seizure responded equally to ruf , and a similar result was reported in other studies.3,7 retention rate is known as an indicator for evaluating both efficacy and adverse effects.10 according to one previous study , the retention rate of ruf at 14.5 months was 41.7%,7 which is consistent with the present study . in this study , the reasons for retention varied and 60% of them were non - responders ( less than 50% in seizure reduction ) . the reasons for retention in those non - responders were as follows : excellent initial response , definite cognitive improvement by parent s record and ruf being more effective than other previous aeds . coppola et al1 reported early effectiveness of ruf in refractory childhood epileptic encephalopathy patients with or without vpa . similarly , in our study , seven patients showed a good initial response to ruf during titration . in our study , two patients reported noticeable improvement in cognitive function even though seizure reduction was less than 50% . one study suggested that ruf has a favorable cognitive profile , increasing its appropriateness for many patients , especially those with cognitive impairment . their cognitive ability was rather improved in the seizure free - state and no worsening had been reported.11 in our study , 27% of patients reported a mild to moderate degree of adverse effects . the most commonly observed adverse events were insomnia ( n=3 ) , loss of appetite ( n=3 ) somnolence ( n=2 ) , irritability ( n=2 ) , vomiting and dizziness . none of previous studies have reported life - threatening adverse effects.3,7,8 the previous studies reported that vomiting and drowsiness were major symptoms of adverse effects.3,12 in our study , most adverse events , such as insomnia and loss of appetite , were mild to moderate in severity . children with lgs and other intractable epilepsy syndromes showed 21.6% response rate and 54% retention rate during ruf treatment . also , 27% of children showed adverse effects of ruf and 10.8% children stopped ruf due to adverse effects . the present data suggest that ruf provides good efficacy and tolerability in pediatric patients with refractory generalized or localization related epilepsy syndromes as well as lgs . cluster headache is a form of primary neurovascular headache , which consists of unilateral head pain that occurs in association with cranial autonomic features and , in most patients , has a striking circannual and circadian periodicity . excruciating painful attacks are accompanied by restlessness or agitation , usually last less than 3 h , and occur in bouts for a few months during which the patient has one or more crisis per day . the international headache society defines cluster headache as attacks of severe , strictly unilateral pain which is orbital , supraorbital and temporal or in any combination of these sites , lasting for 15180 min and occurring from once every other day to 8 times a day . the attacks are associated with one or more of the following , all of which are ipsilateral : conjunctival injection , lacrimation , nasal congestion , rhinorrhoea , forehead and facial sweating , miosis , ptosis , eyelid oedema . epidemiological data for the general population are scarce ; only five studies have been carried out until now on cluster headache prevalence in the general population , with conflicting results ( prevalence rates vary between 56 and 326 cases in every 100,000 inhabitants ) . a recent study on a sample representative of the italian general population aged over 14 years , reported an estimated prevalence rate of 279/100,000 ( 95% ci : 173427 ) , 227/100,000 ( 95% ci : 104431 ) in women and 338/100,000 ( 95% ci : 175592 ) in men . cataract is clouding ( opacity ) of the lens of the eye that causes a progressive , painless loss of vision . the first symptom of cataract is usually blurred vision . other symptoms that may progressively appear are : glare , halos , double vision and perception of colours as more yellow and less vibrant . three different types of cataract can be identified on a histological basis : nuclear , cortical and posterior subcapsular . investigations [ 58 ] on possible risk factors for the development of cataract , showed positive correlation with myopia , diabetes , smoking [ 911 ] , use of systemic corticosteroids , exposition to uv - b , and other environmental factors , to which has to be added genetic predisposition . however , it should be underlined that different degrees of correlation between distinct risk factors and the three types of cataract have been reported [ 6 , 15 ] . prevalence studies of age - related cataracts are hampered by the absence of a uniform grading system for cataract opacities , by differing definitions of visual impairment , and by additional coexisting ocular pathologies causing loss of vision . nonetheless , the framingham eye study in 1977 reported that the proportion of people with age - related cataracts causing loss of vision of 20/30 ( 6/9 ) or worse was 15.5% for all ages and 45.9% for those older than 75 years . in the beaver dam eye study in 1992 , using a similar definition of loss of vision reported proportions were 38.8% for men and 45.9% for women older than 74 years . however , it is not clear if variations in frequency reflect methodological diversity or true differences between populations . interestingly , most of the cited studies analyzed the cataract prevalence in age groups which are substantially different from those of our two cases . one single study reported the prevalence of cataract at a younger age ( 40 years and older ) with identified risk factors for the three different histological types of cataract . this study showed age - specific rates of the different histological cataract type , by stratifying the population sample by age . prevalence of 1% ( cortical type ) , of 0.2% ( nuclear type ) and of 2.0% ( posterior subcapsular ) was reported in individuals between 40 and 49 years and a prevalence of 3.9% ( cortical type ) of 0.2% ( nuclear type ) and of 2.6% ( posterior subcapsular ) was reported in individuals between 50 and 59 years . here , we report two cases that both developed cataract before the age of 50 years on the side affected from the cluster headache and reviewed published similar cases and potential mechanisms for comorbidity . we present a 44-year - old man with a history of cluster headache , diagnosed in accordance with ihs guidelines , since he was 21 years old . daily pain attacks , lasting about 90 min , were localized to the left periorbital area with ipsilateral lacrimation and rinorrhea . the cluster headache that initially occurred sporadically , from the age of 43 years became chronic . at the age of 39 , the patient was diagnosed of cataract on the left eye that was in the same year treated by surgery . the sole risk factor for developing cataract at anamnesis was smoking habit ( 30 cigarettes / day ) . a detailed pharmacological anamnesis excluded any kind of exposure to prolonged treatment with steroid drugs . the second case was a 53-year - old man who experienced his first cluster headache episode when he was 44 years old . cluster periods were characterized by 34 attack / day of severe right - sided pain localized to the periorbital region , lasting about 45 min and associated to ipsilateral lacrimation . the reported risk factor for developing cataract was smoking habit ( 10 cigarettes / day ) . also in this case , there was no report of chronic treatment with steroid drugs . references were identified by searches of pubmed from 1966 until october 2007 with the terms cluster headache and cataract . our search in pubmed shows only one report of a patient with cluster - like headache , which began after surgical removal of the crystalline for cataract and intraocular lens implant . therefore , to our knowledge , this is the first report of cataract occurring in cluster headache patients and , interestingly , on the same eye affected by the pain attack . it is of interest that both patients developed cataract in a young age , after suffering for years from chronic cluster headache attacks and in the absence of main risk factors for the development of early onset cataract , including prolonged steroid drugs exposure . it is noteworthy that the two patients , although not relative , had the same , rather uncommon , last name that we found to be specifically represented in a defined area of north - eastern italy . however , because we could not investigate the pedigree of one patient , any hypothetical genetic link between the uncommon occurrence of early onset cataract and cluster headache can not be proposed . in addition , it was not possible to perform in either of the two patients any investigation on ocular inflammation . thus , no speculation can be made on a possible causal relationship between repeated episodes of pain , inflammation and autonomic abnormalities that are considered to contribute to the cluster headache attack , and the precocious occurrence of the cataract . however , this first evidence of the occurrence of cataract in relatively young adult men with a history of chronic cluster headache is of importance , because underlining this uncommon type of comorbidity may favour the report of additional similar cases .

background and purpose : rufinamide ( ruf ) is a novel antiepileptic drug ( aed ) and its efficacy has been proven in lennox - gastaut syndrome ( lgs ) . however , there is a lack of data regarding the efficacy in pediatric intractable epilepsies other than lgs . the purpose of the study was to explore the efficacy and tolerability of ruf in pediatric patients with intractable epilepsies as well as lgs.methods:this retrospective observation study was conducted in samsung medical center from august 2010 to september 2011 . thirty seven patients ( 27 males , 10 females , aged between 1.8 and 18.4 years ) , with refractory epilepsies or lgs were treated with ruf as an adjunctive drug . efficacy was represented by the response rate and retention rate over the study period . tolerability was measured as the number of patients who showed adverse effects.results:the overall response rate was 21.6% during the 12 months of the study period with 5.4% of seizure - free patients . the retention rate was 54% and ineffectiveness was the most common reason for discontinuation of ruf . the most common adverse effects were insomnia and somnolence.conclusions:ruf may be considered to be an efficacious and safe aed for pediatric patients with intractable epilepsies as well as lgs . cluster headache ( ch ) consists of attacks of severe , unilateral orbital / supraorbital / temporal pain , lasting for 15180 min , occurring once or more times a day , and associated with ipsilateral conjunctival injection , lacrimation and other symptoms . cataract is clouding of the lens of the eye causing a progressive and painless loss of vision . we describe the cases of two men ( not relative , but with the same last name , which originates from north - eastern italy ) that in young adult age , after years of suffering from chronic ch , developed cataract on the same side of the pain attacks . patient 1 was diagnosed as having cataract 18 years after the onset of episodic ( and subsequently chronic ) ch . patient 2 began suffering from chronic ch at the age of 44 years and after 8 years he developed cataract . this is the first report of cataract in patient suffering of ch and occurring in the eye affected by the pain attack .

obesity is not only a risk factor for cancer development but also a factor affecting treatment outcomes . endometrial cancer ( ec ) was the first malignancy to be recognized as related to obesity . it has been associated with lower quality of life values among ec survivors and higher body mass index ( bmi ) , measured before ec diagnosis , has been associated with higher all - cause and endometrial cancer specific mortality . early stage ec treatment involves surgery ( total abdominal hysterectomy and bilateral salpingo - oopherectomy with or without pelvic lymph node dissection ) followed by adjuvant radiotherapy in selected cases . randomized studies have shown that radiotherapy ( rt ) reduces the risk of pelvic relapse . vaginal cuff is the most common site of relapse and the postoperative radiation therapy for endometrial carcinoma 2 ( portec-2 ) trial demonstrated that patients with intermediate - risk ec can be treated safely with postoperative vaginal cuff brachytherapy ( vcb ) in the absence of whole pelvic external beam radiotherapy , thus decreasing toxicity . although vcb is one most commonly used adjuvant gynaecological treatments , there is a lack of studies analysing whether dosimetric factors are influenced by overweight . our aim , therefore , was to evaluate the effect of bmi and perivaginal fat on dose in organs at risk during fractionated vcb . retrospective analysis of 220 consecutive brachytherapy fractions derived from 59 patients who underwent postoperative vcb for gynecological cancer was conducted . fiftysix patients underwent vcb due to endometrial cancer and three patients due to cervical cancer . twenty - six endometrial cancer patients underwent postoperative vcb alone using six fractions ; the remaining patients were treated with whole pelvis external beam radiotherapy ( wprt ) followed by three or four vcb fractions ( 15 patients underwent each fractionation ) . patients were assessed weekly during treatment and every 4 or 6 months afterwards according to the clinical stage . brachytherapy was performed with the largest diameter cylinder that could fit comfortably into the vaginal vault ; and the cylinders were positioned to remain parallel to the cranio - caudal axis of the patient . no instructions were given prior to the vcb procedure apart from the need to evacuate prior to coming to the hospital . all patients underwent a ct planning scan at every application , with 2-mm thick slice at 2-mm overlapping intervals in the supine position using a foley bladder catheter instilling dilute contrast medium ( 5 ml of omnipaque350 [ ge healthcare bio - sciences , madrid , spain ] into 45 ml of saline solution ) in order to increase bladder visibility during segmentation and volume reproducibility during treatment . cts were transferred to a 3d treatment planning system ( oncentra v.4.1 , nucletron , an elekta company , elekta ab , stockholm , sweden ) . to improve comparisons , the same physician re - contoured and re - planned every image set under the same conditions for an iridium-192 remote afterloading unit ( microhdr nucletron , nucletron , an elekta company ) , regardless of the treatment administered . the entire bladder volume was delineated , and the rectum was defined from 1 cm above the cylinder tip to 1.5 cm below the last activated source dwell position . an active length of 2.5 cm was used to deliver a fraction dose of 5 gy at 5 mm depth to the vaginal surface . d0.1cc , d1cc , and d2cc for bladder and rectum were assessed from dvhs for each fraction . the angle of the vaginal cylinder applicator related to the horizontal plane parallel to the craneo - caudal patient axis was calculated , positive values indicating a tip directed to the bladder and negative values indicating a posterior displacement of the cylinder tip toward the rectum . cylinder size and rectum and bladder volumes were also noted . the bmi formula was weight in kilograms divided by the square of the height in metres . categories were underweight < 18.5 kg / m ; normal 18.5 - 24.9 kg / mm ; overweight 25.0 - 29.9 kg / mm ; obese i 30.0 - 34.9 kg / mm ; obese ii 35.0 - 39.9 kg / mm ; and obese iii > 40.0 kg / mm . we considered fat that was confined within the 100% isodose on the first ct planning scan as the perivaginal fat . it was segmented by thresholding on the hounsfield units ( hu ; 190 to 30 hu ) and the volume was recorded . perivaginal fat analysis was performed only in the first ct planning scan because the most common practice for vcb is to perform only one ct and to translate this plan to the next fractions . patient mean dose - volume metrics , volumes , and cylinder angle were calculated from the different fractions . chi - square tests , kruskal - wallis analysis , and univariate regression analysis were performed . a stepwise multiple regression analysis was used to model organs at risk ( oars ) dvh parameters as a function of other variables ( the significance levels for addition to and removal from the model were set at 0.05 and 0.10 , respectively ) . all patients underwent a ct planning scan at every application , with 2-mm thick slice at 2-mm overlapping intervals in the supine position using a foley bladder catheter instilling dilute contrast medium ( 5 ml of omnipaque350 [ ge healthcare bio - sciences , madrid , spain ] into 45 ml of saline solution ) in order to increase bladder visibility during segmentation and volume reproducibility during treatment . cts were transferred to a 3d treatment planning system ( oncentra v.4.1 , nucletron , an elekta company , elekta ab , stockholm , sweden ) . to improve comparisons , the same physician re - contoured and re - planned every image set under the same conditions for an iridium-192 remote afterloading unit ( microhdr nucletron , nucletron , an elekta company ) , regardless of the treatment administered . the entire bladder volume was delineated , and the rectum was defined from 1 cm above the cylinder tip to 1.5 cm below the last activated source dwell position . an active length of 2.5 cm was used to deliver a fraction dose of 5 gy at 5 mm depth to the vaginal surface . d0.1cc , d1cc , and d2cc for bladder and rectum were assessed from dvhs for each fraction . the angle of the vaginal cylinder applicator related to the horizontal plane parallel to the craneo - caudal patient axis was calculated , positive values indicating a tip directed to the bladder and negative values indicating a posterior displacement of the cylinder tip toward the rectum . cylinder size and rectum and bladder volumes were also noted . the bmi formula was weight in kilograms divided by the square of the height in metres . categories were underweight < 18.5 kg / m ; normal 18.5 - 24.9 kg / mm ; overweight 25.0 - 29.9 kg / mm ; obese i 30.0 - 34.9 kg / mm ; obese ii 35.0 - 39.9 kg / mm ; and obese iii > 40.0 kg / mm . we considered fat that was confined within the 100% isodose on the first ct planning scan as the perivaginal fat . it was segmented by thresholding on the hounsfield units ( hu ; 190 to 30 hu ) and the volume was recorded . perivaginal fat analysis was performed only in the first ct planning scan because the most common practice for vcb is to perform only one ct and to translate this plan to the next fractions . patient mean dose - volume metrics , volumes , and cylinder angle were calculated from the different fractions . chi - square tests , kruskal - wallis analysis , and univariate regression analysis were performed . a stepwise multiple regression analysis was used to model organs at risk ( oars ) dvh parameters as a function of other variables ( the significance levels for addition to and removal from the model were set at 0.05 and 0.10 , respectively ) . a mean of 3.7 vcb fractions per patient were retrieved . according to the who bmi classification , 6.8% of patients were classified as normal , 35.6% as overweight , and 57.6% as obese i. there were no severely obese patients ( class ii or iii ) . the median age ( iqr ) and bmi for the overall group was 64.3 years ( 18.4 ) and 32 years ( 9.5 ) , respectively . median bmi values for every who classification were 22 , 27.8 , and 36.6 for normal - weight , overweight , and obese i patients , respectively . patients were significantly older as the bmi class increased ( kruskal - wallis test , p=0.043 ) . the fact that obese patients were treated with larger cylinder applicators ( 82.4% ) than patients in the normal range ( 25% ) ( chi - square test , p=0.037 ) is significant . a positive , significant correlation was observed between bmi and perivaginal fat volume ( r=0.556 , p=0.001 ) ( fig . bmi also showed positive correlation with age ( r=0.272 , p=0.037 ) and negative correlation with the bladder dvh metrics ( d0.1cc , r=0.366 , p=0.004 ; d1cc , r=0.454 , p=0.001 ; d2cc , r= 0.451 , p < 0.001 ) a low positive correlation was observed between the perivaginal fat volume and cylinder angle ( r=0.288 , p=0.027 ) , vaginal cylinders tilted towards the bladder as perivaginal fat volume increased ( fig . 2 ) . univariate regression showed no significant association between bladder dose metrics and cylinder angle . multivariate regression was performed to determine the effect of variables analysed on the bladder dvh metrics ( table 3 ) . age of patients and the use of a 3.5 cm cylinder diameter showed significance for d2cc . when a reanalysis was performed , replacing bmi with perivaginal fat volume , models were unable to retain the perivaginal fat volume among the predictors . patients developed grade 1 or 2 acute toxicity , no higher acute toxicity levels were observed . late toxicity was uncommon , only one patient developed g3 bladder toxicity and two patients developed late rectal toxicity , one of them g1 and the other g2 . the main finding from this study is that higher bmi values showed an association with lower bladder dose parameters but not with rectal dose . adipose tissues are commonly classified as subcutaneous or visceral fat , the latter being associated with metabolic disease . visceral fat located in the periprostatic area has been associated with prostate cancer aggressiveness and its volume showed direct correlation with bmi . a reduction in rectal doses among patients with higher bmi during prostate brachytherapy this dose reduction is considered to be a result of the inverse square law due to the increase in fatty tissue at the prostate - rectum interface . our results , from an analysis of a large sample and a high number of brachytherapy insertions , confirm those of a recent study that reported higher bladder doses in women with lower bmi during vcb . a positive , significant correlation was observed between bmi and perivaginal fat volume ( r=0.5561 , p=0.001 ) ( fig . unfortunately , due to the limited field of view ( fov ) used during ct scan acquisition , we were unable to determine whether correlation existed with visceral fat . the small fov left some part of the visceral fat outside the scanning area precluding this analysis . our results do not support an increase in perivaginal fat or application of the inverse square law as the main cause of lower doses in oars , due to the inability of perivaginal fat to remain in the multivariate regression models . if that was the main explanation , rectal doses would also decrease with higher perivaginal fat volumes . obesity class i patients are strongly associated ( hazard ratio , 3.93 ) with ec , but no association with stage or grade of disease was found in the large women s health initiative study . the relative risk of death from cancer among women with a bmi of 32 or higher was 2.1 compared with the risk for women with bmi < 19 . although obesity is described as a risk factor for adverse outcomes after treatment for many malignancies , the effect of bmi on ec treatment outcomes has been a subject of debate . while analysis of the national institutes of health - aarp diet and health study demonstrated an increased risk of overall and disease - specific mortality among women with higher pre - diagnosed bmi , the analysis of the gog 99 study data associated obesity with higher mortality from causes other than ec but not disease recurrence . the analysis of the astec trial failed to find an association of bmi with a reduced post - treatment survival . a recent systematic review did not suggest an association between bmi and the recurrence of cancer . vaginal brachytherapy ( vbt ) has been one of the main adjuvant treatments for ec since the results of randomized trials that showed vbt was not inferior to pelvic irradiation in high intermediate risk patients , and its use is extended after external beam radiotherapy in high risk patients . there have been concerns regarding the methods for delivery of vbt , whether to use a single plan approach or a customized plan at every fraction . the first approach requires some level of homogeneity between insertions because several variables can affect dose deposition to oars [ 17 - 20 ] . our data are not influenced by bladder filling due to the routinely placed bladder catheter . perivaginal fat volume was associated with an anterior cylinder tilt which , paradoxically , did not go with higher bladder dose ; therefore , we believe that anatomical visceral fat distribution could be the cause of differences in bladder dose according to bmi ( fig . a limitation of our study is the lack of a complete pelvic visceral fat segmentation , which was not possible due to the limited pelvic volume scanned in some insertions . strong correlation was observed between perivaginal fat volume , which was used as a surrogate for visceral fat volume , and bmi . this measure has not been validated , abdominal adiposity at the umbilicus level being the usual measure of visceral and subcutaneous fat . to study these relationships the fact that there were no severe or extremely obese patients ( bmi obese class ii and iii ) in this report is noteworthy , and reflects the bmi distribution among the general population in mediterranean countries compared with the population in the united states . in addition , the population attributable fraction is greater for the us population ( 56.8% ) than for the european population ( 45.2% ) . population differences in bmi obese classes ii or iii could modify these results . although the study did not focus on clinical results , we believe that is important to know , as much as possible , which variables influence the absorbed doses to organs at risk . this knowledge is more important in a clinical setting where only the first vbt application is planned and the remaining are assumed to be similar due to a fixed geometry , according to the american brachytherapy society consensus . in conclusion , the results reported in this study demonstrate a significant inverse correlation between bmi and bladder dose deposition during vcb . the impact of these findings on late toxicity needs to be evaluated in clinical practice . hepatitis b virus ( hbv ) is an important risk factor for developing hepatocellular carcinoma ( hcc ) ( 1 ) . hcc is the fifth most common cancer worldwide , and it causes significant public health problems , especially in association with chronic hepatitis b ( 2 ) . morphologic lesions during hepatocarcinogenesis include dysplastic lesions ( dysplastic foci ( df ) and dysplastic nodules ( dns ) ) with low- and high - grade dysplasia and small cancerous lesions ( 2 cm in diameter ; early hcc ) ( 4 - 7 ) . the most common cause of cirrhosis in shiraz transplant center , which is the largest liver transplant center in the country , is hbv related , and majority of patients with hcc in this center are also hbv related ( 1 ) . therefore , we attempted to determine the incidence of hcc and its precursors in this group of cirrhotic patients through a thorough examination of explanted hbv - related cirrhotic livers . a two - year cross - sectional study was performed on 103 explanted hbv - related cirrhotic livers from the pathology department of shiraz university of medical sciences . during the study period ( 2014 - 2015 ) , explanted cirrhotic hbv - related livers ( recipient cirrhotic liver after liver transplantation ) received in the department of pathology were fixed in formalin for 24 - 48 hours . histologic sections from any hepatic nodule larger than 1 cm in diameter or those with any kind of difference with background parenchyma , such as color or consistency , were studied ( figures 1 and 2 ) . sections were stained with hematoxylin and eosin method , and slides were examined under a double - headed microscope by two pathologists . df were classified into having either small cell changes ( scc ) , formerly called small cell dysplasia , or large cell changes ( lcc ) , formerly called large cell dysplasia . nodules were studied and classified as low - grade dysplastic nodules ( lgdn ) , high - grade dysplastic nodules ( hgdn ) , and hepatocellular carcinoma ( hcc ) ( 5 ) . the following criteria were used to classify the different nodules in cirrhotic livers : cellularity , thickness of hepatocellular plates , nuclear atypia ( either pleomorphism or irregular contour ) , nucleocytoplasmatic ratio , cytoplasmic staining ( eosinophilic , basophilic , amphophilic , and clear ) , pseudoacinar pattern , stromal invasion , portal tracts , biliary pigment , lipid vacuoles , and iron status of nodules ( 8) . in lgdn , hepatocytes are not completely abnormal . that is , they show normal or slightly increased nucleocytoplasmic ( n / c ) ratio , minimal nuclear atypia , and no mitotic activity , but portal tracts are still present . compared with lgdn , hgdn have all of their features except the n / c ratio is higher , nuclear atypia is more obvious , cytoplasmic basophilia is more significant , and liver plates ( more than two cells thick ) are thickened with mitotic figures . in hcc , cell size is usually decreased , nuclear density is at least twice that of normal , nuclear atypia is definite , mitotic figures are present , and pseudoacinar formation may be present ( 9 ) . the term early hcc mean hcc in an early stage that are small in size ( usually < 2 cm ) . histologically , hcc are well differentiated and lack prominent cellular and structural atypia ( 10 - 13 ) . for convenience , we used the term advanced hcc ( ahcc ) for hccs that are greater than 2 cm in size in this article . the chi - square test was used to evaluate the significance of the relation between the nodules and their categorical variables . a total of 103 patients were included in the study . among the patients , 14 ( 13.16% ) were women , 89 ( 86.4% ) were men , age ranged from 20 to 73 , and mean age was 51.4 10.5 . among all explanted cirrhotic livers , 92 ( 89.3% ) had df with lcc , 57 ( 55.3% ) of which showed scc as well . out of these 103 explanted livers , 39 ( 37.9% ) had lgdn , 38 ( 36.9% ) had hgdn , 19 ( 18.4% ) had early hepatocellular carcinoma ( ehcc ) , and 21 ( 20.4% ) had ahcc ( figures 3 - 6 ) . note that all the cases with ehcc and ahcc also had scc , lcc , hgdn , and lgdn . thirteen cases of ehcc were accompanied with ahcc , and 6 cases of ehcc did not show any ahcc ( size larger than 2 cm ) . all of the 21 ahcc cases were transplanted with the preoperative diagnosis of malignancy and hepatitis b - related cirrhosis . however , among livers with ehcc , 6 were not accompanied with ahcc , 2 of which were unsuspected before surgery and diagnosed on the explanted liver . the two ahcc cases were smaller than 2 cm in diameter ( table 1 ) . our results showed that the presence of scc , lgdn , and hgdn has a statistically significant association with ehcc ( p < 0.05 ) and hcc ( p < 0.05 ) . moreover , the presence of ehcc is associated with ahcc ( p < 0.05 ) . hcc is a poor prognostic cancer , and it is one of the most common causes of cancer death around the globe . hcc is also common in countries with high incidence of hbv - related cirrhosis ( 1 ) . despite the improvements in treatment modalities for hcc , the survival rate remains low even after liver transplantation because hcc can not be easily diagnosed before the advanced stage ( 14 ) . therefore , studies on the incidence and diagnosis of precancerous lesions of hcc are important , especially for patients who develop hcc secondary to hbv infection and cirrhosis . therefore , pathologists and clinicians should be able to diagnose ahccs and ehccs from their precursor lesions using various techniques preoperatively ( 10 ) . hepatic precancerous lesions are currently divided into two categories depending on cytological and histological changes : microscopic df and macroscopic dns ( 11 ) . df can be recognized only in microscopic examination as they are smaller than 1 mm . dns are recognizable both in gross examination of hepatic specimens and in microscopic examination as well - defined nodule lesions that are different from the background cirrhotic liver tissue in size , color , texture , or degree of bulging on the cut surface . hccs can be either ehcc ( less than 2 cm ) or ahcc ( larger than 2 cm ) ( 8 , 9 ) . globally , hbv is the most common cause of cirrhosis and hcc in iran ( 12 ) . in this study , we attempted to identify the true prevalence of hcc and its precursors in 103 explanted livers through thorough sectioning of at least 15 sections for each liver after precise inspection of the gross specimens . among all 103 explanted cirrhotic livers in our study , 92 ( 89.3% ) had df with lcc . according to this result , lcc is a common finding in cirrhotic livers of patients with chronic hepatitis b , but it does not seem to be associated with any malignant lesion , thus confirming the lack of premalignant potential in theses lesions . that is , some studies similar to our own found that lcc ( formerly called lcd ) had no histogenetic association with hcc ( 13 ) . others concluded that lcc in relation to hbv might not only be an innocent bystander but might be closely related to hepatocarcinogenesis ( 15 ) . it can be diagnosed by the presence of clusters of hepatocytes with a small size , minimal nuclear atypia , high n / c ratio , and high nuclear crowding . the report by plentz et al . indicated that severely decreased expression of p16 and p21 , telomere shortening , and accumulation of dna damage in scc and hcc compared with lcc and cirrhotic nodules were all suggestive of the malignant potential of scc compared with lcc ( 17 ) . in our study of 103 patients with hepatitis b - associated liver cirrhosis , 57 ( 55.3% ) had scc . out of these 57 livers with scc , 19 ( 70.4% ) had ehcc and 21 ( 36.8% ) had hcc . our results showed that the presence of scc is associated with ehcc ( p < 0.05 ) and hcc ( p < 0.05).therefore , scc may be associated with the presence of ehcc or hcc . according to our results , a thorough sampling is advised for every patient with scc to detect any malignant lesion . for example , in one study , scc was proved to be associated with aneuploidy and elevated dna index , and it was found to be a moderate - to - high risk for progression to hcc ( 18 ) . among the 103 explanted livers , 39 ( 37.9% ) had lgn , 38 ( 36.9% ) had hgdn , 19 ( 18.4% ) had ehcc , and 21 ( 20.4% ) had ahcc . thus , 70% and 53% of the cases with lgdn and 50% and 55% of the cases with hgdn showed ehcc and hcc , respectively . this association was statistically significant ( p value < 0.05 ) . according to previous studies , lgdn and hgdn are indicators of increased risk of hcc in cirrhotic livers . hcc nodules are commonly seen within dns ( i.e. , nodule - within - nodule pattern ) ( 14 ) . in conclusion , scc , lgdn , and hgdn seem to be common associated findings and precursors of hcc in livers infected with hepatitis b. a strict follow - up and a precise and thorough sampling and sectioning of livers with scc and any abnormal dns , especially those larger than 1 cm , are highly recommended because of their association with malignancy .

purposeassociation between body mass index ( bmi ) and doses in organs at risk during postoperative vaginal cuff brachytherapy ( vcb ) treatment has not been evaluated . the aim of this study was to analyse the impact of bmi on the dose delivered to bladder and rectum during high - dose - rate vcb using computed tomography ( ct ) scans at every fraction.materials and methodsa retrospective analysis of 220 planning ct sets derived from 59 patients was conducted . every planning ct was re - segmented and re - planned under the same parameters . rectum and bladder dose - volume histogram values ( d0.1cc , d1cc , and d2cc ) were extracted and evaluated . the mean values for all applications per patient were calculated and correlated with bmi , as well as other factors influencing rectal and bladder doses . multiple regression analysis performed to model organ at risk dose - volume parameters.resultsaccording to world health organization ( who ) , 6.8% of patients were normal , 35.6% were overweight , and 57.6% were class i obese . median rectal doses were 133.5% , 110.9% , and 99.3% for d0.1cc , d1cc , and d2cc , respectively . the corresponding median bladder doses were 96.2% , 80.6% , and 73.3% , respectively . bmi did not show significant association with rectal doses . however , bmi did show a significant association with evaluated bladder dose metrics ( d0.1cc , r=0.366 , p=0.004 ; d1cc , r=0.454 , p < 0.001 ; d2cc , r=0.451 , p < 0.001 ) . bmi was retained in the multivariate regression models ( d0.1cc , p=0.004 ; d1cc , p < 0.001 ; d2cc , p=0.001).conclusionin this group of mediterranean , overweight , and moderately obese patients , bmi showed association with lower bladder dose values , but not with rectal doses . backgroundthe most common cause of liver transplantation in iran is hepatitis b positive cirrhosis , and it also one of the major and important causes of hepatocellular carcinoma ( hcc ) . most cases with hcc follow a multistep sequence . morphologic lesions during hepatocarcinogenesis include dysplastic lesions and small cancerous lesions ( 2 cm in diameter ; early hcc ) . however , insufficient information is available on the incidence of hcc and its precursors in hepatitis b - related cirrhosis.objectivesin this study , we determined the incidence of hcc and its precursors in hepatitis b - related cirrhosis in the largest liver transplant center in iran.methodsin a two - year study , all explanted livers of patients with hepatitis b virus ( hbv)-positive cirrhosis were completely sectioned and examined . each specimen was investigated grossly and microscopically to determine any abnormal nodule or cellular changes ( at least 15 sections from each liver).resultsamong all explanted cirrhotic livers ( 103 livers ) during the study period ( 2014 - 2015 ) , 92 ( 89.3% ) had dysplastic foci with large cell changes ( lcc ) , 57 ( 55.3% ) of which showed small cell changes ( scc ) as well . thirty - nine cases ( 37.9% ) had low - grade dysplastic nodules ( lgdn ) , 38 ( 36.9% ) high - grade dysplastic nodules ( hgdn ) , 19 ( 18.4% ) were early hepatocellular carcinoma ( ehcc ) , and 21 ( 20.4% ) were hepatocellular carcinoma more than 2 cm . all the cases with ehcc and hcc of more than 2 cm also had scc , lcc , hgdn , and lgdn . thirteen cases of ehcc were accompanied with hccs more than 2 cm , and 6 cases of ehcc did not show any hcc ( larger than 2 cm).conclusionsscc , lgdn , and hgdn are common associated findings and precursors of hcc in livers infected with hepatitis b. a strict follow - up and a precise and thorough sampling of livers with scc and any abnormal dysplastic nodules ( dns ) , especially those larger than 1 cm , are highly recommended because these dns are highly associated with malignancy .

dry eye disease ( ded ) continues to present clinicians with a diagnostic dilemma , primarily due to its multiple causative factors . as a result of the disease s complexity , there is currently no test or set of tests considered a gold standard for its diagnosis and monitoring . without such a diagnostic standard of care , however , a recent study showed that 43% of asymptomatic patients had clinical signs of dry eyes.1 if left untreated , ded can significantly impact a person s vision and quality of life . a diagnostic tool that can globally diagnose ded , regardless of the cause , may be valuable in helping physicians establish an earlier and more accurate dry eye diagnosis . based on the definition from the dry eye workshop ( dews ) in 2007 , ded is defined as follows : dry eye is a multifactorial disease [ .. ] that results in symptoms of discomfort , visual disturbance , and tear film instability with potential damage to the ocular surface . it is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.2 dry eye is a multifactorial disease [ .. ] that results in symptoms of discomfort , visual disturbance , and tear film instability with potential damage to the ocular surface . it is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.2 as can be seen from the definition , the symptoms are likely non - specific , and ocular surface damage may actually be a late complication . the dews identified increased tear osmolarity and tear film instability as core mechanisms of ded , regardless of the etiology.2 tests that accurately measure tear osmolarity and tear film instability should therefore , theoretically be best for identifying and determining the severity of ded . tear osmolarity is attractive , because it offers an objective numerical output that can be monitored ; other commonly used diagnostic tests rely heavily on subjective grading criteria . in the past , the two most commonly used instruments to measure tear osmolarity were the clifton and vapor pressure osmometers . studies have demonstrated their accuracy , high sensitivity and specificity.3,4 the main downside to these instruments is that they require a significant amount of time and involve numerous steps , increasing the potential for tear evaporation ; as such , they are not practical , especially for dry eye patients with low tear volumes.3,4 a newer technology produced by tearlab uses a micro - electrode to measure the number of charged particles in a tear sample ; this electrode is designed to avoid direct contact with the ocular surface , thereby reducing the chance for reflex tearing.3 the tearlab measurement method appears as accurate as the more involved osmometers , differing on average by only 2 mosm / l in both normal and dry eye patients.3 the tear - lab device requires only a small tear sample ( ~0.2 l ) and is able to provide results almost instantly , reducing the level of tear evaporation.3,5 when compared with a vapor pressure osmometer , the tearlab osmolarity system was able to measure more patients with lower volume tear samples.4 compared to the clifton osmometer , the tearlab device was reported to have similar sensitivity but slightly better specificity and positive predictive value.3 the aim of the current review is to establish whether published studies support the use of tear osmolarity testing , primarily using the tearlab device , as a tool for ded diagnosis and treatment monitoring . comments from the literature related to the role of tear osmolarity in the pathophysiology of ded and how ocular or systemic conditions and pharmaceuticals impact tear osmolarity are also discussed . a literature search was conducted using pubmed in june 2014 , using the search term tear osmolarity to identify all potentially relevant articles to that date . a review of the abstract of each article was used to identify those related or unrelated to tear osmolarity , or to identify those articles where tear osmolarity was not a primary focus of the paper . where there was any doubt , the paper was classified as related to tear osmolarity . a more detailed review of the remaining papers related to tear osmolarity tear osmolarity research before the year 2000 was limited in scope , methodology , and applicability . more recent research generally reflects any relevant earlier results , so a decision was made that articles published in the year 1999 or earlier would not be included in the review . discussions of tear osmolarity that did not include a diagnostic component were also eliminated ; while they provide a research basis for the use of osmolarity in evaluating the tear film , diagnostic papers are indicative of the clinical application of this more basic research . a rating of all relevant articles was performed using the grading of recommendations assessment , development and evaluation ( grade ) which is based on the likelihood that further research is required to confirm the significance of reported results,6 as well as the university of michigan practice guideline which is based on the study design.7 the grade scoring included consideration of both study sample size and financial interest disclosures in evaluating the quality of the evidence . the financial interest categorization was not to suggest that sponsored studies are lower quality than independent research , but only to reflect the potential for bias in reporting results . relevant data were entered into an access database ( microsoft corporation , redmond , wa , usa ) for analysis . an article was rated in the high category if it fulfilled both the a criteria in the university of michigan practice guideline and fulfilled the definition in the high requirement under the grade system . similarly , b and moderate , c and low , as well as d and very low were used from the university of michigan practice guideline and the grade rating systems , respectively , to identify the rating of the relevant articles . the articles were also subjectively reviewed for the impression they left with the reader , whether positive , neutral , or table 1 contains the summary of preliminary search data ; a total of 407 articles were listed as potentially relating to tear osmolarity . the results from this preliminary analysis identified 164 papers in the peer - reviewed literature as having some reference to tear osmolarity and its use as a diagnostic tool . of the 164 articles identified as relevant peer - reviewed articles , an impression of the results ( positive / neutral / negative ) was possible for 163 of them ( one foreign language study was excluded ) . all but four of the remaining studies could be graded as well , using the combination of the two grading systems outlined previously . table 2 shows the breakdown of articles by grading and impression . as can be seen , only 32% ( 52/163 ) of the studies available qualified as moderate or high quality ; sample size , a lack of randomization , and the lack of control populations were the primary limiting design factors that led to lower quality ratings for studies . in this subgroup of moderate or high quality articles , 73% ( 38/52 ) had a positive impression , 17% had a neutral impression , and 10% had a negative impression . this breakdown by impression was similar to that for the studies graded low or very low ( 71% positive , 24% neutral , and 5% negative , respectively ) . table 3 summarizes the financial interest breakdown , with a percentage breakdown in each financial interest column . approximately two - thirds of the articles were independent , with the remaining third sponsored ( by tearlab or others ) . the impressions for 93% of the much larger group of independent articles were positive or neutral . while not shown in table 3 , the results were independent of study quality ; of the 104 independent studies that were rated , 31% ( 32/104 ) had a high or moderate rating , and of those 32 articles , 72% ( 23/32 ) were positive , indicating that tear osmolarity was a useful diagnostic tool in the evaluation of dry eye and its severity . a further 19% ( 6/32 ) were neutral , while 9% ( 3/32 ) of the articles suggested there was little value to the use of tear osmolarity . the following discussion outlines the results of the literature review based primarily on studies rated as moderate or high . we discuss how tear osmolarity testing is performed , the cut - off value for dry eye diagnosis , the accuracy and repeatability of tear osmolarity testing , and how tear osmolarity compares with other commonly used diagnostic tests . finally , articles discussing the effect of external factors , ocular and/or systemic disease , and pharmaceuticals on tear osmolarity are reviewed . evaluating the diagnostic ability of tear osmolarity requires specifying a value that discriminates a healthy eye from an eye with ded ; this value is known as the threshold value . in the literature , tear osmolarity threshold values have varied from 305 mosm / l8 to 316 mosm / l.5 one reported reason for variability in tear osmolarity threshold values is tear film instability , a hallmark characteristic of the disease.9,10 normal , mild / moderate , and severe dry eyes had average tear osmolarity values of approximately 302+/8 mosm / l , 315+/10 mosm / l and 336+/022 mosm / l , respectively.10,11 another study had lower reported mean values , but the study noted that they included more patients with mild and moderate ded than severe ded.8 it can be seen that the distribution of ded severity in different populations may explain some of the variability in threshold values . studies have demonstrated that tear osmolarity is most influenced by , and correlated with , disease severity.10,11 one study found that using a tear osmolarity threshold of 305 mosm / l gave a 98.4% positive predictive value.8 using a tear osmolarity threshold of 316 mosm / l to 317 mosm / l , sensitivity varied from 59%12 to 81%,13 specificity varied from 78%14 to 94%,12 with a positive predictive value of 85%,3,13 a negative predictive value of 74%,13 and an overall predictive accuracy of 89%.12 a meta - analysis that used a 316 mosm / l threshold noted that tear osmolarity may be more accurate than lactoplate , the schirmer s test , and rose bengal testing.12 currently , the 316 mosm / l threshold is believed to better discriminate between mild and moderate / severe dry eye , while 308 mosm / l is now considered to be a widely accepted threshold.5 a tear osmolarity threshold of 308 mosm / l correctly diagnosed severe dry eye and normal patients 90.7% and 81.3% of the time , respectively ; this value appeared to be the most sensitive for discriminating between normal eyes and those presenting with early stages of ded.10 variability in tear osmolarity can also be a diagnostic indicator ; in one study , variability between the two eyes in normal , mild , or moderate dry eye patients and severe dry eye patients was 6.95.9 mosm / l , 11.710.9 mosm / l , and 26.522.7 mosm / l , respectively.10 variability in inter - eye measurements and repeat measurements in the same eye appears to increase with the severity of dry eye . mild ded may also manifest as variability with repeat measurement of the same eye or between the two eyes . measured osmolarity is low and stable in those without ded.9 tear osmolarity has been shown to have good repeatability8 in normal subjects , with no significant difference in osmolarity values when using up to four readings taken 1 to15 minutes apart.9 a concern with using osmolarity as a diagnostic test is the observed overlap in measured values between normal and dry eyes ; this has been documented as especially high in two studies.15,16 however , in one of these studies,15 dry eye patients were being treated with eye drops or systemic medications , which would be expected to lower osmolarity . the second study16 may have suffered from a selection bias , where other diagnostic tests , but not tear osmolarity , were used to categorize patients into a dry eye and a control group . this categorization may have skewed the results in favor of the tests used in the categorization process.16 different studies have suggested that tear osmolarity does not provide a sharp diagnostic cut - off , but rather can serve as a guide to indicate disease severity or progression.8,10 while tear osmolarity testing is beneficial at providing a numerical value to the current state of dry eyes , other complementary diagnostic tests are valuable in discriminating the etiology of the disease.10 when compared with schirmer s test results , meibomian gland grading , ocular surface disease index ( osdi ) , tear break - up time ( tbut ) , corneal and conjunctival staining , tear osmolarity was better at predicting dry eye severity ; as tear osmolarity increased , dry eye severity also increased in a largely linear pattern.8,11 another study also demonstrated that tear osmolarity had higher sensitivity when compared with corneal staining , conjunctival staining , and meibomian grading , and better specificity when compared with tbut and schirmer s test results.10 the higher the severity of ded , the greater the correlation between tear osmolarity and other diagnostic tests;8 this result may indicate that other diagnostic tests are less sensitive to mild ded diagnosis . given the intermittency of disease severity in the mild dry eye patient , the performance of all diagnostic tests will be more variable in this population segment . for instance , meibomian gland dysfunction may not increase tear osmolarity , as demonstrated in one study.17 one explanation for this is that meibomian gland dysfunction alone may not be sufficient at overwhelming the homeostatic control in most patients . examining all such studies in the present review , there is an indication that osmolarity is correlated with some other diagnostic tests , but the correlation is less apparent when looking only at those studies rated moderate quality or higher . one factor here is that the general correlation between objective tests and subjective tests for dry eye is poor.1 this issue is likely due to the multiple etiologies and presentations of ded ; when particular tests are specific to one etiology , then there may be a lack of overall correlation.1 like other diagnostic signs , tear osmolarity may not correlate with symptoms in a general population,18 although it has been shown to correlate over time with effective therapy , as described in the pharmacological effects and treatment section of the present review . dry eye symptoms can be non - specific or can be a result of a psychiatric disorder such as depression or post - traumatic stress disorder ; both of these conditions increased dry eye symptoms but were not associated with other dry eye signs such as increased c - reactive protein or tear osmolarity.19 one study9 found that for the majority ( 92% , or 23/25 ) of patients , when the patient was symptomatic , his or her maximum tear osmolarity was > 308 mosm / l , and when the patient was asymptomatic , his or her maximum tear osmolarity was < 308 mosm / l . in the same study,9 a minority of patients ( 8% , or 2/25 ) had increased tear osmolarity and no symptoms or low tear osmolarity and symptoms ; this result may be due to them having only mild ded in the former case , and low osmolarity due to a condition other than ded in the latter case . this study9 concluded that increased tear osmolarity is likely a precursor for dry eye complaints , despite the lack of correlation between tear osmolarity and dry eye symptoms . certain factors , diseases , or drugs have been noted to be associated with ded and increased tear osmolarity . it is likely that not all patients exposed to the same external factors , diseases , or drugs will have dry eyes or increased tear osmolarity . such reactions will be a function of the individual s response to these stresses and whether or not their eyes are able to adjust . the effect of external factors such as humidity on tear osmolarity is not clear . in one study , subjects exposed to low relative humidity of 20% for 10 minutes.20 another study found that exposure to humidity of 5% for 2 hours did not increase or result in more variability in tear osmolarity.21 both studies included subjects who were using artificial tears before the study , although they discontinued use more than 1 hour in advance of testing . one study did not specify the type of artificial tear used , and the other did not state the airflow velocity . such differences may account for the conflicting results.20,21 in addition , the use of less sensitive diagnostic tests may have resulted in the inclusion of some mild dry eye patients in the normal group , which would have further confounded results . the literature is divided over whether or not contact lens wear increases2,22,23 or has no effect on tear osmolarity.10,24,25 where osmolarity was measured in some of these studies,10,24 there was an appropriate delay between contact lens removal and osmolarity measurement . the differences in reported results are likely a function of the wearer s ability to maintain homeostasis of their tear film , overcoming the drying effect of contact lenses.10 it appears that certain procedures may influence tear osmolarity more than others . in a recent 2013 article , no change in tear osmolarity or any other dry eye diagnostic test was observed after refractive surgery , despite a decrease in corneal sensitivity.26 after cataract surgery , tear osmolarity has been noted to increase.27 the difference here may be explained by the subjects ages ; average age was 26 years in the refractive surgery study26 and 71 years in the cataract surgery study.27 older patients may take longer to recover from surgically induced ocular trauma.27 trabeculectomy surgery performed on a patient population with an average age of 71 years also had higher tear osmolarity than a control group ; this result was attributed to the presence of the bleb , the use of mitomycin c ( which may cause epithelial damage ) , or the residual effects of long - term use of glaucoma medications before surgery.28 no difference in tear osmolarity was noted between the eye that underwent eyelid reconstruction surgery and the un - operated eye of the same patient ; this result may be related with corrective tearing , as evidenced by the higher schirmer s test results in the operated eye.29 table 4 shows studies that investigated the effect of various ocular and systemic conditions on tear osmolarity . of the systemic conditions , diabetes was found to increase tear osmolarity.30,31 an increase in tear osmolarity was correlated with an increase in diabetes duration . the relation between diabetes and increased tear osmolarity is most likely due to a decrease in the amount of aqueous secretion as a result of injury to the lacrimal gland , the small blood vessels of the lacrimal gland , and/or the corneal nerve.31 thyroid disease that results in an increase in palpebral aperture and proptosis has also been reported to significantly increases tear osmolarity when compared to a control.32 increased pain sensation , as evident in patients with fibromyalgia , was related to increased tear osmolarity.33 traumatic brain injury patients had higher tear osmolarity values than a healthy control group.34 patients with increased tear osmolarity may also be dehydrated , so water intake may be an important consideration for dry eye diagnosis and treatment.35 an increase in estrogen and/or progesterone ( in the form of birth control pills)25 or the excess of androgens ( as noted in patients with polycystic ovary syndrome)36 seemed to have no effect on tear osmolarity . another study found that low levels of 17-beta estradiol , estrone , and testosterone were associated with increased tear osmolarity ; treatment with phytoestrogen decreased tear osmolarity when compared to a control group.37 more studies in this area appear warranted to further understand the correlation between sex hormones and tear osmolarity ; adequate control of hydration or measurement of hydration status may be important in such studies . different ocular conditions also seem to have different effects on tear osmolarity ( table 4 ) . for instance , eyes with pseudoexfoliation syndrome , a condition that is thought to alter goblet cell activity and mucin production , have been reported to have increased tear osmolarity when compared to a control group.38 patients with pterygia , a predominantly inflammatory condition , also show increased tear osmolarity when compared with a control group.39 keratoconus has been demonstrated to reduce corneal sensitivity , but tear osmolarity was similar to a control group . the reason for this similarity is not clear , though it may be that keratoconus is not generally clinically considered an inflammatory disease.40 herpetic keratitis was associated with higher tear osmolarity in both the affected and unaffected eyes when compared to a control group ; the reason the unaffected eye had higher tear osmolarity may be due to the type of keratitis.41 patients with nasolacrimal duct obstruction are likely to compensate by decreasing tear production , resulting in a tear osmolarity value similar to a control group.42 table 5 summarizes the findings of different studies on the effect of certain drugs on tear osmolarity . the use of oral mucolytics , often to treat respiratory conditions , increases tear osmolarity . this increase is most likely due to alterations to the mucin layer of the tear film.43 glaucoma medications increased tear osmolarity , perhaps due to the preservatives , while such medications did not have a significant effect on tbut or schirmer s test results.28 artificial tears have been noted by several studies to decrease tear osmolarity,20,44,45 but one study found no correlation between tear osmolarity and the use of artificial tears.16 the latter study did not disclose the type of artificial tear used . this may be an important factor , because the type of eye drop can impact tear osmolarity.45 for instance , lower osmolarity eye drops have been associated with lower tear osmolarity.46,47 it has been noted that the decrease in osmolarity observed with the use of artificial tears may be comparable to the effects of heat and massage application to the eyelids using a thermal massager.44 the effect of anti - inflammatory medication on tear osmolarity is less clear , especially because the studies evaluating this effect suffer from limitations such as small sample size and the lack of a control group or randomization.4850 in one recent randomized , double - blind , parallel study in finland , osmolarity was observed to increase through the dry winter months.51 participants taking oral sea buckthorn oil , containing n-3 and n-6 fatty acids and antioxidants , had less of an increase in tear osmolarity when compared to a control group , presumed to have been a function of the oil s anti - inflammatory mechanism.51 evaluating the diagnostic ability of tear osmolarity requires specifying a value that discriminates a healthy eye from an eye with ded ; this value is known as the threshold value . in the literature , tear osmolarity threshold values have varied from 305 mosm / l8 to 316 mosm / l.5 one reported reason for variability in tear osmolarity threshold values is tear film instability , a hallmark characteristic of the disease.9,10 normal , mild / moderate , and severe dry eyes had average tear osmolarity values of approximately 302+/8 mosm / l , 315+/10 mosm / l and 336+/022 mosm / l , respectively.10,11 another study had lower reported mean values , but the study noted that they included more patients with mild and moderate ded than severe ded.8 it can be seen that the distribution of ded severity in different populations may explain some of the variability in threshold values . studies have demonstrated that tear osmolarity is most influenced by , and correlated with , disease severity.10,11 one study found that using a tear osmolarity threshold of 305 mosm / l gave a 98.4% positive predictive value.8 using a tear osmolarity threshold of 316 mosm / l to 317 mosm / l , sensitivity varied from 59%12 to 81%,13 specificity varied from 78%14 to 94%,12 with a positive predictive value of 85%,3,13 a negative predictive value of 74%,13 and an overall predictive accuracy of 89%.12 a meta - analysis that used a 316 mosm / l threshold noted that tear osmolarity may be more accurate than lactoplate , the schirmer s test , and rose bengal testing.12 currently , the 316 mosm / l threshold is believed to better discriminate between mild and moderate / severe dry eye , while 308 mosm / l is now considered to be a widely accepted threshold.5 a tear osmolarity threshold of 308 mosm / l correctly diagnosed severe dry eye and normal patients 90.7% and 81.3% of the time , respectively ; this value appeared to be the most sensitive for discriminating between normal eyes and those presenting with early stages of ded.10 variability in tear osmolarity can also be a diagnostic indicator ; in one study , variability between the two eyes in normal , mild , or moderate dry eye patients and severe dry eye patients was 6.95.9 mosm / l , 11.710.9 mosm / l , and 26.522.7 mosm / l , respectively.10 variability in inter - eye measurements and repeat measurements in the same eye appears to increase with the severity of dry eye . mild ded may also manifest as variability with repeat measurement of the same eye or between the two eyes . measured osmolarity is low and stable in those without ded.9 tear osmolarity has been shown to have good repeatability8 in normal subjects , with no significant difference in osmolarity values when using up to four readings taken 1 to15 minutes apart.9 a concern with using osmolarity as a diagnostic test is the observed overlap in measured values between normal and dry eyes ; this has been documented as especially high in two studies.15,16 however , in one of these studies,15 dry eye patients were being treated with eye drops or systemic medications , which would be expected to lower osmolarity . the second study16 may have suffered from a selection bias , where other diagnostic tests , but not tear osmolarity , were used to categorize patients into a dry eye and a control group . this categorization may have skewed the results in favor of the tests used in the categorization process.16 different studies have suggested that tear osmolarity does not provide a sharp diagnostic cut - off , but rather can serve as a guide to indicate disease severity or progression.8,10 while tear osmolarity testing is beneficial at providing a numerical value to the current state of dry eyes , other complementary diagnostic tests are valuable in discriminating the etiology of the disease.10 when compared with schirmer s test results , meibomian gland grading , ocular surface disease index ( osdi ) , tear break - up time ( tbut ) , corneal and conjunctival staining , tear osmolarity was better at predicting dry eye severity ; as tear osmolarity increased , dry eye severity also increased in a largely linear pattern.8,11 another study also demonstrated that tear osmolarity had higher sensitivity when compared with corneal staining , conjunctival staining , and meibomian grading , and better specificity when compared with tbut and schirmer s test results.10 the higher the severity of ded , the greater the correlation between tear osmolarity and other diagnostic tests;8 this result may indicate that other diagnostic tests are less sensitive to mild ded diagnosis . given the intermittency of disease severity in the mild dry eye patient , the performance of all diagnostic tests will be more variable in this population segment . for instance , meibomian gland dysfunction may not increase tear osmolarity , as demonstrated in one study.17 one explanation for this is that meibomian gland dysfunction alone may not be sufficient at overwhelming the homeostatic control in most patients . examining all such studies in the present review , there is an indication that osmolarity is correlated with some other diagnostic tests , but the correlation is less apparent when looking only at those studies rated moderate quality or higher . one factor here is that the general correlation between objective tests and subjective tests for dry eye is poor.1 this issue is likely due to the multiple etiologies and presentations of ded ; when particular tests are specific to one etiology , then there may be a lack of overall correlation.1 like other diagnostic signs , tear osmolarity may not correlate with symptoms in a general population,18 although it has been shown to correlate over time with effective therapy , as described in the pharmacological effects and treatment section of the present review . dry eye symptoms can be non - specific or can be a result of a psychiatric disorder such as depression or post - traumatic stress disorder ; both of these conditions increased dry eye symptoms but were not associated with other dry eye signs such as increased c - reactive protein or tear osmolarity.19 one study9 found that for the majority ( 92% , or 23/25 ) of patients , when the patient was symptomatic , his or her maximum tear osmolarity was > 308 mosm / l , and when the patient was asymptomatic , his or her maximum tear osmolarity was < 308 mosm / l . in the same study,9 a minority of patients ( 8% , or 2/25 ) had increased tear osmolarity and no symptoms or low tear osmolarity and symptoms ; this result may be due to them having only mild ded in the former case , and low osmolarity due to a condition other than ded in the latter case . this study9 concluded that increased tear osmolarity is likely a precursor for dry eye complaints , despite the lack of correlation between tear osmolarity and dry eye symptoms . when compared with schirmer s test results , meibomian gland grading , ocular surface disease index ( osdi ) , tear break - up time ( tbut ) , corneal and conjunctival staining , tear osmolarity was better at predicting dry eye severity ; as tear osmolarity increased , dry eye severity also increased in a largely linear pattern.8,11 another study also demonstrated that tear osmolarity had higher sensitivity when compared with corneal staining , conjunctival staining , and meibomian grading , and better specificity when compared with tbut and schirmer s test results.10 the higher the severity of ded , the greater the correlation between tear osmolarity and other diagnostic tests;8 this result may indicate that other diagnostic tests are less sensitive to mild ded diagnosis . given the intermittency of disease severity in the mild dry eye patient , the performance of all diagnostic tests will be more variable in this population segment . for instance , meibomian gland dysfunction may not increase tear osmolarity , as demonstrated in one study.17 one explanation for this is that meibomian gland dysfunction alone may not be sufficient at overwhelming the homeostatic control in most patients . examining all such studies in the present review , there is an indication that osmolarity is correlated with some other diagnostic tests , but the correlation is less apparent when looking only at those studies rated moderate quality or higher . one factor here is that the general correlation between objective tests and subjective tests for dry eye is poor.1 this issue is likely due to the multiple etiologies and presentations of ded ; when particular tests are specific to one etiology , then there may be a lack of overall correlation.1 like other diagnostic signs , tear osmolarity may not correlate with symptoms in a general population,18 although it has been shown to correlate over time with effective therapy , as described in the pharmacological effects and treatment section of the present review . dry eye symptoms can be non - specific or can be a result of a psychiatric disorder such as depression or post - traumatic stress disorder ; both of these conditions increased dry eye symptoms but were not associated with other dry eye signs such as increased c - reactive protein or tear osmolarity.19 one study9 found that for the majority ( 92% , or 23/25 ) of patients , when the patient was symptomatic , his or her maximum tear osmolarity was > 308 mosm / l , and when the patient was asymptomatic , his or her maximum tear osmolarity was < 308 mosm / l . in the same study,9 a minority of patients ( 8% , or 2/25 ) had increased tear osmolarity and no symptoms or low tear osmolarity and symptoms ; this result may be due to them having only mild ded in the former case , and low osmolarity due to a condition other than ded in the latter case . this study9 concluded that increased tear osmolarity is likely a precursor for dry eye complaints , despite the lack of correlation between tear osmolarity and dry eye symptoms . certain factors , diseases , or drugs have been noted to be associated with ded and increased tear osmolarity . it is likely that not all patients exposed to the same external factors , diseases , or drugs will have dry eyes or increased tear osmolarity . such reactions will be a function of the individual s response to these stresses and whether or not their eyes are able to adjust . the effect of external factors such as humidity on tear osmolarity is not clear . in one study , subjects exposed to low relative humidity of 20% for 10 minutes.20 another study found that exposure to humidity of 5% for 2 hours did not increase or result in more variability in tear osmolarity.21 both studies included subjects who were using artificial tears before the study , although they discontinued use more than 1 hour in advance of testing . one study did not specify the type of artificial tear used , and the other did not state the airflow velocity . such differences may account for the conflicting results.20,21 in addition , the use of less sensitive diagnostic tests may have resulted in the inclusion of some mild dry eye patients in the normal group , which would have further confounded results . the literature is divided over whether or not contact lens wear increases2,22,23 or has no effect on tear osmolarity.10,24,25 where osmolarity was measured in some of these studies,10,24 there was an appropriate delay between contact lens removal and osmolarity measurement . the differences in reported results are likely a function of the wearer s ability to maintain homeostasis of their tear film , overcoming the drying effect of contact lenses.10 it appears that certain procedures may influence tear osmolarity more than others . in a recent 2013 article , no change in tear osmolarity or any other dry eye diagnostic test was observed after refractive surgery , despite a decrease in corneal sensitivity.26 after cataract surgery , tear osmolarity has been noted to increase.27 the difference here may be explained by the subjects ages ; average age was 26 years in the refractive surgery study26 and 71 years in the cataract surgery study.27 older patients may take longer to recover from surgically induced ocular trauma.27 trabeculectomy surgery performed on a patient population with an average age of 71 years also had higher tear osmolarity than a control group ; this result was attributed to the presence of the bleb , the use of mitomycin c ( which may cause epithelial damage ) , or the residual effects of long - term use of glaucoma medications before surgery.28 no difference in tear osmolarity was noted between the eye that underwent eyelid reconstruction surgery and the un - operated eye of the same patient ; this result may be related with corrective tearing , as evidenced by the higher schirmer s test results in the operated eye.29 table 4 shows studies that investigated the effect of various ocular and systemic conditions on tear osmolarity . of the systemic conditions , diabetes was found to increase tear osmolarity.30,31 an increase in tear osmolarity was correlated with an increase in diabetes duration . the relation between diabetes and increased tear osmolarity is most likely due to a decrease in the amount of aqueous secretion as a result of injury to the lacrimal gland , the small blood vessels of the lacrimal gland , and/or the corneal nerve.31 thyroid disease that results in an increase in palpebral aperture and proptosis has also been reported to significantly increases tear osmolarity when compared to a control.32 increased pain sensation , as evident in patients with fibromyalgia , was related to increased tear osmolarity.33 traumatic brain injury patients had higher tear osmolarity values than a healthy control group.34 patients with increased tear osmolarity may also be dehydrated , so water intake may be an important consideration for dry eye diagnosis and treatment.35 an increase in estrogen and/or progesterone ( in the form of birth control pills)25 or the excess of androgens ( as noted in patients with polycystic ovary syndrome)36 seemed to have no effect on tear osmolarity . another study found that low levels of 17-beta estradiol , estrone , and testosterone were associated with increased tear osmolarity ; treatment with phytoestrogen decreased tear osmolarity when compared to a control group.37 more studies in this area appear warranted to further understand the correlation between sex hormones and tear osmolarity ; adequate control of hydration or measurement of hydration status may be important in such studies . different ocular conditions also seem to have different effects on tear osmolarity ( table 4 ) . for instance , eyes with pseudoexfoliation syndrome , a condition that is thought to alter goblet cell activity and mucin production , have been reported to have increased tear osmolarity when compared to a control group.38 patients with pterygia , a predominantly inflammatory condition , also show increased tear osmolarity when compared with a control group.39 keratoconus has been demonstrated to reduce corneal sensitivity , but tear osmolarity was similar to a control group . the reason for this similarity is not clear , though it may be that keratoconus is not generally clinically considered an inflammatory disease.40 herpetic keratitis was associated with higher tear osmolarity in both the affected and unaffected eyes when compared to a control group ; the reason the unaffected eye had higher tear osmolarity may be due to the type of keratitis.41 patients with nasolacrimal duct obstruction are likely to compensate by decreasing tear production , resulting in a tear osmolarity value similar to a control group.42 table 5 summarizes the findings of different studies on the effect of certain drugs on tear osmolarity . the use of oral mucolytics , often to treat respiratory conditions , increases tear osmolarity . this increase is most likely due to alterations to the mucin layer of the tear film.43 glaucoma medications increased tear osmolarity , perhaps due to the preservatives , while such medications did not have a significant effect on tbut or schirmer s test results.28 artificial tears have been noted by several studies to decrease tear osmolarity,20,44,45 but one study found no correlation between tear osmolarity and the use of artificial tears.16 the latter study did not disclose the type of artificial tear used . this may be an important factor , because the type of eye drop can impact tear osmolarity.45 for instance , lower osmolarity eye drops have been associated with lower tear osmolarity.46,47 it has been noted that the decrease in osmolarity observed with the use of artificial tears may be comparable to the effects of heat and massage application to the eyelids using a thermal massager.44 the effect of anti - inflammatory medication on tear osmolarity is less clear , especially because the studies evaluating this effect suffer from limitations such as small sample size and the lack of a control group or randomization.4850 in one recent randomized , double - blind , parallel study in finland , osmolarity was observed to increase through the dry winter months.51 participants taking oral sea buckthorn oil , containing n-3 and n-6 fatty acids and antioxidants , had less of an increase in tear osmolarity when compared to a control group , presumed to have been a function of the oil s anti - inflammatory mechanism.51 the effect of external factors such as humidity on tear osmolarity is not clear . in one study , subjects exposed to low relative humidity of 20% for 10 minutes.20 another study found that exposure to humidity of 5% for 2 hours did not increase or result in more variability in tear osmolarity.21 both studies included subjects who were using artificial tears before the study , although they discontinued use more than 1 hour in advance of testing . one study did not specify the type of artificial tear used , and the other did not state the airflow velocity . such differences may account for the conflicting results.20,21 in addition , the use of less sensitive diagnostic tests may have resulted in the inclusion of some mild dry eye patients in the normal group , which would have further confounded results . the literature is divided over whether or not contact lens wear increases2,22,23 or has no effect on tear osmolarity.10,24,25 where osmolarity was measured in some of these studies,10,24 there was an appropriate delay between contact lens removal and osmolarity measurement . the differences in reported results are likely a function of the wearer s ability to maintain homeostasis of their tear film , overcoming the drying effect of contact lenses.10 it appears that certain procedures may influence tear osmolarity more than others . in a recent 2013 article , no change in tear osmolarity or any other dry eye diagnostic test was observed after refractive surgery , despite a decrease in corneal sensitivity.26 after cataract surgery , tear osmolarity has been noted to increase.27 the difference here may be explained by the subjects ages ; average age was 26 years in the refractive surgery study26 and 71 years in the cataract surgery study.27 older patients may take longer to recover from surgically induced ocular trauma.27 trabeculectomy surgery performed on a patient population with an average age of 71 years also had higher tear osmolarity than a control group ; this result was attributed to the presence of the bleb , the use of mitomycin c ( which may cause epithelial damage ) , or the residual effects of long - term use of glaucoma medications before surgery.28 no difference in tear osmolarity was noted between the eye that underwent eyelid reconstruction surgery and the un - operated eye of the same patient ; this result may be related with corrective tearing , as evidenced by the higher schirmer s test results in the operated eye.29 the effect of external factors such as humidity on tear osmolarity is not clear . in one study , subjects exposed to low relative humidity of 20% for 10 minutes.20 another study found that exposure to humidity of 5% for 2 hours did not increase or result in more variability in tear osmolarity.21 both studies included subjects who were using artificial tears before the study , although they discontinued use more than 1 hour in advance of testing . one study did not specify the type of artificial tear used , and the other did not state the airflow velocity . such differences may account for the conflicting results.20,21 in addition , the use of less sensitive diagnostic tests may have resulted in the inclusion of some mild dry eye patients in the normal group , which would have further confounded results . the literature is divided over whether or not contact lens wear increases2,22,23 or has no effect on tear osmolarity.10,24,25 where osmolarity was measured in some of these studies,10,24 there was an appropriate delay between contact lens removal and osmolarity measurement . the differences in reported results are likely a function of the wearer s ability to maintain homeostasis of their tear film , overcoming the drying effect of contact lenses.10 it appears that certain procedures may influence tear osmolarity more than others . in a recent 2013 article , no change in tear osmolarity or any other dry eye diagnostic test was observed after refractive surgery , despite a decrease in corneal sensitivity.26 after cataract surgery , tear osmolarity has been noted to increase.27 the difference here may be explained by the subjects ages ; average age was 26 years in the refractive surgery study26 and 71 years in the cataract surgery study.27 older patients may take longer to recover from surgically induced ocular trauma.27 trabeculectomy surgery performed on a patient population with an average age of 71 years also had higher tear osmolarity than a control group ; this result was attributed to the presence of the bleb , the use of mitomycin c ( which may cause epithelial damage ) , or the residual effects of long - term use of glaucoma medications before surgery.28 no difference in tear osmolarity was noted between the eye that underwent eyelid reconstruction surgery and the un - operated eye of the same patient ; this result may be related with corrective tearing , as evidenced by the higher schirmer s test results in the operated eye.29 table 4 shows studies that investigated the effect of various ocular and systemic conditions on tear osmolarity . of the systemic conditions , diabetes was found to increase tear osmolarity.30,31 an increase in tear osmolarity was correlated with an increase in diabetes duration . the relation between diabetes and increased tear osmolarity is most likely due to a decrease in the amount of aqueous secretion as a result of injury to the lacrimal gland , the small blood vessels of the lacrimal gland , and/or the corneal nerve.31 thyroid disease that results in an increase in palpebral aperture and proptosis has also been reported to significantly increases tear osmolarity when compared to a control.32 increased pain sensation , as evident in patients with fibromyalgia , was related to increased tear osmolarity.33 traumatic brain injury patients had higher tear osmolarity values than a healthy control group.34 patients with increased tear osmolarity may also be dehydrated , so water intake may be an important consideration for dry eye diagnosis and treatment.35 an increase in estrogen and/or progesterone ( in the form of birth control pills)25 or the excess of androgens ( as noted in patients with polycystic ovary syndrome)36 seemed to have no effect on tear osmolarity . another study found that low levels of 17-beta estradiol , estrone , and testosterone were associated with increased tear osmolarity ; treatment with phytoestrogen decreased tear osmolarity when compared to a control group.37 more studies in this area appear warranted to further understand the correlation between sex hormones and tear osmolarity ; adequate control of hydration or measurement of hydration status may be important in such studies . different ocular conditions also seem to have different effects on tear osmolarity ( table 4 ) . for instance , eyes with pseudoexfoliation syndrome , a condition that is thought to alter goblet cell activity and mucin production , have been reported to have increased tear osmolarity when compared to a control group.38 patients with pterygia , a predominantly inflammatory condition , also show increased tear osmolarity when compared with a control group.39 keratoconus has been demonstrated to reduce corneal sensitivity , but tear osmolarity was similar to a control group . the reason for this similarity is not clear , though it may be that keratoconus is not generally clinically considered an inflammatory disease.40 herpetic keratitis was associated with higher tear osmolarity in both the affected and unaffected eyes when compared to a control group ; the reason the unaffected eye had higher tear osmolarity may be due to the type of keratitis.41 patients with nasolacrimal duct obstruction are likely to compensate by decreasing tear production , resulting in a tear osmolarity value similar to a control group.42 table 5 summarizes the findings of different studies on the effect of certain drugs on tear osmolarity . the use of oral mucolytics , often to treat respiratory conditions , increases tear osmolarity . this increase is most likely due to alterations to the mucin layer of the tear film.43 glaucoma medications increased tear osmolarity , perhaps due to the preservatives , while such medications did not have a significant effect on tbut or schirmer s test results.28 artificial tears have been noted by several studies to decrease tear osmolarity,20,44,45 but one study found no correlation between tear osmolarity and the use of artificial tears.16 the latter study did not disclose the type of artificial tear used . this may be an important factor , because the type of eye drop can impact tear osmolarity.45 for instance , lower osmolarity eye drops have been associated with lower tear osmolarity.46,47 it has been noted that the decrease in osmolarity observed with the use of artificial tears may be comparable to the effects of heat and massage application to the eyelids using a thermal massager.44 the effect of anti - inflammatory medication on tear osmolarity is less clear , especially because the studies evaluating this effect suffer from limitations such as small sample size and the lack of a control group or randomization.4850 in one recent randomized , double - blind , parallel study in finland , osmolarity was observed to increase through the dry winter months.51 participants taking oral sea buckthorn oil , containing n-3 and n-6 fatty acids and antioxidants , had less of an increase in tear osmolarity when compared to a control group , presumed to have been a function of the oil s anti - inflammatory mechanism.51 a systematic review of 164 peer - reviewed articles related to the diagnostic value of tear osmolarity found that 72% of them have a positive impression of the value of tear osmolarity as a diagnostic tool for ded . these results were largely independent of financial interest and article quality rating . there were not as many studies graded high or moderate as might be expected in a review of the literature of this type . it is worth noting that the grading systems used in the current review are typically applied to therapeutic trials where a gold standard control often exists . dry eye is a multifactorial disease with no clear gold standard diagnostic test , so randomized masked trials and comparisons to a relevant control are more difficult to design . performing tear osmolarity testing in a clinical setting appears to be more feasible with the availability of new instruments such as the tearlab osmolarity system . reflex tearing is not a concern when tear osmolarity is collected as per the label instructions.3,5,9 test results have been shown to be repeatable and accurate , with good sensitivity and specificity . they appear to be better than other commonly used diagnostic tests , especially in the early stages of the disease.8,10,12,13 correlations between different diagnostic tests remain an issue , but the lack of correlation is not surprising given the multi - factorial nature of ded and the fact that different diagnostic tests reveal different aspects of the disease.1 the seemingly inconsistent threshold values reported in the literature may be a reflection of the disease s characteristic of tear film instability . it may also be due to the difference in disease severity of the tested groups , given the strong correlation between tear osmolarity and disease severity.911 the more severe the ded , the higher the threshold and the greater the variability in the tear osmolarity value.9 the currently agreed - upon value that discriminates between healthy and dry eyes is when the higher tear film osmolarity value of the two eyes is 308 mosm / l.5,10 as with any diagnostic tool , interpretation must be based on the complete clinical picture , including other diagnostic tests that might be helpful in determining the dry eye etiology.10 certain commonly encountered external factors such as contact lens wear may or may not increase tear osmolarity , depending on the individual response.2,24 the impact of environmental factors , such as temperature , humidity , and airflow on tear osmolarity appears unclear , as is the effect of sex hormones ; these factors would benefit from further investigation.20,21,25,36,37 the effect of ocular surgery on tear osmolarity may be partially dependent on the patient s age , the type of surgery performed , and the ability of the eye to recover.2629 systemic conditions commonly believed to result in dry eye , such as dehydration , diabetes , and thyroid ophthalmopathy , have been shown to increase tear osmolarity , as expected.31,32,35 ocular conditions , such as herpetic keratitis , pterygia , and pseudoexfoliation syndrome , have also been demonstrated to increase tear osmolarity.38,39,41 keratoconus , largely viewed clinically as a non - inflammatory condition , was not associated with increased tear osmolarity.40 some pharmaceuticals , such as mucin - altering drugs and glaucoma medications , appear to increase tear osmolarity , while others , such as artificial tears , likely lower tear osmolarity.20,28,43 research into the effect of anti - inflammatory medications on tear osmolarity has produced varied results ; the topic deserves further investigation.48,49,51 there are limitations in any review of this nature , and consequently to any of our comments related to the factors associated with osmolarity . by confining the review to tear osmolarity , related discussions of some of the dry eye issues ( such as the effect of hormones ) in some papers will have been missed . additionally , in limiting the review to more recent papers , some of the foundational work in this field has been omitted . in summary , the findings in the articles evaluated in the present review appear to confirm the central role of increased tear osmolarity in the pathophysiology of ded , as described by the dews.2 the majority of the studies reviewed here , and more specifically those rated as moderate or high quality , support the use of tear osmolarity as a tool to diagnose , grade severity , and track therapeutic response in ded . anemia , which can be defined as hemoglobin less than 13 g / dl or hematocrit less than 36% , is a relatively common preoperative finding . approximately one third of patients are anemic before they undergo joint arthroplasty , with baseline hemoglobin levels between 10 and 13 g / dl . in veterans undergoing noncardiac surgery , the incidence of preoperative anemia ( hematocrit < 36% ) was 34% , and in surgical patients who refused transfusions because of religious reasons , 28% had preoperative hemoglobin levels less than 12 g / dl . the incidence of anemia also increases with age . in a recent study of 296 elderly patients with hip fractures , the mean hemoglobin on hospital admission was 12.1 1.7 g / dl . anemia is especially prevalent in patients with cancer , and treatment plans often include surgery . a low initial hemoglobin level measured at disease presentation is a risk factor for further development of anemia throughout the course of treatment . in patients with colorectal cancer following radical mastectomy for breast cancer , 38% of women were anemic ( hemoglobin < 12 g / dl ) before their first chemotherapy cycle , and 59% became anemic after surgery and chemotherapy . ensuring adequate tissue oxygenation in the surgical setting frequently involves the administration of blood transfusions . patients with preoperative hemoglobin levels between 10 and 13 g / dl may be three or more times as likely to receive allogeneic blood than those with baseline hemoglobin greater than 14 g / dl . although it has been documented that patients prefer to receive autologous blood , those who are anemic before surgery are not candidates for preoperative autologous donation ( pad ) . patients with coronary artery disease have impaired hemodynamic and nonhemodynamic responses to hypoxia , and autologous blood donation is not feasible because the hemodilution that ensues may not be tolerated . endogenous erythropoietin normally stimulates the bone marrow to increase red blood cell ( rbc ) mass under hypoxic conditions , but if this nonhemodynamic response to hypoxia is impaired then hemoglobin levels do not rebound sufficiently . if hemoglobin levels fall even further during surgery due to blood losses , as commonly occurs , the likelihood of receiving an allogeneic blood transfusion increases . rbc transfusions are associated with an increased risk for postoperative infections and tumor recurrence , as well as with a variety of immunologic complications that may adversely affect surgical patients . longer hospital stays and higher health care costs have also been linked to blood transfusions . although allogeneic blood is now reportedly safer than ever , safety issues with the blood supply still exist . furthermore , blood is a scarce resource that is becoming even more so . as the ' baby boomer ' population ages , two factors are converging that will limit blood availability and drive up its cost . first , the eligible donor pool is shrinking and , second , surgery in these patients , whether elective or otherwise , is on the increase . because of these quality - of - care and economic factors , the decision to transfuse should be carefully weighed by all members of the surgical team , and transfusion alternatives considered . the importance of correcting preoperative anemia can not be overemphasized because this condition is known to be associated with poor outcomes after surgery . coronary artery disease patients with anemia exhibit higher mortality rates and experience more cardiac , abdominal , and renal complications after cardiac or noncardiac surgery than do patients with normal hemoglobin levels . myocardial ischemia is one of the major complications that occur in anemic patients without overt cardiac disease . in elderly men undergoing radical prostatectomy , st - segment changes and postoperative ischemic episodes were seen more frequently in those with lower hematocrit . in another study of noncardiac surgical patients of various types comprising a large veterans administration database , perioperative anemia and blood transfusions were associated with an increased risk for infection , higher mortality rates , and substantial consumption of health care resources . dunne and coworkers recently documented a relationship between preoperative anemia and stage of colorectal cancer at diagnosis , and a study of 198 patients with rectal cancer showed that low preoperative hemoglobin levels were an independent risk factor for mortality ( p < 0.0001 ) . taken together , these reports indicate that preoperative anemia is prevalent , that anemia and blood transfusions are associated with risks and poor outcomes , and that anemia should be treated , preferably with strategies that exclude allogeneic blood . the human recombinant form of endogenous erythropoietin ( rhuepo ; epoetin alfa ) is indicated for the treatment of anemic patients who are scheduled to undergo elective surgery ( noncardiac and nonvascular ) to reduce the need for allogeneic blood transfusions . multiple clinical studies involving many different surgical types have shown that preoperative rhuepo is safe and effective . patients with initial hematocrits between 33% and 39% and who stand to lose up to 3000 ml blood during surgery are among those who can benefit the most [ 21 - 23 ] . the positive impact of rhuepo is readily apparent in patients undergoing orthopedic surgery , which is often elective and associated with substantial blood losses . oral iron supplementation and treatment with rhuepo should be considered in any anemic patient ( hemoglobin > 10 and 13 g / dl ) whose risk for transfusion is estimated to exceed 10% . patients undergoing major orthopedic surgery who received rhuepo 300 units / kg per day for 10 days preoperatively , on the day of surgery , and 4 days postoperatively had a nearly six - fold reduction in allogeneic transfusion risk as compared with patients who received placebo . in another study that compared rhuepo administered daily ( 300 units / kg per day ) with rhuepo administered weekly ( 600 units / kg per week ) for four doses beginning 21 days before surgery , transfusion requirements were comparable , indicating that the weekly rhuepo regimen also reduces transfusion requirements while improving patient convenience . rhuepo has also been demonstrated to reduce transfusion requirements in elderly patients undergoing major hip or knee reconstruction , in more heterogeneous populations of patients needing total joint arthroplasty or major hip or knee surgery , and even in children requiring craniosynostosis repair . in cardiac surgery , all of seven randomized studies included in a meta - analysis provided evidence that rhuepo , with or without pad , produced significant decreases in the proportion of patients transfused with allogeneic blood . in another single - blind , randomized study of patients undergoing coronary artery bypass graft surgery , patients treated with low dose ( 100 units / kg ) rhuepo for 4 days before surgery received less than half the amount of autologous blood ( p < 0.01 ) as compared with the control group . surgery in cancer patients represents a particular challenge because these patients may suffer severe anemia caused by their cancer or its treatment . in patients with a hemoglobin under 8.5 g / dl and with colorectal cancer , 300 iu / kg rhuepo on day 4 before surgery followed by 150 iu / kg for the next 7 days thereafter stimulated erythropoiesis and reduced transfusion requirements compared with placebo . transfusion requirements were reduced and hemoglobin levels were improved with preoperative rhuepo treatment in patients with gastrointestinal tract cancer and in those with rectal cancer undergoing surgery . other patient groups for whom surgery is indicated and in whom preoperative rhuepo exerts beneficial effects include those with prostate cancer , head and neck cancer , and women undergoing gynecologic surgery for benign conditions . when surgical blood losses are anticipated to exceed 3000 ml , rhuepo is effectively used in conjunction with pad . in patients who were to undergo total hip replacement , rhuepo facilitated dose - dependent increases in pad amounting to 4.3 units ( 300 iu / kg ) , 3.4 units ( 150 iu / kg ) , and 3.0 units ( 75 iu / kg ) , as compared with 2.1 units with placebo , and it minimized reductions in hematocrit associated with repeated phlebotomy . treatment with rhuepo resulted in rapid increases in rbc production ( 3.5 days ) in nonanemic ( hematocrit > 39% ) patients who were participating in an aggressive program for autologous blood donation . this treatment allowed for higher rbc volumes to be donated compared with placebo - treated patients . even in anemic gastrointestinal cancer patients , rhuepo facilitated pad compared with patients who were supplemented with iron alone . other examples of the utility of rhuepo in pad before gynecologic surgery and orthopedic surgery can be found . although a field of medicine unto itself , the practice of bloodless surgery is gaining attention and therefore deserves brief mention . jehovah 's witnesses refuse transfusions on the basis of religious convictions , but are known to survive trauma and surgery with remarkably low hemoglobin levels . in many cases , these patients do so with the pharmacological support of erythropoietic agents [ 49 - 51 ] . in a case study involving 48 jehovah 's witness patients , rhuepo was successfully used to avoid transfusions completely during and after elective coronary and heart valve surgery . when used in combination with products that can substitute for blood ( e.g. the hemoglobin - based oxygen carriers , also known as hbocs ) , there may be complementary effects . thus , patients wishing to avoid transfusions may have even more options in the future . in conclusion , preoperative anemia can be effectively managed with minimal exposure to allogeneic transfusions in virtually all surgical specialties . rhuepo reduces transfusion requirements , facilitates collection of preoperative autologous blood , and is effective alone or with other blood conservation strategies in severe anemia . surgeons should enthusiastically adopt available therapies that help to avoid transfusions and their accompanying risks , conserve blood , and treat preoperative anemia , with the goal of improving surgical outcomes . tgm has received research support and honoraria for lectures from ortho biotech products , l.p . pad = preoperative autologous donation ; rbc = red blood cell ; rhuepo = recombinant human erythropoietin .

objectiveto evaluate the evidence in the peer - reviewed literature regarding the use of tear osmolarity as a physiological marker to diagnose , grade severity , and track therapeutic response in dry eye disease ( ded ) . in addition , to review the evidence for the role of tear osmolarity in the pathophysiology of ded and ocular surface disease.methodsa literature review of all publications after the year 2000 , which included the keywords tear osmolarity , was conducted . relevant articles were graded according to quality of evidence and research , using the university of michigan practice guideline and the grading of recommendations assessment , development , and evaluation ( grade ) rating systems . articles were further categorized by the nature of any reported financial support and by the overall impression they provided related to tear osmolarity.resultsa total of 164 articles were identified as relevant to the search criteria , although some were editorials , and some were written in a foreign language . of the total , it was possible to grade 159 , and an overall impression was generated for 163 . a positive impression of tear osmolarity in ded diagnosis was evident in 72% ( 117/163 ) of all articles , with a neutral impression in a further 21% ( 35/163 ) ; 7% had a negative impression . the percentage of positive impressions appeared independent of the quality of research ; 73% ( 38/52 ) of articles graded high / moderate quality supported the use of tear film osmolarity measurement in ded diagnosis . impressions were also independent of the source of financial support , with 72% ( 75/104 ) of independent studies positive.conclusionthe literature broadly supports the use of tear film osmolarity as an objective numerical measure for diagnosing , grading severity , and managing treatment of ded . preoperative anemia in a surgical patient predisposes to poor outcomes and allogeneic blood transfusions . as an alternative to transfusions , pharmacologic management of preoperative anemia with recombinant human erythropoietin ( rhuepo ) has been well studied in many different types of surgery . rhuepo , when used alone or in combination with preoperative autologous blood donation before elective surgery , stimulates erythropoiesis and helps to avoid or reduce the need for allogeneic blood transfusions . the clinical evidence on preoperative use of rhuepo in orthopedic , cardiac , and cancer surgery , as well as in bloodless surgery , is reviewed .

curcumin,1,7-bis(4-hydroxy-3-methoxypheny l)- 1- 6- heptadiene- 3 , 5-dione , commonly known as diferuloylmethane , is the yellow pigment component of the curry or turmeric ( curcuma longa ) ( 1 ) . turmeric extracts have been extensively used for the treatment of several diseases in ayurvedic medicine in india for several centuries . curcumin was first extracted from turmeric in its impure form in 1815 , but it was not until 1910 when it was crystallized and its structure was elucidated ( 2 ) . it has antimicrobial , antioxidant , immunomodulatory , anti - inflammatory , anti - alzheimer and anticancer activity ( 38 ) . it has shown no toxicity in vitro in numerous cell culture systems , and in vivo in animal models and over 13 phase i human clinical trials . it is generally recognized as safe by the united states food and drug administration ( fda ) ( 2 , 4 , 5 ) . curcumin has been shown to affect several targets in somatic cells for its biological activity ( 38 ) . it inhibits nf-b activity , cox-2 , and 5-lox expression and modulates release of several cytokines ( 3 , 4 ) . it also binds to a number of other proteins including thioredoxin reductase , protein kinases and several receptors ( 3 , 4 ) . however , most of these proteins / factors may not be expressed/ present in terminally non - transcriptional sperm . also , the sperm has a unique characteristic , the motility , that is not present in other cells . thus , curcumin may have different molecules/ mechanism(s ) for its action that are unique to sperm . recently , our laboratory reported , for the first time ever , that the curcumin affects sperm function ( motility / capacitation and acrosome reaction / fertilization ) in vitro and fertility in vivo . intravaginal administration of curcumin caused a significant , but reversible reduction in fertility ( 9 ) . the molecular mechanism(s ) by which curcumin inhibits / blocks sperm motility has not been delineated . since modulation of intracellular ph ( phi ) and plasma membrane polarization has been shown to be involved in sperm motility and capacitation / acrosome reaction of several mammalian species ( 1021 ) , the present study was conducted to investigate the effect of curcumin on sperm intracellular ph and plasma membrane polarization . it was hypothesized that the curcumin - mediated effect on sperm motility is caused by modulation of phi and/or membrane polarization . the long - term objective of the study was to understand the molecular mechanism(s ) by which curcumin affects sperm motility and function and to develop a novel non - steroidal contraceptive with spermicidal properties . semen was liquefied and analyzed for volume , sperm concentration and percent and progressive motility . only those semen samples that had sperm concentration of > 50x10sperm / ml , percent motility of > 60% , progressive motility of > + 3 ( on a scale of 0 to + 5 ) , and contamination of immature germ cells and immune cells of < 1% were used to collect a pure swim - up sperm population ( 22 ) . the study was approved by the west virginia university - institutional review board ( irb ) for human studies . mouse sperm were collected from cauda epididymis and vas deferens of mature balb / c or cd-1 males . motile sperm were isolated by the swim - up procedure and washed by centrifugation ( 500 g , 10 min ) with ham 's f-10 medium supplemented with human serum albumin ( 5 mg / ml ) or modified sperm washing medium ( irvine scientific , santa ana , ca , usa ) . the study was approved by the west virginia university - animal care and use committee ( acuc ) for animal studies . # c27727 ) was obtained from sigma - aldrich ( st . louis , mo , usa ) . curcumin was dissolved in dimethylsulf - oxide ( dmso ) ( 25 - 50 mm stock ) and then diluted in medium to the desired concentrations . the effect of curcumin on sperm forward motility was examined by incubating 10 - 100 l of sperm suspension ( 100 - 250x10 motile sperm / ml ) with various concentrations of curcumin ( 50 - 400 m , final concentration ) up to 1 hr . the percentage of forward moving sperm was recorded every 5 - 20 min , before and after incubation . each experiment , including various treatment groups , was performed 3 - 5 times using sperm from 3 - 5 men or mice on different days to take care of the inter - individual variation among various sperm specimens from different men and mice . the intracellular ph ( phi ) of human and mouse sperm was measured by fluorescent ph - indicator 2,7-bicarb - oxyethy l-5 , 6-carboxyfluorescein- acetoxymethylester ( bcecf - am ) ( molecular probes , eugene , or , usa ) following the manufacturer 's protocol as described by hamamah et al . bcecf is a neutral lipophilic form of bis - carboxyfluorescein which diffuses freely through the plasma membrane . in the cell , it is hydrolyzed by esterases , releasing the bcecf which is retained within the cytoplasm . the motile sperm , isolated by the swim - up procedure , were centrifuged and the pellet was washed and resuspended in 1 ml of phosphate - buffered saline ( pbs , ph=7.4 ) . sperm ( 8 - 15x10/ml ) were then loaded with 2 m bcecf ( final concentration ) and incubated ( 37c , 35 min ) in dark . following incubation , sperm were centrifuged , washed ( x2 ) and resuspended in pbs . for the intracellular ph calibration curve , sperm were loaded with bcecf at various extracellular phs ( phe ; 6.8 , 7.0 , 7.2 , 7.4 , 7.6 and 7.8 ) and then treated with 0.1% triton - x100 . subsequently , the fluorescence intensity was measured as described below , and the calibration curve was constructed by plotting fluorescence intensity versus extracellular ph ( 24 ) . using calibration curve , the fluorescence intensity of bcecf is dependent upon the ph with a maximum response at an excitation of =535 nm ( f1 ) , while at =490 nm ( f2 ) , the intensity is independent of ph . the phi was determined graphically using the ratio f1/f2 from a calibration curve obtained after permeabilization of spermatozoa with 0.1% triton by measuring the maximum fluorescence intensity after adding naoh and the minimum after adding hcl . the f1/f2 ratio represents a pseudo - linear function of the ph ( 23 ) . to examine the effect of curcumin , bcecf - loaded sperm were incubated ( 37c , 5 - 10 min ) with various concentrations of curcumin ( 50 - 400 m ) and then washed . the treated / control sperm were transferred into wells ( 200 l / well ) of a black 96-well microplate and the fluorescence intensity was measured ( biotek synergy2 multiplatform automated plate reader ) using excitation and emission wavelengths of 490 and 535 nm , respectively . each experiment , including various treatment groups , was performed 3 - 5 times using sperm from 3 - 5 men or mice on different days to take care of the inter - individual variation among various sperm specimens from different men and mice . moreover , it was pertinent to examine the effect of cur - cumin per se on fluorescence intensity . similar experiments carried out with various concentrations ( 50 - 400 m ) of curcumin without sperm did not affect the fluorescence intensity , indicating curcumin per se , without sperm , does not affect fluorescence intensity . the changes in sperm plasma membrane polarization were examined using the fluorescence sensitive dye bis ( 1,3-dibarbituric acid)-trimethine oxanol [ dibac4(3 ) ] ( molecular probes , eugene , or , usa ) following the manufacturer 's protocol as described by rossato et al . the motile sperm in the swim - up fraction were centrifuged , washed ( x2 ) with pbs and incubated ( 37 c , 1 hr ) with 2 m dibac4(3 ) . dibac4(3 ) can enter depolarized cells and then bind to intracellular proteins or membrane and exhibits enhanced fluorescence . increased depolarization results in additional influx of the anionic dye and an increase in fluorescence . sperm ( 8 - 15x 10/ml ) were then centrifuged and washed twice with pbs . the dibac4(3)-incubated sperm were aliquoted in different tubes at equal volumes and incubated ( 5 - 10 min ) with various concentrations of curcumin ( 100 m , 200 m , 300 m , and 400 m ) . after final washing with pbs , the sperm were transferred into wells ( 200 l / well ) of a black 96-well microplate and the fluorescence intensity was measured ( biotek synergy2 multiplatform automated plate reader ) using excitation and emission wavelengths of 485 and 530 nm , respectively . each experiment , including various treatment groups , was performed 3 - 5 times using sperm from 3 - 5 men or mice on different days to take care of the inter - individual variation among various sperm specimens from different men and mice . similar experiments carried out with various concentrations ( 50 - 400 m ) of curcumin without sperm did not affect the fluorescence intensity , indicating curcumin per se , without sperm , does not affect fluorescence intensity . the significance of difference among various groups was analyzed using analysis of variance ( anova ) and tukey kramer multiple comparison test . semen was liquefied and analyzed for volume , sperm concentration and percent and progressive motility . only those semen samples that had sperm concentration of > 50x10sperm / ml , percent motility of > 60% , progressive motility of > + 3 ( on a scale of 0 to + 5 ) , and contamination of immature germ cells and immune cells of < 1% were used to collect a pure swim - up sperm population ( 22 ) . the study was approved by the west virginia university - institutional review board ( irb ) for human studies . mouse sperm were collected from cauda epididymis and vas deferens of mature balb / c or cd-1 males . motile sperm were isolated by the swim - up procedure and washed by centrifugation ( 500 g , 10 min ) with ham 's f-10 medium supplemented with human serum albumin ( 5 mg / ml ) or modified sperm washing medium ( irvine scientific , santa ana , ca , usa ) . the study was approved by the west virginia university - animal care and use committee ( acuc ) for animal studies . # c27727 ) was obtained from sigma - aldrich ( st . louis , mo , usa ) . curcumin was dissolved in dimethylsulf - oxide ( dmso ) ( 25 - 50 mm stock ) and then diluted in medium to the desired concentrations . the effect of curcumin on sperm forward motility was examined by incubating 10 - 100 l of sperm suspension ( 100 - 250x10 motile sperm / ml ) with various concentrations of curcumin ( 50 - 400 m , final concentration ) up to 1 hr . the percentage of forward moving sperm was recorded every 5 - 20 min , before and after incubation . each experiment , including various treatment groups , was performed 3 - 5 times using sperm from 3 - 5 men or mice on different days to take care of the inter - individual variation among various sperm specimens from different men and mice . the intracellular ph ( phi ) of human and mouse sperm was measured by fluorescent ph - indicator 2,7-bicarb - oxyethy l-5 , 6-carboxyfluorescein- acetoxymethylester ( bcecf - am ) ( molecular probes , eugene , or , usa ) following the manufacturer 's protocol as described by hamamah et al . bcecf is a neutral lipophilic form of bis - carboxyfluorescein which diffuses freely through the plasma membrane . in the cell , it is hydrolyzed by esterases , releasing the bcecf which is retained within the cytoplasm . the motile sperm , isolated by the swim - up procedure , were centrifuged and the pellet was washed and resuspended in 1 ml of phosphate - buffered saline ( pbs , ph=7.4 ) . sperm ( 8 - 15x10/ml ) were then loaded with 2 m bcecf ( final concentration ) and incubated ( 37c , 35 min ) in dark . following incubation , sperm were centrifuged , washed ( x2 ) and resuspended in pbs . for the intracellular ph calibration curve , sperm were loaded with bcecf at various extracellular phs ( phe ; 6.8 , 7.0 , 7.2 , 7.4 , 7.6 and 7.8 ) and then treated with 0.1% triton - x100 . subsequently , the fluorescence intensity was measured as described below , and the calibration curve was constructed by plotting fluorescence intensity versus extracellular ph ( 24 ) . using calibration curve , the fluorescence intensity of bcecf is dependent upon the ph with a maximum response at an excitation of =535 nm ( f1 ) , while at =490 nm ( f2 ) , the intensity is independent of ph . the phi was determined graphically using the ratio f1/f2 from a calibration curve obtained after permeabilization of spermatozoa with 0.1% triton by measuring the maximum fluorescence intensity after adding naoh and the minimum after adding hcl . the f1/f2 ratio represents a pseudo - linear function of the ph ( 23 ) . to examine the effect of curcumin , bcecf - loaded sperm were incubated ( 37c , 5 - 10 min ) with various concentrations of curcumin ( 50 - 400 m ) and then washed . the treated / control sperm were transferred into wells ( 200 l / well ) of a black 96-well microplate and the fluorescence intensity was measured ( biotek synergy2 multiplatform automated plate reader ) using excitation and emission wavelengths of 490 and 535 nm , respectively . each experiment , including various treatment groups , was performed 3 - 5 times using sperm from 3 - 5 men or mice on different days to take care of the inter - individual variation among various sperm specimens from different men and mice . moreover , it was pertinent to examine the effect of cur - cumin per se on fluorescence intensity . similar experiments carried out with various concentrations ( 50 - 400 m ) of curcumin without sperm did not affect the fluorescence intensity , indicating curcumin per se , without sperm , does not affect fluorescence intensity . the changes in sperm plasma membrane polarization were examined using the fluorescence sensitive dye bis ( 1,3-dibarbituric acid)-trimethine oxanol [ dibac4(3 ) ] ( molecular probes , eugene , or , usa ) following the manufacturer 's protocol as described by rossato et al . the motile sperm in the swim - up fraction were centrifuged , washed ( x2 ) with pbs and incubated ( 37 c , 1 hr ) with 2 m dibac4(3 ) . dibac4(3 ) can enter depolarized cells and then bind to intracellular proteins or membrane and exhibits enhanced fluorescence . increased depolarization results in additional influx of the anionic dye and an increase in fluorescence . sperm ( 8 - 15x 10/ml ) were then centrifuged and washed twice with pbs . the dibac4(3)-incubated sperm were aliquoted in different tubes at equal volumes and incubated ( 5 - 10 min ) with various concentrations of curcumin ( 100 m , 200 m , 300 m , and 400 m ) . after final washing with pbs , the sperm were transferred into wells ( 200 l / well ) of a black 96-well microplate and the fluorescence intensity was measured ( biotek synergy2 multiplatform automated plate reader ) using excitation and emission wavelengths of 485 and 530 nm , respectively . each experiment , including various treatment groups , was performed 3 - 5 times using sperm from 3 - 5 men or mice on different days to take care of the inter - individual variation among various sperm specimens from different men and mice . similar experiments carried out with various concentrations ( 50 - 400 m ) of curcumin without sperm did not affect the fluorescence intensity , indicating curcumin per se , without sperm , does not affect fluorescence intensity . the significance of difference among various groups was analyzed using analysis of variance ( anova ) and tukey kramer multiple comparison test . curcumin caused a concentration - dependent decrease in human sperm forward motility ( table 1 ) . at 50 m concentration , there was no apparent ( p>0.05 ) effect on sperm forward motility in 5 - 10 min ( with a slight decrease over time ) and in 1 hr it decreased by up to 25% ( p < 0.001 ) . at 100 m concentration , there was a significant ( p<0.001 ) decrease in sperm forward motility in 5 - 10 min which decreased by up to 80% in 1 hr . at concentrations 200 m , there was a complete block of sperm forward motility within 5 - 10 min . dmso vehicle did not affect ( p>0.05 ) sperm forward motility as compared to medium control . effect of curcumin on human sperm forward motility ( % ) * versus control , significantly different ( p<0.001 ) ; all others non - significant curcumin caused a similar effect on mouse sperm forward motility ( table 2 ) . at 50 m concentration , there was no apparent ( p>0.05 ) effect on sperm forward motility in 5 - 10 min ( with a slight decrease over time ) and in 1 hr , it decreased by up to 40% ( p<0.001 ) . at 100 m concentration , there was a significant ( p<0.001 ) decrease in sperm forward motility in 5 - 10 min , which decreased by up to 70% in 1 hr . at concentrations 200 m , there was a complete block of forward motility within 5 - 10 min . dmso vehicle did not affect ( p>0.05 ) sperm forward motility as compared to the medium control . effect of curcumin on murine sperm forward motility ( % ) * versus control , significantly different ( p<0.001 ) ; all others non - significant there was a linear relationship between the extracellular ph and the fluorescence intensity both in human ( r=0.9513 ) and mouse ( r=0.9835 ) sperm ( figure 1 , panel a and b , respectively ) . with an increase in extracellular ph , there was a corresponding increase in fluorescence intensity . the ph=7.4 was selected in our subsequent experiments to examine the effect of curcumin on intracellular sperm ph ( phi ) . calibration curve of human ( panel a ) and mouse ( panel b ) sperm fluorescence intensity at various extracellular ph . bcecf - loaded sperm were treated with 0.1% triton - x 100 at various extracellular ph ( phe ) and the fluorescence intensity was measured . fluorescence intensity values were then plotted against phe values to obtain the calibration curve . in both human and mouse sperm , curcumin caused a concentration - dependent decrease in intracellular ph ( figure 2 ) , in human ( panel a ) and mouse ( panel b ) sperm . in human sperm , control sperm had phi of 7.30.003 which was not significantly different ( p>0.05 ) from dmso - treated sperm , which had a phi of 7.30.105 ( figure 3 , panel a ) . treatment with curcumin significantly ( p<0.001 ) decreased intracellular ph in a concentration - dependent manner as compared to control/ dmso - treated sperm . at 50 m curcumin concentration , phi was 7.210.010 , at 100 m phi was 7.040.008 , at 200 m phi was 6.950.02 , at 300 m phi was 6.880.0067 and at 400 m phi was 6.810.014 . comparing the change in phi within the curcumin - treated groups , the difference was significant ( p<0.05 ) only among 50 m , 100 m and 400 m groups . human ( panel a ) and mouse ( panel b ) sperm intracellular ph ( phi ) after curcumin treatment . bcef - loaded sperm were treated with increasing concentrations of curcumin and the fluorescence intensities were measured and converted to intracellular ph using the calibration curve . intracellular ph ( phi ) values ( meanse ) from 3 - 5 independent experiments using sperm from 3 - 5 different men and mice are shown . there was no effect of any concentration of curcumin tested per se on fluorescence intensity without sperm . effect of curcumin on plasma membrane potential in human ( panel a ) and mouse ( panel b ) sperm . fluorescence intensity values ( meanse ) from 3 - 5 independent experiments using sperm from 3 - 5 different men and mice are shown . fluorescence intensity values lower than the control indicate hyperpolarization . there was no effect of any concentration of curcumin tested per se on fluorescence intensity without sperm . similar results were obtained in mouse sperm ( figure 2 , panel b ) . in mouse sperm , control sperm had phi of 7.150.005 which was not significantly different ( p>0.05 ) from dmso - treated sperm which had a phi of 7.150.00 ( figure 2 , panel b ) . treatment with curcumin significantly ( p<0.001 ) decreased intracellular ph in a concentration - dependent manner as compared to control / dmso - treated sperm . at 50 m curcumin concentration , phi was 7.070.017 , at 100 m phi was 6.970.017 , at 200 m phi was 6.91 0.012 , at 300 m phi was 6.880.02 and at 400 m phi was 6.830.017 . comparing the changes in phi within the curcumin - treated groups , the difference was significant ( p<0.05 ) only among 50 m , 100 m and 400 m groups . the effect of various concentrations of curcumin on plasma membrane polarization was evaluated using both human and mouse sperm . in human sperm , treatment with dmso did not significantly ( p>0.05 ) affect fluorescence intensity as compared to control sperm ( control : 286143.016 ; dmso : 3252 215.860 ) ( figure 3 , panel a ) . treatment with curcumin caused significant ( p<0.001 ) hyperpolarization of sperm plasma membrane as compared to control / dmso - treated sperm ( figure 3 , panel a ) . at 50 m curcumin , fluorescence intensity were 60151.052 , at 100 m , 59720.664 , at 200 m , 52272.421 , at 300 m , 45847.576 and at 400 m 41429.464 . comparing the changes in fluorescence intensities within the curcumin - treated groups , the differences were not - significant . similar results were obtained in mouse sperm ( figure 3 , panel b ) . in mouse sperm , treatment with dmso did not significantly affect fluorescence intensity as compared to control ( control : 5592.0852.186 ; dmso : 5130.905130.770 ) ( figure 3 , panel b ) . treatment with curcumin caused significant ( p<0.001 ) hyperpolarization of sperm plasma membrane as compared to control / dmso - treated sperm ( figure 3 , panel b ) . at 50 m curcumin , fluorescence intensity was 997.50222.229 , at 100 m was 877.35641.328 , at 200 m was 624.33142.116 , at 300 m was 578.92943.245 and at 400 m was 522.43157.900 . comparing the changes in fluorescence intensity within the curcumin - treated groups , the difference was significant ( p<0.05 ) only between 50 m and 400 m groups . curcumin caused a concentration - dependent decrease in human sperm forward motility ( table 1 ) . at 50 m concentration , there was no apparent ( p>0.05 ) effect on sperm forward motility in 5 - 10 min ( with a slight decrease over time ) and in 1 hr it decreased by up to 25% ( p < 0.001 ) . at 100 m concentration , there was a significant ( p<0.001 ) decrease in sperm forward motility in 5 - 10 min which decreased by up to 80% in 1 hr . at concentrations 200 m , there was a complete block of sperm forward motility within 5 - 10 min . dmso vehicle did not affect ( p>0.05 ) sperm forward motility as compared to medium control . effect of curcumin on human sperm forward motility ( % ) * versus control , significantly different ( p<0.001 ) ; all others non - significant curcumin caused a similar effect on mouse sperm forward motility ( table 2 ) . at 50 m concentration , there was no apparent ( p>0.05 ) effect on sperm forward motility in 5 - 10 min ( with a slight decrease over time ) and in 1 hr , it decreased by up to 40% ( p<0.001 ) . at 100 m concentration , there was a significant ( p<0.001 ) decrease in sperm forward motility in 5 - 10 min , which decreased by up to 70% in 1 hr . at concentrations 200 m , there was a complete block of forward motility within 5 - 10 min . dmso vehicle did not affect ( p>0.05 ) sperm forward motility as compared to the medium control . effect of curcumin on murine sperm forward motility ( % ) * versus control , significantly different ( p<0.001 ) ; all others non - significant there was a linear relationship between the extracellular ph and the fluorescence intensity both in human ( r=0.9513 ) and mouse ( r=0.9835 ) sperm ( figure 1 , panel a and b , respectively ) . with an increase in extracellular ph the ph=7.4 was selected in our subsequent experiments to examine the effect of curcumin on intracellular sperm ph ( phi ) . calibration curve of human ( panel a ) and mouse ( panel b ) sperm fluorescence intensity at various extracellular ph . bcecf - loaded sperm were treated with 0.1% triton - x 100 at various extracellular ph ( phe ) and the fluorescence intensity was measured . fluorescence intensity values were then plotted against phe values to obtain the calibration curve . in both human and mouse sperm , curcumin caused a concentration - dependent decrease in intracellular ph ( figure 2 ) , in human ( panel a ) and mouse ( panel b ) sperm . in human sperm , control sperm had phi of 7.30.003 which was not significantly different ( p>0.05 ) from dmso - treated sperm , which had a phi of 7.30.105 ( figure 3 , panel a ) . treatment with curcumin significantly ( p<0.001 ) decreased intracellular ph in a concentration - dependent manner as compared to control/ dmso - treated sperm . at 50 m curcumin concentration , phi was 7.210.010 , at 100 m phi was 7.040.008 , at 200 m phi was 6.950.02 , at 300 m phi was 6.880.0067 and at 400 m phi was 6.810.014 . comparing the change in phi within the curcumin - treated groups , the difference was significant ( p<0.05 ) only among 50 m , 100 m and 400 m groups . human ( panel a ) and mouse ( panel b ) sperm intracellular ph ( phi ) after curcumin treatment . bcef - loaded sperm were treated with increasing concentrations of curcumin and the fluorescence intensities were measured and converted to intracellular ph using the calibration curve . intracellular ph ( phi ) values ( meanse ) from 3 - 5 independent experiments using sperm from 3 - 5 different men and mice are shown . there was no effect of any concentration of curcumin tested per se on fluorescence intensity without sperm . effect of curcumin on plasma membrane potential in human ( panel a ) and mouse ( panel b ) sperm . fluorescence intensity values ( meanse ) from 3 - 5 independent experiments using sperm from 3 - 5 different men and mice are shown . there was no effect of any concentration of curcumin tested per se on fluorescence intensity without sperm . similar results were obtained in mouse sperm ( figure 2 , panel b ) . in mouse sperm , control sperm had phi of 7.150.005 which was not significantly different ( p>0.05 ) from dmso - treated sperm which had a phi of 7.150.00 ( figure 2 , panel b ) . treatment with curcumin significantly ( p<0.001 ) decreased intracellular ph in a concentration - dependent manner as compared to control / dmso - treated sperm . at 50 m curcumin concentration , phi was 7.070.017 , at 100 m phi was 6.970.017 , at 200 m phi was 6.91 0.012 , at 300 m phi was 6.880.02 and at 400 m phi was 6.830.017 . comparing the changes in phi within the curcumin - treated groups , the difference was significant ( p<0.05 ) only among 50 m , 100 m and 400 m groups . the effect of various concentrations of curcumin on plasma membrane polarization was evaluated using both human and mouse sperm . in human sperm , treatment with dmso did not significantly ( p>0.05 ) affect fluorescence intensity as compared to control sperm ( control : 286143.016 ; dmso : 3252 215.860 ) ( figure 3 , panel a ) . treatment with curcumin caused significant ( p<0.001 ) hyperpolarization of sperm plasma membrane as compared to control / dmso - treated sperm ( figure 3 , panel a ) . at 50 m curcumin , fluorescence intensity were 60151.052 , at 100 m , 59720.664 , at 200 m , 52272.421 , at 300 m , 45847.576 and at 400 m 41429.464 . comparing the changes in fluorescence intensities within the curcumin - treated groups , similar results were obtained in mouse sperm ( figure 3 , panel b ) . in mouse sperm , treatment with dmso did not significantly affect fluorescence intensity as compared to control ( control : 5592.0852.186 ; dmso : 5130.905130.770 ) ( figure 3 , panel b ) . treatment with curcumin caused significant ( p<0.001 ) hyperpolarization of sperm plasma membrane as compared to control / dmso - treated sperm ( figure 3 , panel b ) . at 50 m curcumin , fluorescence intensity was 997.50222.229 , at 100 m was 877.35641.328 , at 200 m was 624.33142.116 , at 300 m was 578.92943.245 and at 400 m was 522.43157.900 . comparing the changes in fluorescence intensity within the curcumin - treated groups , the difference was significant ( p<0.05 ) only between 50 m and 400 m groups . the data indicated that curcumin affects sperm forward motility starting at 100 m concentration , with a complete block at 200 m concentration within 5 - 10 min in both human and murine sperm . these findings correlate well with the earlier published data ( 9 ) . in general , forward motility corresponded well with overall motility and progressive motility , as a decrease in forward motility corresponded with decrease in overall and progressive motility . also , there was a loss of sperm viability when there was a decrease in forward motility . the totally immotile sperm were completely non - viable as tested by eosin - nigrosin staining ( data not shown ) . the present study was conducted to examine the mechanism by which curcumin affects sperm forward motility . the findings indicated that curcumin acidified sperm intracellularly and hyperpolarized cell membrane of both human and mouse sperm . there was a concentration - dependent decrease in phi from 7.3 to 6.81 at highest concentration ( 400 m ) of curcumin tested . these findings correlate well with the effect of curcumin on human and murine sperm forward motility . starting at 100 m concentration , there was a significant effect within 5 - 10 min , with a total block at 200 m concentration . the phi plays an important role in modulating mammalian sperm motility ( 12 , 17 , 20 ) . these cumulative findings indicate that the acidic phi inhibits sperm motility , which is in agreement with our findings . the phi of 6.6 - 6.8 has been reported in one study for uncapacitated murine sperm ( 13 ) . in this study , this may be due to different methodologies / media and/or the strain of mice used and/or various degrees of capacitation of sperm preparations . the sodium - proton membrane exchange mechanism can modulate phi and affect motility ( 26 ) . the phi changes are also involved in sperm motility as the sperm cell passes through the epididymis ( 27 , 28 ) . also , the ph of the epididymal cauda fluid is acidic in almost all species ( 29 ) and acts directly on the phi to decrease the sperm motility ( 28 ) . our extensive data search in pubmed and google scholar did not yield any publication which has examined the effect of curcumin on intracellular ph in sperm or any other cell types . the degree of decrease in intracellular florescence intensity is indicative of the degree of hyperpolarization . the change in sperm membrane potential has been shown to be involved in sperm motility and function ( 18 , 19 , 21 , 24 , 25 , 28 , 3033 ) . for mouse sperm , intracellular alkalinization produces a hyperpolarization of sperm membrane potential which may be important for both hyperactivated motility and acrosome reaction . in this study , curcumin induced hyperpolarization and intracellular acidification which correlate with a reduction / block of sperm motility . there was more drastic effect on hyperpolarization than on change in phi . at 50 m concentration , which did not significantly decrease the phi , it showed a significant effect on membrane polarization in both human and mouse sperm . it is possible that curcumin may affect phi and membrane potential by interacting with different molecules and signal transduction cascades . besides sperm , hyperpolarization has also been connected to intracellular phi changes in several other cell systems ( 34 ) . although there is no publication which has examined the effect on sperm , curcumin has been reported to affect membrane potential in other cell systems . curcumin inhibits sarco / endoplasmic reticulum caatpase ( ser ca ) pump that has a major role in maintaining lower levels of intracellular [ ca ] by importing extracellular protons in rabbit skeleton muscle ( 36 ) . a study by cao et al . ( 37 ) examined the effect of curcumin on changes in mitochondrial membrane potential in human hepatomag2 ( hepg2 ) cells . their study showed that hepg2 cells incubated with curcumin induced oxidative damage to mitochondrial dna through mitochondrial membrane hyperpolarization . several molecules / mechanisms are involved in regulation of sperm intracellular ph and membrane polarization . sodium - hydrogen exchange has been suggested as a mediator of ph regulation in various mammalian sperm ( 27 , 38 , 39 ) . at least two sodium - hydrogen exchangers ( nhe1 and nhe5 ) have been shown to be expressed in spermatozoa . the catalytic subunit of protein kinase a ( pka ) is involved in activation of motility evoked by hco3 anion ( 40 ) . capacitation - associated hyperpolarization involves a decrease in intracellular [ na+ ] regulated by pka through activation of the cystic fibrosis transmembrane conductance regulator ( cftr ) ( 41 ) . alkalinization activates the intracellular ph - sensitive i(ksper ) , inducing the membrane potential to approach negative potentials where ca entry via i(catsper ) is maximized ( 33 , 42 ) . the exact molecular mechanism(s ) and signal transduction pathway involved in modulation of sperm intracellular ph and membrane potential by curcumin need further investigation . there are several mechanisms that can affect sperm motility / function leading to infertility ( 43 , 44 ) . this preliminary data indicates that curcumin may inhibit tyrosine phosphorylation of a subset of sperm surface proteins and ca channels ( unpublished data ) . tyrosine phosphorylation has been shown to be involved in sperm motility , capacitation / acrosome reaction and function ( 16 ) . the findings indicate that curcumin causes intracellular acidification and membrane hyperpolarization which may be involved in inhibiting sperm forward motility . this is the first study to our knowledge that examined the effect of curcumin on sperm phi and membrane potential . these exciting findings will have application in deciphering the molecular mechanism(s ) involved in curcumin action and in delineating the signal transduction pathway relevant to sperm motility and function . also , this data may have clinical application in development of a novel non - steroid contraceptive with spermicidal properties . the incidence of splenic abscess is a clinically uncommon disease with current literature reporting a 0.140.7% occurrence rate [ 1 , 2 ] . although splenic abscesses are a clinical rarity , they have the potential to be fatal . the presentation of this disease is often vague and insidious including left upper quadrant abdominal pain , fever and chills . additionally , these patients may present with leukocytosis , left upper quadrant mass and pleural effusion on chest x - ray . splenic abscesses generally occur in patients with underlying comorbidities , which commonly include neoplasia , immunodeficiency , trauma , metastatic infection , splenic infarct or diabetes . the best management of splenic abscesses is still debatable with the various modalities including antibiotics , percutaneous drainage or splenectomy . the current literature supports a 67100% success rate with percutaneous drainage ; however , tung et al . states that the most optimal treatment for splenic abscess is splenectomy . of the 600 cases of splenic abscess documented in the current literature , most have been described as air confined to the left upper quadrant on chest x - ray . we report a young patient with a ruptured splenic abscess resulting in an acute abdomen and pneumoperitoneum . to our knowledge , there have only been four other reported cases of ruptured splenic abscess causing pneumoperitoneum [ 3 , 4 ] . a 48-year - old female with the past medical history of diabetes , coronary artery disease and psoriasis , presented with acute onset of diffuse abdominal pain 2 days prior to admission to the hospital . upon presentation , she was tachycardic with a heart rate of 130 and hypotensive with a blood pressure of 78/45 . she was resuscitated in the emergency department with intravenous fluids and her blood pressure and heart rate responded appropriately . her initial labs showed a normal wbc level of 8.3 thou / mcl ; however , the patient was acidotic with a ph of 7.29 and a lactic acid level of 3.5 mmol / l . an acute abdominal series was obtained demonstrating free air below the right hemidiaphragm ( fig . 1 ) . the patient was subsequently boarded for an exploratory laparotomy with repair of perforated viscus , as that is the usual cause of pneumoperitoneum , especially under the right hemidiaphragm . because the patient 's vital signs stabilized after being resuscitated , the decision was made to obtain a ct scan to better assess the location of the perforated viscus . much to our surprise , the patient did not have a perforated viscus , but a splenic abscess that had ruptured causing the pneumoperitoneum ( fig . 2 ) . figure 2:ct scan demonstrating gas - forming splenic abscess and free air in the peritoneal cavity . ct scan demonstrating gas - forming splenic abscess and free air in the peritoneal cavity . the patient then became increasingly confused and her vital signs again deteriorated demonstrating worsening sepsis . the patient was taken to the operating room where a laparoscopic splenectomy was attempted but was quickly converted to laparotomy with splenectomy due to the gross contamination of the abdomen . the patient was continued on antibiotics and taken to the intensive care unit for post - operative care . the splenic abscess grew prevotella intermedia , a bacterium commonly found in the oral flora . the patient underwent a full work - up looking for the source of the splenic abscess . a transesophageal echocardiogram was performed but was negative for any masses , thrombus or vegetation . furthermore , a panorex was performed and was negative as the patient had reported tooth pain 1 week prior to her admission . she returned to the clinic on post - operative day 14 to receive her splenectomy vaccinations . although a rare disease , splenic abscess should be included on the differential diagnosis of a patient presenting to the hospital with peritonitis or pneumoperitoneum . this is especially true for patients who are immunocompromised or have underlying comorbidities including neoplasia , diabetes , trauma or history of splenic infarct or embolization . in our case , ct scan provided important information about the cause of our patient 's pneumoperitoneum , as we had assumed it was due to a perforated ulcer or diverticulitis . this did change our operative management , as we attempted to perform a less invasive surgery on our patient by starting with a laparoscopic technique . however , we had to convert to an open splenectomy due to the gross contamination and adhesions from the splenic abscess . in retrospect , an open laparotomy was the best approach to this patient 's care as it allowed for better visualization and irrigation of the abdominal cavity . it is unknown if we had successfully performed the splenectomy laparoscopically if it would have decreased recovery time . the current literature supports the laparoscopic splenectomy as a safe and effective procedure in patients with splenic abscess demonstrating an average length of stay of 14 days . in our case , the patient remained in the hospital for only 10 days after converting to the open procedure . the current literature reports that the most common organisms found in splenic abscesses are aerobic microbes , particularly streptococci and escherichia coli . this microbe is commonly found in the oral flora and is associated with periodontal disease . although our transesophageal echocardiogram and panorex were both negative , we believe this splenic abscess developed as a result of translocation of the oral flora as the patient was complaining of a tooth ache 1 week prior to her presentation . in summary , we have presented a case of ruptured splenic abscess as a cause of pneumoperitoneum and peritonitis . although rare , we encourage splenic abscess be included on the differential diagnosis of patients presenting with peritonitis and pneumoperitoneum . although there is still controversy in the current literature when managing splenic abscesses , our recommendation is splenectomy , especially in the case of ruptured splenic abscess resulting in hemodynamic instability . whether it is better to perform the splenectomy laparoscopically or open is still debatable , the important thing is to obtain source control . furthermore , a full work - up is required for patients presenting with a splenic abscess as the underlying cause could cause more serious illnesses for the patient in the future . there are no conflicts of interests or financial funding to disclose for any of the contributing authors .

backgroundcurcumin has shown to affect sperm motility and function in vitro and fertility in vivo . the molecular mechanism(s ) by which curcumin affects sperm motility has not been delineated . since modulation of intracellular ph ( phi ) and plasma membrane polarization is involved in sperm motility , the present study was conducted to investigate the effect of curcumin on these sperm ( human and murine ) parameters.methodsthe effect of curcumin on sperm forward motility was examined by counting percentages of forward moving sperm . the effect of curcumin on intracellular ph ( phi ) was measured by the fluorescent ph indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein - acetoxymethyl ester ( bcecf - am ) . the effect of curcumin on plasma membrane polarization was examined using the fluorescence sensitive dye bis ( 1,3-dibarbituric acid)-trimethine oxanol [ dibac4(3)].resultscurcumin caused a concentration - dependent ( p<0.05 ) decrease in forward motility of both human and mouse sperm . it also caused a concentration - dependent decrease in intracellular ph ( phi ) in both human and mouse sperm . curcumin induced significant ( p<0.05 ) hyperpolarization of the plasma membrane in both human and mouse sperm.conclusionthese findings indicate that curcumin inhibits sperm forward motility by intracellular acidification and hyperpolarization of sperm plasma membrane . this is the first study to our knowledge which examined the effect of curcumin on sperm phi and membrane polarization that affect sperm forward motility . these exciting findings will have application in deciphering the signal transduction pathway involved in sperm motility and function and in development of a novel non - steroidal contraceptive for infertility . we encountered a case of ruptured splenic abscess presenting as peritonitis and pneumoperitoneum . our patient did not have an underlying neoplasm nor was she immunosuppressed . in our case , splenectomy was the treatment of choice in combination with antibiotics , which proved to be a good outcome for the patient . work - up for the cause of the abscess was negative , although bacteria predominately found in the oral flora were isolated from the abscess . we strongly encourage that splenic abscess be considered on the differential diagnosis of patients presenting with pneumoperitoneum and peritonitis , although a clinical rarity .

squamous cell carcinoma of the head and neck ( scchn ) is the fifth most common neoplasm with an estimated annual global incidence of more than 500,000 cases diagnosed worldwide . the treatment is usually interdisciplinary and mainly involves surgeons , radiation oncologists , medical oncologists , clinical nurse specialists , speech and language specialists , and dieticians . dependent on the situation , goals of the treatment can be to obtain ( i ) a high locoregional control and survival rates in patients with limited disease , ( ii ) an increased survival in patients with advanced disease ( improved locoregional control , reduced probability of distant metastasis , and second malignancies ) , ( iii ) an increased organ - function preservation in resectable and unresectable tumors , and ( vi ) an increased therapeutic ratio ( cure / toxicity ratio ) . single modality treatment is recommended for the patients with early - stage disease ( stage i or stage ii , approximately 40% of the patients with scchn ) and combined modality treatment for patients with locally advanced disease . the combined modality treatment may include surgery followed by adjuvant radiotherapy or radiochemotherapy , concomitant radiochemotherapy ( using conventional or alternative fractionation regimen ) , and induction chemotherapy followed by radiotherapy or radiochemotherapy [ 3 , 4 ] . a precise understanding of prognostic factors is important to select the optimal treatment for the individual patient or to stratify patients for clinical trials or statistical analyses . in this retrospective single - institutional study , the role of potential prognostic factors was evaluated and compared in patients with squamous cell carcinoma of the oro- and hypopharynx after treatment with definitive radiotherapy / radiochemotherapy ( drt ) versus surgery followed by postoperative radiotherapy ( prt ) . between 1992 and 2000 , 288 patients with squamous cell carcinoma of the oropharynx or hypopharynx received a radiation therapy as definitive ( n = 162 ) or as postoperative ( n = 126 ) treatment . eligibility criteria for this retrospective single - institutional study were histologically proven squamous cell carcinoma of the oropharynx or hypopharynx , no distant metastasis or synchronous cancer at time of diagnosis , and definitive or postoperative radiotherapy with a minimum total dose of 60 gy . one hundred and thirty - eight of 162 ( 85% ) patients treated with definitive radiotherapy received a concomitant boost fractionation regimen , and 24 ( 15% ) were treated with conventional fractionation ( single fraction dose of 2 gy , one fraction a day , five fractions a week ) . two concomitant boost regimen were used : regimen 1 consisted of a total dose of 66 gy in five weeks with a daily fraction dose of 2 gy and a concomitant boost of 1.6 gy during the last two weeks ( n = 76 ) and regimen 2 of a total dose of 69.9 gy in 5.5 weeks with a daily fraction dose of 1.8 gy and a concomitant boost of 1.5 gy during the last 2.5 weeks ( n = 62 ) . ninety - four of 138 ( 68% ) patients treated with concomitant boost fractionation regimen received a simultaneous chemotherapy as well as five ( 4% ) patients treated with conventional fractionation . the simultaneous chemotherapy consisted of 70 mg / m carboplatin on days 15 and 2933 ( n = 73 ) or 70 mg / m carboplatin and 600 mg / m 5-fluorouracil on days 15 and 2933 ( n = 26 ) . carboplatin was administered as a daily short - term intravenous infusion and 5-fluorouracil as an intravenous continuous infusion for 120 hours . for the postoperative radiotherapy , only conventional fractionation without simultaneous chemotherapy was used ( n = 123 ) . the radiotherapy was performed with opposed lateral fields for the upper neck and one anterior field for the lower neck using 6 mev photons . patients were treated in a thermoplastic mask for immobilization , and individual blocks were used to spare normal tissue where possible . after a dose of 30 to 36 gy to the reference point , the spinal cord was spared out of the photon fields and the uninvolved posterior neck treated with electrons of selected energy according to ct findings with daily doses of 2.5 gy five times a week to the prescribed total dose . target volumes were defined on ct scans , and the dose was calculated to midplane . in selected patients , pre - treatment ct scans of all patients were digitized with an automatic laser scanner ( fips plus ) . the stored images were transferred to a personal computer . the macroscopic tumor shape ( primary tumor and locoregional lymph node metastases ) was defined in every ct slice ( slice thickness 5 mm or 8 mm ) using a drawing tool ( software : photostyler ) . the number of pixels n enclosed by this contour was determined with a custom - shaped image processing program ( software : interactive data language ) . the area ai of the ith slice was determined as ai = pixel size ( length)pixel size ( width)n . the pixel size was determined using the scaling as given on the ct - hardcopy . the determined tumor area of each slice was multiplied with its slice thickness di . the ttv was approximated by ( 1)vtumor(cm3)=i1mai(mm2)di(mm)/1000 . repeated measurements using irregularly shaped tumor phantoms showed a difference between the reference volumes and ct - based volume measurements depending of the slice thickness ( 5 mm or 8 mm ) of 1.4% to 4.5% . in addition to the quantitative tumor volumetry based on digitised pretreatment ct scans , ttv was estimated based on the postoperative histopathological report in patients treated with adjuvant radiotherapy . the ttv was approximated using the equation v = 4/3 abc , where a , b , and c represent the orthogonal maximal tumor diameters . the overall survival was defined as the time between the first day of the treatment and death of any cause . the overall survival was estimated using the kaplan - meier method , and treatment groups were compared using a two - sided log rank test . the locoregional failure - free survival was defined as the time between the first day of the treatment and a locoregional failure . the distant metastasis - free survival was defined as the time between the first day of the treatment and a distant failure . the locoregional failure - free survival and the distant metastasis - free survival were estimated by the cumulative incidence method , and treatment groups were compared using the gray test [ 5 , 6 ] . the simultaneous relationship of multiple prognostic factors to overall survival was assessed using cox 's proportional hazard regression analysis . the simultaneous relationship of multiple prognostic factors to locoregional failure - free or distant metastasis - free survival was assessed using the hazards of the cumulative incidence function model . to estimate the reliability of the tumor volumetry , the ttv derived from digitised pretreatment ct scans was compared with the ttv derived from calculations based on tumor diameters provided by the postoperative histo - pathological report of the same patients . for the method comparison , the median follow - up time for the censored patients treated with definitive radiotherapy / radiochemotherapy was 28.5 months and for the patients treated with adjuvant postoperative radiotherapy 36.5 months . the 5-year overall survival for the patients treated with definitive radiotherapy / radiochemotherapy was 0.27 ( 95% ci 0.180.35 ) and for the patients treated with postoperative radiotherapy , 0.69 ( 95% ci 0.590.79 ) . patients treated with definitive radiotherapy / radiochemotherapy had a greater proportion of stage 4 tumors ( 96.9% versus 66.9% ) , a much larger median ttv ( 68.4 cm versus 21.2 cm ) , and a lower proportion of preradiotherapy hemoglobin level 12 g / dl ( 17.6% versus 42.6% ) . the performance status was not considered in this study because it was not consistently documented in the patient files . on univariate analysis , the ttv ( figures 1 and 2 ) and the pre - radiotherapy hemoglobin level had a statistically significant impact on the overall survival and on the locoregional control of patients treated with definitive radiotherapy / radiochemotherapy . simultaneous chemotherapy had a statistically significant effect on the overall survival and on the incidence of distant metastasis . in addition , there was a statistical trend of an association of simultaneous chemotherapy with the locoregional control ( p = 0.08 ) . the n - classification had a statistically significant impact on locoregional control but no significant impact on the overall survival or metastasis - free survival . on multivariate analysis , only the ttv and the simultaneous chemotherapy maintained their statistical significance ( table 3 ) . on univariate analysis , the ttv had a statistically significant impact on the locoregional control ( figure 3 ) . the n - classification had a statistically significant impact on the incidence of distant metastasis and a statistical trend on the locoregional control ( p = 0.06 ) . on multivariate analysis , only the ttv ( p = 0.05 ) maintained its statistical significance ( table 5 ) . the ttv was estimated using quantitative tumor volumetry of digitized pre - treatment ct scans in all patients . in addition to the ct - based volumetry , in 34 patients , the ttv was also estimated based on tumor diameters reported in the histo - pathological report . the tumor volumes based on the two methods were compared to estimate the precision of the tumor volume measurements . the method comparison of the 34 patients showed that the 95% limit of agreement between the two total tumor volume measurements was approximately 150% of the average total tumor volume measurement of both methods ( figure 4 ) . this retrospective single institution analysis investigated possible prognostic factors of patients with squamous cell cancer of the oro- and hypopharynx treated with ( i ) definitive radiotherapy / radiochemotherapy or ( ii ) surgery followed by postoperative radiotherapy . the two patient groups were analysed separately because they differed considerably in respect to patient- and treatment - related characteristics and prognosis . the tumor volume has been stated to be one of the most precise and most relevant predictor of radiotherapy outcome . for patients with oro- and hypopharyngeal cancer treated with definitive radiotherapy or radiochemotherapy , the quantitative tumor volume was identified as significant prognostic factor in the majority of studies [ 1020 ] , and in few studies , as a prognostic factor of marginal [ 21 , 22 ] or no significance . in the multivariate analysis of our study , the total tumor volume had a statistically highly significant impact on the overall survival and locoregional control on patients treated with definitive radiotherapy / radiochemotherapy . a new finding of our study is that the total tumor volume also had a statistically significant impact on the locoregional control in patients treated with surgery followed by postoperative radiotherapy . for this patient group , our data suggest that the total tumor volume should be used to select patients for an intensified definitive or postoperative adjuvant treatment . patients treated with adjuvant simultaneous radiochemotherapy compared to adjuvant radiotherapy alone [ 24 , 25 ] . a comparative analysis of both studies revealed the extracapsular extension of tumor from neck nodes and/or microscopically involved surgical margins as significant clinical risk factors for poor outcome . the only other significant prognostic factor in the multivariate analysis for patients treated with definitive radiotherapy in our study was the application of a simultaneous chemotherapy . the simultaneous chemotherapy was significantly associated with an improved overall survival and distant metastasis - free survival and showed a statistical trend of an improved locoregional control ( p = 0.15 ) . randomized clinical trials [ 2735 ] and meta - analyses [ 36 , 37 ] have shown a significantly improved local control and survival with definitive simultaneous radiochemotherapy compared to definitive radiotherapy alone in patients with advanced squamous cell cancer of the head and neck . interestingly , in our study , the preradiotherapy hemoglobin level was a significant prognostic factor in the univariate analysis of patients treated with definitive radiotherapy / radiochemotherapy but lost its statistical significance in the multivariate analysis . if the total tumor volume was removed from the multivariate model , the pre - radiotherapy hemoglobin level retained its significance . our data suggest that the total tumor volume is the stronger of both prognostic factors . a significant association of the pre - radiotherapy hemoglobin concentration with the treatment outcome after definitive radiotherapy / radiochemotherapy in the absence of quantitative tumor volume data has been reported by several studies in the literature [ 3846 ] . for patients treated with postoperative radiotherapy , the pre - radiotherapy hemoglobin level showed no prognostic significance in our study . in the literature , one study found a significant impact of the pre - radiotherapy hemoglobin level on survival in patients with head and neck cancer treated with adjuvant radiotherapy . other studies evaluated the prognostic significance of the hemoglobin level at different time points during the treatment . in these studies , no prognostic significance was found for the pre - radiotherapy hemoglobin level , but for the hemoglobin level before surgery , after surgery [ 49 , 50 ] , duration of low hemoglobin level during the interval between surgery and radiotherapy , or for the difference of the hemoglobin concentration before and after adjuvant radiotherapy . several studies using po2 histography have shown an impact of the tumor oxygenation on the survival of patients with head and neck cancer after radiotherapy [ 5255 ] , but no clear correlation was found between the tumor oxygenation by means of po2 histography and the hemoglobin concentration [ 56 , 57 ] . on multivariate analysis , the potential prognostic factors , gender , age , pre - radiotherapy hemoglobin level , tumor site , t- and n- , and r - status , rt - interruptions > 5 days , and interval surgery - rt > 32 days , showed no statistical significance in our study . other prognostic factors on multivariate analysis for locally advanced head and neck cancers reported by other studies were the performance status [ 45 , 46 ] , high - grade acute organ toxicity , or the uicc stage . our data suggest that the total tumor volume is the predominant prognostic factor in patients with squamous cell cancer of the oro- and hypopharynx treated with definitive radiotherapy / radiochemotherapy or surgery followed by postoperative radiotherapy . the total tumor volume should be used to identify high - risk patients and to stratify patients in clinical trials or statistical analyses . measures of intellectual abilities and vocabulary , the crystallized abilities , are more resistant , to age effects than fluid abilities , such as attention and executive functions . memory functions seem most affected , particularly those related to source memory ( episodic or explicit ) . women perform better on verbal and memory tasks , whereas men excel in spatial tasks . however , sex differences in aging effects have not . some evidence suggests that , women show less age - associated cognitive decline than men . our data , on young adults ( age 1 8 - 45 years ) indicate that men show significant , decline in several neurocognitivc domains while women evince no decline . we have initially studied sex difference in neurocognitivc measures with a standardized battery that , examines 8 neurobehavioral domains . in a sample of 241 healthy young adults , aged 18 to 45 ( 124 men , 117 premenopausal women ) , women had better verbal memory , and men performed better on spatial and motor tasks . however , we did not . observe better performance in women for the language domain ( figure 1 ) . in examining components of domains that show sex differences , we find that the verbal memory advantage for women is accounted for primarily by performance on the california verbal learning test ( cvlt/ figure 2 ) , whereas the advantage for men in spatial abilities is accounted for by performance on benton 's line orientation test . some sex differences are manifested not in the profile of abilities , but in the effects of aging within the range of young adulthood . in our sample , no significant correlations were observed between age and any of the behavioral measures in women ( correlations ranged from -0.15 to 0.09 ) . for men , however , increased age was associated with decrease in performance on attention ( r=0.43 , p=0.0001 ) , verbal memory ( r=-0.20 , p=0.029 ) , spatial memory ( r=-0.34 , p=0.0001 ) , and spatial abilities ( r=-0.33 , p=0.0002 ) ( all df=122 , all p values are 2-tailed ) ( figure 3 ) . and ( figure 4 ) . the battery is lengthy , and each test provides measures that are difficult to link with current , knowledge on brain systems regulating behavior . furthermore , tests comprising such batteries are not readily applied in functional imaging studies , and few alternative forms are available for repeated testing . to address these limitations our general approach to task development and validation process was detailed in gur et al . advantages of the computerized battery include : ( i ) each measure is designed to probe a narrow and well - defined neurobehavioral domain ; ( ii ) more uniform presentation of test stimuli ; ( iii ) errorless data entry and scoring ; ( iv ) availability of reaction time data ; ( v ) shorter time for administration ; and ( vi ) alternative forms can be readily generated using set algorithms . is more impersonal ; ( ii ) some participants , particularly the elderly , dislike computers or require training ; and ( iii ) tests are not . yet available for some well - validated indices of language functioning , particularly those involving verbal output ( eg , vocabulary , verbal fluency ) . however , our experience with computerized testing indicates that the first two disadvantages can be overcome , and the third can be addressed with available technology . most older adults respond well to computerized testing , if approached properly , and we have developed a short module that trains participants in the use of the mouse to the level required for testing . the normative data have shown very favorable psychometric characteristics such as high inter - item consistency ( cronbach 's alpha ) , test - retest reliability , and comparable levels of difficulty ( at 70% to 80% correct for the normative sample ) and true - score variance . our efforts to generate the kind of sensitivity that , will permit , differentiation within healthy people have also been successful . as can be seen in figure 5 , the summary scores show sex differences in young adults . the pattern of sex differences duplicates that obtained with traditional batteries , but adds the finding that women do better in facial memory , not available in the traditional battery . measures of reaction and testing time provide an efficiency ( accuracy / time ) index , used to calculate comparable z scores across tests . algorithms can generate multiple forms for repeated administration , and error analysis is performed for items and parameters to examine strategy and persistence . perceiving , experiencing , and expressing emotions seem essential capacities , and more recently the study of emotion has benefited from converging methodologies in animals and humans . the face has been the main target of study in humans , and methods were applied to quantify expression of emotion with cross - cultural consistency standardized tools have been developed for measuring emotion discrimination , mood induction , affective valence , and arousal . emotional displays that , can be reliably coded in the face are happiness , sadness , anger , fear , and disgust ( surprise is more controversial ) . seems to be organized , perhaps parallel to the cognitive system , along laterality and anterior - posterior dimensions . ' ittiere is controversy about whether emotional expression is lateralized , although a meta - analysis by borod et al seems to confirm that negative emotions are expressed more intensely on the left side of the face , whereas the opposite holds for positive emotions : there is more agreement , though fewer data , that , receptive , experiential , and expressive aspects of emotion processing can be mapped to frontal , temporal , and parieto - occipital involvement , respectively . this interaction between the emotion and cognitive systems , particularly as it applies to memory , is an issue of current interest . large - scale studies with standardized measures have indicated that elderly people are in better mood than their young counterparts . gross et al examined cross - cultural samples for age differences and concluded that older adults reported fewer negative emotional experiences and greater emotional , control . however , findings regarding emotional expressivity were less consistent , with older participants reporting less expressivity . there is also evidence that the elderly are more vulnerable than the young to adverse effects of negative emotional states on memory and other cognitive abilities . indeed , it has been suggested that depressed mood is the strongest predictor of health decline in the elderly . women perform better in speeded emotion recognition tasks and in tasks requiring facial expression of emotions . in a study of facial recognition , we reported sex differences in sensitivity to happy and sad expressions depending on the poser 's sex . women were more sensitive to opposite than to same - sex expressions , whereas men were differentially poor at detecting sadness in female faces . regarding emotional experience , women are more prone to clinical depression , mood fluctuations associated with phases of the menstrual cycle have been documented , and such phase - associated hormonal changes may relate to cognitive performance . magnetic resonance imaging ( mri ) studies of the brain have documented that , aging is associated with progressive parenchymal volume decrease and cerebrospinal fluid ( csf ) volume increase . some studies suggest that the volume decrease with age is in gray matter ( gm ) tissue , while others report a decrease also in the white matter ( wm . ) compartment . there is more consistency in the regional distribution of effects , with mesolimbic , temporal , and frontal regions showing greater vulnerability . sex differences have been observed in the compartmental composition of intracranial volumes , in the volume and density of language - associated cortex , and in the rate of age - associated changes . while the data indicate less parenchymal loss in women than in men , particularly for frontal and temporal regions , samples were limited in the elderly cohorts . our data in the elderly suggest , similar rates of tissue loss in men and women , perhaps reflecting an acceleration following menopause . henkel et al concluded that age - related decline in source memory affects processes involved in binding features into complex memories and [ ... ] contextual features of memories . neural substrates for the source memory system implicate the mesial temporal and frontal regions . henkel ct al 's hypothesis , based on behavioral data , is supported in raz et al 's study . while the volume of limbic structures was unrelated to cognitive functions across the age range , in older participants reduction predicted declines in explicit memory . neural substrates for age - related changes in affect are less clearly defined , although recent work affords some hypotheses that , should be tested . studies on networks for emotion have implicated the amygdala , hypothalamus , mesocorticolimbic dopaminergic systems , and projections to orbital and dorsolateral frontal , temporal , and parietal cortex . studies in patients with brain lesions support , the role of these regions in emotion regulation . the link between age - related changes in neuroanatomy and performance on such tasks has not been established . to evaluate how these effects extend across agegroups , the correlation between age and total intracranial volume was nil ( r=0.02 ) , indicating no secular drift in head size . for the young ( < 50 years ) sample considered separately , there was a. small yet significant correlation between age and gm volume ( r=-0.17 , df=130 , p<0.05 ) . this correlation was higher in men ( r=-0.27 , df=74 , p<0.01 ) than in women ( r=-0.01 , df=54 , ns ) . age did not correlate significantly with wm or csf volumes in this age - group . for the entire age range , gm continues to decline in volume with aging , r(184)=-0.49 , r(92)=-0.52 , and r(90)=-0.40 for the whole sample , males and females , respectively , all p<0.0001 , whereas the volume of csf continues to increase with age ( the corresponding correlations were 0.31 , 0.45 , and 0.29 , all p<0.0001 ) . wm changes are less clear . although the correlation with age is not significant for the entire sample , r=0.09 , within each sex the correlations were small but positive , r=0.28 , p=0.01 and r=0.24 , p=0.02 . we conclude that gm volume is reduced with aging , csf volume increases concomitantly , while wm volume does not change appreciably and is perhaps increased slightly . while the effects of gm and csf agree with a recent study by guttmann et al , their conclusion regarding wm . was that its percentage of the intracranial compartments , it appears that they examined only percentages and not raw volumes . in the case of guttmann et al 's study , reduced % wm could reflect age effects on another compartment , for example , increased csf . perhaps the paucity of subjects in our elderly group is matched by the paucity of theirs in the young group ( 10 participants < 40 years ) . our sample also enabled examination of whether these volume changes are related to cognitive performance . while the young and elderly participants received age - appropriate neuropsychological batteries , both groups received several identical tests . the total number of items recalled during the memorization trials was correlated with the volumes of brain parenchyma ( gm . as can be seen in figure 6 , parenchymal volume goes down with age , number of words recalled also declines , and the parenchymal volume is associated with memory ( even with age effects partialled out ) . the feasibility of studying neural substrates of behavior is enhanced by functional imaging methods for measuring regional brain activity . activation patterns are linked to performance on cognitive tasks requiring verbal , spatial , attention , memory , and facial processing . the study of age effects on regional brain physiology has been quite extensive , with considerable agreement that cerebral blood flow ( cbf ) shows age - related decline even in people screened for cerebrovascular disorders . similarly , measures of neurotransmitter function have shown reduced availability of both dopaminergic and serotonergic transmitter activity . the decline has been linked to performance on behavioral tasks related to the dopaminergic system . the effects of aging on glucose metabolism are less clear , although it too shows decline , particularly in frontal and temporal regions . of most relevance to the study of the memory and emotion systems , cherrier et al have examined correlations between mood state self - ratings and cerebral metabolism during pet 18f - fdg in 27 persons with age - associated memory impairment ( aged 44 - 81 years ) . specifically , regions involved in both mood and memory had similar abnormalities . there have been reports of boredom related to mesial temporal and parietal asymmetry as well as decreased parietal metabolism . thse findings suggest that , mood changes may influence metabolism in brain regions implicated in emotion and memory function . there are fewer data on the effects of aging on regional brain activation in response to task demands . using the 133xe method , we found substantial age - related decline in overall cbf values , but the pattern of lateralized changes in response to verbal and spatial tasks was identical in young and elderly people . however , we used tasks that are quite resistant to age effects , and the results may differ for tasks showing greater age - related decline . indeed , cabeza ct al found less frontal activation , measured with h2 0 pet cbf , in elderly people for a memory task . madden et al in a cbf study using spect reported that several cbf activations were greater for young than for older adults . however , they observed that , although performance demonstrated a greater age - related slowing for visual encoding than for semantic retrieval , these changes were not associated with corresponding changes in rcbf activation . the association between cbf activation and performance , and the effects of aging on this association , merit further elucidation . several studies examined emotion processing with functional neuroimaging methods in healthy people . with the xe method for measuring cortical cbf during processing emotional facial expressions , we noted increased right temporoparietal activity . with a mood induction procedure , increased cbf was observed during sad induction and correlated with mood change . in a pet study with h2 0 , we reported a cbf increase in the left amygdala and a cbf decrease in the right amygdala during sad mood . subsequent studies with pet and functional mri ( fmri ) have confirmed the lesion data , implicating amygdala and anterior brain regions in mood , while posterior regions seem activated in visual emotion discrimination . unlike neuroanatomic studies that have consistently examined sex differences and age effects , there is a paucity of neurophysiologic studies that have examined these factors . using the xe cbf method , we reported that women have higher rates of cortical cbf , and this finding was replicated and extended to other methods that measure cbf for the entire cranium . we found about equal rates of age - associated reduction in cbf in men and women , and this has also been reported in other studies , although samples were usually small in the elderly range . no studies have linked changes in cbf activation to sex differences in agerelated neuroanatomic changes and to performance of memory and emotion tasks . the method has several potential advantages : higher spatial and temporal resolution , noninvasiveness and lack of ionizing radiation , direct correlation with anatomical imaging , greater repeatability , and economy . the disadvantages of fmri techniques include : loud background noise generated by the gradients ; difficulties in presenting stimuli and performing tasks in the magnet , bore ; claustrophobia ; low signal - to - noise for most methods ; and lack of quantification in physiologic units for most methods . among the various fmri methods , blood oxygenation level - dependent this technique relies on magnetic susceptibility effects of deoxyhemoglobin , which cause regional decreases in signal in imaging sequences sensitive to susceptibility ( eg , echoplanar ) . with regional brain activation studies , a net increase in signal intensity is observed in regions known to be activated by the task . this is attributed to an increase in regional blood flow compared to regional oxygen consumption . a typical response is a 1 % to 25% increase in regional image intensity , which develops over 3 to 8 seconds following task initiation . susceptibility effects are field dependent so , using the 4-t magnet , available to us , an initial decrease in signal intensity is detectable in the first 1 to 2 seconds following stimulation , corresponding to a focal increase in deoxyhemoglobin . when combined with ultrafast echoplanar imaging ( -100 ms per slice ) , the time course of signal change in response to individual stimuli can thus be observed . to evaluate task - induced regional activation , we have applied a verbal and a spatial task previously demonstrating regional activation with other methods . this study examined activation for a spatial task ( judgment , of line orientation ) compared with a. verbal reasoning task ( analogies ) in a sample of 29 healthy participants ( 15 men and 14 women ) . an image - based multisubject analysis was performed by registering the brains of the different subjects . a well - characterized brain registration algorithm was used to register the tl images from the different subjects to that of one particular subject . once registered , the statistics were summed across subjects and divided by the square root of the number of subjects , as is appropriate for independent , normally distributed variables . the statistical images were smoothed by convolution with a gaussian kernel with full width at half maximum of 12 mm and thresholded at a. p value of 0.05 corrected for multiple comparisons using the theory of gaussian random fields . the activation map in figure 7 indicates that the hypothesized left - lateralized changes were seen for the verbal task in posterior temporal and inferior parietal regions , while right - latcralizcd increase was seen for the spatial task in these regions . this imagebased analysis revealed a. distributed network of cortical regions , which expanded for the hard verbal task and became more circumscribed for the hard spatial task . the task by hemisphere interactions for the hypothesized inferior parietal , superior temporal , and planum temporale regions were significant at an order of magnitude comparable to what we have obtained with other methods ( all f<0.001 ) . appears that spatial processing requires , for harder tasks , greater reliance on visual association cortex with minimal activation of other areas . poorer performance in women may relate to continued reliance on supplementary strategies , perhaps verbal , which are ineffective for the success on the harder spatial items . effects of aging on regional activation in relation to cognitive strategics have not yet been examined with fmri . as is generally the case for age - related changes , the hormonal environment , can have pervasive effects that require scrutiny , not only during early development , but also during the perimenopausal phase . menopause is a single event in a progressive process of ovarian aging beginning with increased frequency of menstrual disturbances and anovulation as follicular units are depleted . the process is accelerated after age 37 , ultimately culminating in the virtual absence of follicles and capacity to generate significant quantities of estradiol . the median chronological age at menopause in the usa is 51.4 years ( range 48 to 55 years ) . estrogen decrease is associated with substantial central nervous system ( cns ) alterations including vasomotor instability , insomnia , depression , and cognitive decline . recent , studies suggest that estrogen has a protective effect with respect to onset of alzheimer 's disease and cognitive decline . there is evidence that neurobiologie processes triggered by the hormonal changes exert influence by affecting neurotransmitter availability , cerebral perfusion , and perhaps by eliminating neuroprotective effects of estrogen . in a recent study by matteis et al , using transcranial doppler ultrasonography , they found , as we did , higher flow estimates in women than men overall . however , a subgroup of 15 postmenopausal women aged 48 to 53 years had lower flow values than 15 premenopausal women of the same age , or any other group . there is increasing evidence across behavioral , neuroanatomic , and neurophysiologic domains that sex differences play a prominent role in modulating the effects of aging on brain function . the overall finding is that age - related decline begins earlier in men than in women . the decline is most pronounced in frontotemporal regions associated with attention , inhibition , and memory . more specific tasks using a computerized approach can help better delineate associations between agerelated decline and aspects of cognitive and emotion processing . the sex differences in brain aging may be further investigated on the molecular level and data on other physiologic parameters , such as glucose and oxygen metabolism and receptor function , could help further elucidate mechanisms for explaining these differences . such studies could ultimately help explain a range of phenomena related to sex differences including cognition and emotion processing . although we have focused on findings in healthy people , the effects have implications for brain disorders where gender differences have been observed across the life span . for example , neurodevelopmental disorders such as attention deficit and learning disabilities are more common in boys , schizophrenia is more severe in young men , and depression is more common in women . understanding the neural basis of these disorders can be advanced by considering sex differences in brain function . the clinical implications of these findings need to be examined in relation to disease presentation and course . in view of the greater vulnerability of males in prefrontal regions , one would expect , brain disorders affecting these regions to be more severe and perhaps requiring multimodal therapeutic interventions . for females , with improved understanding of regional brain activity during emotion processing , we maybe in a position to explain the neurobiology of increased vulnerability to depression . finally , the measures employed in this work seem sensitive to variability in healthy people and may therefore serve as endophenotypic markers of vulnerability to neuropsychiatrie disorders in which sex differences are evident and may contribute to developing genetic models .

purpose . to compare the impact of prognostic factors of patients treated with definitive radio(chemo)therapy versus patients treated with surgery and postoperative radiotherapy for squamous cell carcinoma of the oro- and hypopharynx . patients and methods . 162 patients treated with definitive radiotherapy and 126 patients treated with postoperative radiotherapy were retrospectively analysed . the impact of the prognostic factors gender , age , total tumor volume ( ttv ) , pre - radiotherapy hemoglobin level ( hb - level ) , tumor site , t- and n - classification , radiotherapy interruptions > 5 days , radiotherapy versus simultaneous radiochemotherapy , r - status and time interval between surgery and radiotherapy were investigated . results . the median follow - up time for the censored patients treated with definitive radio(chemo)therapy was 28.5 months and for postoperative radiotherapy 36.5 months . on univariate analysis , the ttv , hb - level , and simultaneous radiochemotherapy had a significant impact on the survival of patients treated with definitive radio(chemo)therapy . for patients treated with postoperative radiotherapy , only the ttv showed a statistical trend for the survival ( p = 0.13 ) . on multivariate analysis , the ttv and simultaneous radiochemotherapy maintained their statistical significance for patients treated with definitive raditherapy , and the ttv , the statistical trend for patients treated with postoperative radiotherapy ( p = 0.19 ) . conclusions . the ttv was the predominant prognostic factor for both , patients treated with definitive or postoperative radiotherapy . gender and aging moderate brain - behavior relationships . advances in neuroscience enable integration of neurobehavioral , neuroanatomic , and neurophysiology measures . here we present neurobehavioral studies thai examine cognitive and emotion processing in healthy men and women and highlight the effects of sex differences and aqinq . neuroanatomic studies with maqnetic resonance imaging ( mri ) indicate that the progressive decrease in brain volume affects froniotemporal brain regions in men more than in vi / omen , functional imaging methods suggest sex differences in rate of blood flow , pattern of glucose metabolism , and receptor activity . the role of ovarian hormones is important in elucidating the observed relationships . a life span perspective on gender differences through the integration of available methodologies will advance understanding healthy people and the effects of brain disorders .

the purpose of this process is to destroy and remove the injurious agents and injured tissues , thereby promoting tissue repair . when this beneficial response occurs in an uncontrolled manner , the result is excessive cellular / tissue damage that results in chronic inflammation and destruction of normal tissue . moreover , inflammatory airway and lung diseases , such as asthma or chronic obstructive pulmonary disease ( copd ) , are characterized by chronic inflammation . many of the known inflammatory target proteins , such as matrix metalloproteinase-9 ( mmp-9 ) , intercellular adhesion molecule-1 ( icam-1 ) , vascular cell adhesion molecule-1 ( vcam-1 ) , cyclooxygenase-2 ( cox-2 ) , and cytosolic phospholipase a2 ( cpla2 ) , are associated with inflammatory signaling pathways induced by various stimuli , including tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , adenosine-5-triphosphate ( atp ) , cigarette smoke extract ( cse ) , lipoteichoic acid ( lta ) , or lipopolysaccharide ( lps ) [ 26 ] . airway smooth muscle is considered as an end - response effector regulating regional differences in ventilation by contracting in response to various proinflammatory mediators and exogenous substances released under homeostatic or pathologic conditions , such as asthma . lung cells , in particular alveolar epithelial type ii cells , are susceptible to the injurious effects of oxidants . it has been shown that lung cells release inflammatory mediators and cytokines / chemokines , such as il-1 , il-6 , il-8 , and tnf- in response to oxidative stress . moreover , the sfks , pkc , growth factor tyrosine kinase receptors , nadph oxidase / ros , pi3k / akt , and mapks are components of signaling cascades that respond to extracellular stimuli by targeting transcription factors , such as nf-b and ap-1 , resulting in the modulation of inflammatory gene expression . thus , this review will focus on some general aspects of inflammatory signaling regulation and summarize current knowledge regarding the presence and functional roles of these inflammatory signal molecules within the respiratory system , and their proposed involvement in the expression inflammatory target proteins in response to proinflammatory mediators during airway and lung inflammation . the pharmacological interventions protect against inflammation - induced airway and lung diseases will be discussed . leukocytes continuously circulate throughout the body in order to come in contact with antigens sequestered within tissues . to enter tissues , circulating leukocytes migrate from the blood , between vascular endothelial cells and into the tissue . during this migration , leukocytes initially bind to endothelial cells via low affinity adhesion molecules . the low affinity adhesion in combination with the force of the blood flow results in rolling of leukocytes on endothelial cells . vcam-1 is one of the inducible cell transmembrane glycoproteins of the immunoglobulin supergene family expressed on several cell types and plays an important role in a number of inflammatory and immune responses . it was first identified as an adhesion molecule induced on endothelial cells by proinflammatory cytokines or lps [ 11 , 12 ] . in normal processes , vcam-1 is important during development since a vcam-1 knockout is an embryonic lethal . in pathogenesis , vcam-1 expression is induced on endothelial cells during inflammatory bowel disease , atherosclerosis , infection , and asthmatic responses [ 1315 ] . upregulation of vcam-1 expression on cytokine - triggered vascular endothelial cells enhances the targeted transmigration of pmns into extravascular space of inflammation . in airways , to reach the submucosa and airway lumen , circulating pmns must first be recruited across the vascular endothelium and then migrate through the interstitial matrix before interacting with airway epithelium . accumulation of inflammatory cells within the airways can be influenced by expression of adhesion molecules on airway epithelium . thus , similar processes that govern pmns adhesion to lung airway resident cells may occur and contribute to the damage to these cells seen in inflammatory responses of asthma . this event is crucial in the development of allergic inflammation and is mediated by adhesion molecules and cytokines . icam-1 is an endothelial- and leukocyte - associated transmembrane protein long known for its importance in stabilizing cell - cell interactions and facilitating leukocyte endothelial transmigration . more recently , icam-1 has been characterized as a site for the cellular entry of human rhinovirus . because of these associations with immune responses , many researchers have hypothesized that icam-1 could function in signal transduction . earlier studies showed that icam-1 gene is highly expressed in pulmonary fibroblasts of copd patients . in addition , blocking pulmonary icam-1 expression ameliorates lung injury in established diet - induced pancreatitis . thus , adhesion molecules play a key role in regulating inflammation in respiratory disorders ( figure 1 ) the first class of pla2 is secretary pla2 ( spla2 ) that is expressed in a variety of cell types and it has no preference for aa at sn-2 position , requires millimolar amounts of ca for activity and is sensitive to sulfhydryl reducing agents , such as dithiothreitol ( dtt ) , and is resistant to heat or acid conditions . the second class of pla2 is calcium - independent pla2 ( ipla2 ) that does not require ca for catalytic activity . ipla2 prefers plasmalogen substrates and does not appear to have a preference for the type of fatty acid at the sn-2 position . the third class is the novel , high molecular weight ( 85 kda ) cytosolic pla2 ( cpla2 ) . cpla2 enzymes catalyze the hydrolysis of the sn-2 position of membrane glycerophospholipids , leading to production of free fatty acids and lysophospholipids . this reaction is of particular importance if the esterified fatty acid is arachidonic acid ( aa ) , which is converted by downstream metabolic enzymes to various bioactive lipophilic compounds called eicosanoids , including prostaglandins ( pgs ) and leukotrienes ( lts ) . the increase in cpla2 activation and expression following external stimuli , including proinflammatory cytokines , growth factors , and microbial toxin , is often observed in several systems . the implication of cpla2 in inflammatory diseases has been confirmed by that the airway anaphylactic response in the cpla2 knockout mice is markedly reduced compared with that in the wild - type mice . moreover , cpla2-deficient mice have provided the most definitive evidence for the central role of cpla2 in eicosanoid as well as in the pathogenesis of several inflammatory diseases , such as acute respiratory distress syndrome ( ards ) due to bacterial sepsis [ 30 , 31 ] . these studies have demonstrated that there was a reduction in the bronchial lumen and alveolar thickening in the control mice that was remarkably absent in the cpla2 knockout mice . this outcome also appeared in 5-lipoxygenase ( lo)-knockout mice and mice with pgd2 receptor deficiency . thus , cpla2 seems to function as a crucial upstream regulator of the production of eicosanoids for airway resistance during allergic inflammation and is correlated to the process of asthma ( figure 1 ) . the inhibition of cpla2-mediated pathways may also provide a therapeutic approach to airway and lung injury . cox metabolites have diverse effects in the lung and are known to modify airway tone as well as inflammatory responses . cox-1 is constitutively expressed in most tissues and considered to be the housekeeping isoform that produces pgs which are required for maintenance of normal cell and organ function . in contrast , cox-2 is primarily an inducible isoform whose expression can be upregulated in many cell types by cytokines , mitogens , and endotoxin [ 3 , 4 ] . it is highly expressed in inflamed tissues and believed to produce pgs involved in inflammatory processes . cox-2 has multiple transcriptional regulatory sequences in its promoter region , including a tata box , an nf - il6 motif , two ap-2 sites , three sp1 sites , two nf-b sites , a cre motif , and an e - box . cox-2 gene expression can be induced by multiple cytokines and growth factors , via activation of transcriptional regulatory proteins that act on these promoter sites . thus , cox-2 appears to be the primary cox controlling pge2 synthesis in response to inflammation ( figure 1 ) . cox effects are widespread and extremely complex ; however , studies in knockout mice for cox-1 versus cox-2 reveal sometimes overlapping , not altogether predictable roles for these two enzymes . the levels of prostanoids in bronchoalveolar lavage fluid are increased in asthma , and several studies have found enhanced expression of both cox-1 and cox-2 in the airways of asthmatics [ 36 , 37 ] . a recent research has renewed interest in the role of prostanoids in allergic airway disease . moreover , the expression of cox-2 protein induced by lactobacillus rhamnosus gg ( lgg ) , endotoxin , and lipoteichoic acid ( lta ) in t84 epithelial cells . the presence of cox-3 mrna transcript , with a size of approximately 5.2 kb , was subsequently confirmed in human cells ; cox-3 was in highest concentrations in the cerebral cortex and heart tissue . however , the retention of intron 1 in cox-3 seems to slow its enzymatic activity in comparison to cox-1 and cox-2 . thus , the inhibition of cox-2-mediated inflammatory pathway may provide a therapeutic approach to respiratory diseases . mmps are proteolytic enzymes that are able to degrade extracellular matrix ( ecm ) components and , thus , play a role in cell migration and tissue remodeling . moreover , they can splice and ( in)activate cytokines and chemokines , thereby influencing the recruitment and function of inflammatory cells . to date , 24 mmps have been identified in mammals ; cellular sources include inflammatory , stromal , and epithelial cells . some mmps are anchored to the cell surface , whereas others are secreted into the extracellular space . they are released as inactive proenzymes and are activated by proteolytic cleavage of the n - terminal domain . most mmps are constitutively secreted once they become translated . in gelatinase subfamily of mmps ( mmp-2 and mmp-9 ) , the catalytic domain that includes the zn binding site also contains repeats of fibronectin motifs allowing the ability to bind gelatin , their major substrate . patients with asthma have an increased gelatinolytic activity linked to mmp-2 and mmp-9 and higher levels of tissue inhibitor of metalloproteinase-1 ( timp-1 ; a natural inhibitor of mmps ) in their sputum . the activated form of mmp-9 ( 85 kda ) was found in the sputum from 60% of asthmatics , but was absent from that of control subjects . although less frequently detectable than pro - mmp-9 ( pro - mmps are catalytically inactive and are activated into the active mmp after cleaving of the prodomain ) , pro - mmp-2 ( 72 kda ) was also found more frequently in asthmatics ( 50% ) than in control subjects ( 5% ) . in addition , patients with copd have an increased gelatinolytic activity in sputum linked to mmp-2 and mmp-9 . in smokers with emphysema , mmp-8 and mmp-9 levels in bronchoalveolar lavage ( bal ) fluid were significantly higher than in smokers without emphysema . in vitro cultured human airway smooth cells and a549 cells , tnf- and il-1 induce mmp-9 expression and cell migration [ 2 , 43 , 44 ] via various signaling pathways , such as pkc , mapks , nf-b , and ap-1 . thus , mmps and their inhibitors ( timps ) play multiple functions in physiological processes and interact with many other mediators regulating inflammatory processes , cell behavior , and angiogenesis . these mediators are implicated in many intricate loops of reciprocal interactions rendering the understanding of the role of mmps in regulatory processes difficult . in many respiratory diseases , mmps are overexpressed or oversecreted leading to both deregulation of physiological homeostatic processes and ecm degradation and disorganization ( figure 1 ) . pkcs are important in many cellular responses in the lung , including permeability , contraction , migration , hypertrophy , proliferation , apoptosis , and secretion . pkc is a family of serine / threonine kinases characterized by at least eleven different isotypes . pkc isotypes are differentially regulated by calcium ( ca ) , diacylglycerol , and phospholipids and differ in structure , expression , intracellular localization , substrate utilization , and mechanisms of activation . the pkc isotypes are subdivided into three groups : the classical , novel , and atypical . this subdivision is based on the structural and functional differences in the conserved domains c1c4 . the classical pkc , pkci / ii , and pkc isotypes are ca and diacylglycerol dependent . the novel pkc , pkc , pkc , pkc , and pkc isotypes contain c2 domains that lack ca - binding ability but still retain functional c1a and c1b domains that can bind the endogenous diacylglycerol and exogenous phorbol esters . the atypical pkc , pkc , and pkc isotypes lack a functional c2 domain and contain a single c1 domain that lacks the ability to bind diacylglycerol and phorbol esters . therefore , the mechanism of activation of the atypical pkc isotypes is both ca and diacylglycerol independent . moreover , pkcs are important signaling intermediates in chronic airway diseases like asthma and copd . resident airway epithelial cells produce proinflammatory mediators under the regulation of pkc . increased pkc activity increases nf-b - dependent proinflammatory cytokine generation in human airway epithelial cells , while expression of a dominant negative pkc mutant has inhibiting effects . in human airway smooth muscle cells , pkc , i , , , , , and are found in the cytosol and ii in the membrane under basal conditions . the proinflammatory neuropeptide bradykinin ( bk ) causes activation of pkc , i , , and when applied to airway smooth muscle cells . bk also induces cox-2 protein expression and pge2 accumulation in human airway smooth muscle cells via a pkc-dependent signaling . pkc is increased in the lungs of patients with copd and is thought to be important in the hypertrophy and proliferation of airway smooth muscle cells . pkc activity is also increased in proliferating human airway smooth muscle cells . on the other hand , pkc is important in mediating the effects of proinflammatory cytokines by phosphorylating cpla2 leading to the release of aa from phospholipids with subsequent production of bioactive eicosanoids in activated cells . at least three pkcs are expressed in interstitial fibroblasts , including pkc , , and . activation of pkc causes decreased collagen expression via the extracellular signal - regulated kinase kinase ( mek)/erk signaling cascade , a response that is opposed by pkc . selected pkcs are activated by lps , leading to the production of the proinflammatory cytokines , such as tnf- , il- , and il-6 . in addition , thrombin causes an increase in cytosolic [ ca ] and activation of selected pkcs . tnf- has been shown to induce mmp-9 expression via a pkc-dependent pathway in a549 cells . taken together , these studies indicated that pkcs play a critical role in mediating inflammation and respiratory diseases ( figure 2 ) . because multiple signaling pathways contribute to the key cellular responses important in lung biology , therapeutic strategies targeting pkcs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect . mechanisms that regulate pkc activity , including phosphorylation and interaction with isozyme - specific binding proteins , are also potential therapeutic targets in the respiratory diseases . ros are products of normal cellular metabolism and are known to act as second messengers . under physiological conditions , ros participate in maintenance of cellular redox homeostasis to protect cells against oxidative stress . in addition , regulation of redox state is important for cell activation , viability , proliferation , and organ function . however , overproduction of ros , most frequently due to excessive stimulation of either reduced nadph by proinflammatory cytokines or the mitochondrial electron transport chain and xanthine oxidase , results in oxidative stress . oxidative stress is a deleterious process that leads to airway and lung damage and consequently to several respiratory inflammatory diseases / injuries . ros are intracellularly generated from several sources , including mitochondrial respiration , cytochrome p450 , the nadph oxidase system , and xanthine / xanthine oxidase . however , the major ros generating enzyme is nadph oxidase , a membrane - bound multicomponent enzyme complex that is present in phagocytes as well as nonphagocytic cells . first , superoxide produced by nadph oxidase 2 is required for respiratory burst that occurs in phagocytes , leading to microbial killing . ros derived from nadph oxidase can specifically and reversibly react with proteins , altering their activity , localization , and half - life . activated phagocytic cells generate ros via assembly and activation of the nadph oxidase complex , which comprises membrane - associated flavocytochrome b558 ( gp91 ) and p22 and various cytosolic cofactors ( p47 , p67 , and p40 , and the gtpase , rac1 ) and mediates transmembrane electron transfer from the major cellular electron donor , nadph , to reduce molecular o2 to superoxide anion ( o2 ) and hydrogen peroxide ( h2o2 ) . a number of homologs of the main business end of nadph oxidase , gp91 , have been discovered , and mammalian systems are now known to contain seven nadph oxidase homologs , comprising nadph oxidase 15 ( nadph oxidase 2 being the new name for gp91 ) and two larger dual oxidases , duox1 and duox2 , which are widely expressed in many cell types to mediate a variety of biological functions , such as cell mitosis , differentiation , migration , and immune regulation . in in vitro studies , using macrophages , alveolar and bronchial epithelial cells , ros have been shown to induce gene expression of inflammatory mediators , such as il-1 and tnf- [ 57 , 58 ] . patients with asthma demonstrate increased generation of ros , such as superoxide anion , hydrogen peroxide , and hydroxyl radicals . increased production of ros has been demonstrated by many cell types within the lung in asthma , including macrophages , antigen - presenting cells ( apcs ) , neutrophils , and eosinophils . excessive production of ros correlates with the degree of airway hyperresponsiveness , as quantified by methacholine challenge . in addition , oxidative stress also contributes to a proteinase - antiproteinase imbalance , both by inactivating antiproteinases , such as 1-antitrypsin and secretory leukocyte proteinase inhibitor , and by activating proteinases , such as mmps . on the other hand , oxidants also promote inflammation by activating nf-b or ap-1 , which orchestrates the expression of multiple inflammatory genes recognized to be important in copd , such as tnf-. recently , phagocytic nadph oxidase - ros signaling has been shown to play a critical role in promoting tnf--induced , nf-b - dependent acute inflammatory responses , and tissue injury specifically in the lungs , which is effected by preferential leukocyte infiltration . thus , oxidative stress plays a critical role in inflammatory responses in airway and lung diseases via the upregulation of redox - sensitive transcription factors ( such as ap-1 or nf-b ) and thereby proinflammatory genes ( such as mmp-9 , vcam-1 , icam-1 , cox-2 , or cpla2 ) expression . taken together , nadph oxidase / ros play a critical role during development of airway and lung diseases ( figure 2 ) . the pi3k family are central signaling elements in a diverse array of cellular functions , including growth , proliferation , migration , and survival . it is , therefore , understandable that dysregulation of pi3k has been implicated in the induction and/or progression of a variety of disease states , including those of the respiratory tract , ranging from asthma to cancer . pi3ks type i pi3ks are activated by cell surface receptors , such as growth factors , insulin , and g - protein - coupled receptors ( gpcrs ) . class ii pi3ks comprised , , and isoforms , which are characterized by the presence of a c2 domain at the c terminus . structurally , pi3ks ia exist as heterodimeric complexes in which a catalytic p110 subunit ( designated as , , or ) is in association with a particular regulatory subunit ( designated as p85 , p55 , and p50 ) . importantly , pi3k ia signals downstream of receptor tyrosine kinase and ras . the single class pi3k ib consists of the p110 catalytic subunit complexed to the p101 regulatory subunit and signals downstream of gpcrs and ras , which is activated by subunits from gpcrs , such as the receptors for chemokines . despite limitations in selectivity , the two commercially available pi3k inhibitors , wortmannin and ly294002 , have contributed greatly to our understanding of the biological role of pi3k in lung inflammation . moreover , previous study indicated that intratracheal administration of ly294002 significantly reduced ovalbumin-(ova- ) induced increases in total cell counts , eosinophil counts , and il-5 , il-13 , and ccl11 ( eotaxin ) levels in bal fluid and dramatically inhibited ova - induced tissue eosinophilia and airway mucus production . this study confirmed that ly294002 markedly attenuated ova - induced serine phosphorylation of akt , a direct downstream substrate of pi3k . in addition , other studies also showed that ly294002 and wortmannin attenuated eosinophilic airway inflammation and airway hyperresponsiveness in a murine asthma model . thus , pi3k inhibition was indicated to have therapeutic potential for the treatment of asthmatic airway inflammation . ly294002 was shown to reduce chemokine - induced ros generation in phagocytes , which was further confirmed by studies using pi3k knockout mice . it was also reported that serum withdrawal ( sw ) killed human u937 blood cells by elevating cellular ros levels , which occurred through pi3k activation . thus , pi3k family plays a prominent role in various airway and lung inflammation ( figure 2 ) . moreover , inhibitors of pi3k / akt may prove to be useful novel therapies in the treatment of respiratory diseases . sfks are signaling enzymes that have long been recognized to regulate critical cellular processes , such as proliferation , survival , migration , and metastasis . src protein tyrosine kinase ( ptk ) family is categorized into nonreceptor tyrosine kinases and consists of nine members . src , fyn , yes , and yrk are ubiquitously expressed , whereas blk , fgr , hck , lck , and lyn are expressed in more restricted patterns . src ptk family members are activated in response to the stimulation of a variety of cell surface receptors , such as tyrosine kinase receptors , integrin receptors , and g protein - coupledreceptors , and by cellular stress . moreover , we reported that tnf- or il-1 induces vcam-1 and icam-1 expression via a c - src - dependent pathway in human airway smooth muscle cells . in addition , c - src has been shown to regulate cox-2/pge2/il-6-dependent airway inflammation via nadph oxidase / ros . in human lung epithelial cells , in addition to activating nf-b - inducing kinase ( nik ) via traf2 , tnf- could activate c - src through pkc . systemic inhibition of these kinases using specific small molecule inhibitors for src ptks ( either pp2 or su-6656 ) significantly attenuated lps - induced lung injury and capillary permeability and reduced lps - dependent cytokine and chemokine levels in the lung and the serum . thus , the role of src family ptks in inflammatory responses is a rising area of research ( figure 2 ) . however , application of small chemical inhibitors to effectively and specifically block src ptks could have a great clinical implication for airway and lung diseases with inflammatory responses as underlying mechanisms . cell - surface tyrosine kinases receptors play pivotal roles in development , tissue repair , and normal cellular homeostasis . aberrant expression or signaling patterns of these receptors have also been linked to the progression of a diversity of diseases , including asthma . two major families of tyrosine kinases receptors , the epidermal growth factor receptor ( egfr ) and platelet - derived growth factor receptor ( pdgfr ) families , have received a great deal of attention as potentially therapeutic targets for respiratory diseases , as these receptors have been shown to play key roles in chronic tissue remodeling in asthma , bronchitis , and pulmonary fibrosis . the egfr system on epithelial cells and underlying mesenchymal cells ( fibroblasts , myofibroblasts , and smooth muscle cells ) drives numerous phenotypic changes during the progression of these pulmonary diseases , including mesenchymal cell hyperplasia , differentiation , and ecm production . the pdgfr system , located primarily on mesenchymal cells , transduces signals for cell survival , growth , and chemotaxis . the variety of egfr and pdgfr ligands produced by the airway epithelium or adjacent mesenchymal cells allows for intimate epithelial - mesenchymal cell communication . in humans , the airway epithelium expresses egfr ligands constitutively , including egf , tgf- , hb - egf , amphiregulin , heregulin , and betacellulin . expression of several egfr ligands has also been investigated in diseases , such as copd and asthma . showed that p. aeruginosa bacterial supernatant induces mucin production in human airway epithelial cells ( nci - h292 ) via egfr activation . multiple studies have also reported that stimulation of airway epithelial cells by lps induces the secretion of il-8 via a cellular cascade involving a tlr4/myeloid differentiation primary response gene ( myd)88/nf-b - dependent pathway . in addition , ros have been shown to stimulate pdgfr activation via c - src family kinases . there is accumulating evidence that pkc - dependent phosphorylation of p47 is essential for pdgf - stimulated ros generation , which is important for pdgf - induced mapks activation . taken together , these studies suggest that growth factor tyrosine kinase receptors may also play a key role in mediating expression of inflammatory genes ( figure 2 ) . mapks are important components of signaling modules activated by neurotransmitters , cytokines , and growth factors , as well as chemical and mechanical stressors . in the airway , these external signals produce acute responses that modify smooth muscle contraction and may also induce chronic responses that modify airway structure . both acute and chronic events in airway remodeling result from altered expression of multiple genes encoding protein mediators of cell - cell signaling , ecm remodeling , cell cycle control , and intracellular signaling pathways . in mammals , three groups of mapks have been identified : the extracellular signal - regulated protein kinases ( erks ) , the c - jun nh2-terminal kinases ( jnks ) , and the p38 mapk . the p38 mapk is activated by cellular stresses , including uv radiation , lps , growth factors , and cytokines . the jnk is activated by many of the same stimuli that activate p38 mapk , such as cellular stresses and numerous cytokines . thus , the inhibition of mapks activity via pharmacological or genetic approaches blocks allergic inflammation of airways . moreover , asthmatic patients demonstrated increased immunostaining for phospho ( p)-erk , p - p38 mapk , and p - jnk . the phosphorylation of p38 mapk was primarily observed in the basal layer of the columnar epithelium . it is likely that p38 mapk drives basal metabolic processes for this particular cell type . there was significant correlation between clinical severity of asthma and intensity of immunostaining for p - erk and p - p38 mapk and between p - erk and the number of tissue eosinophils and neutrophils in the airways . early studies of p38 mapk demonstrated that il-1 and tnf- activate the p38 mapk in monocytes . furthermore , inhibition of the p38 mapk pathway was shown to exert anti - inflammatory effects through inhibition of il-1 , il-6 , and tnf- expression . in airway smooth muscle cells , there is solid evidence that both erk and p38 mapk pathways contribute to il-1-induced cox-2 expression and pge2 synthesis [ 76 , 77 ] . in a mouse model of chronic lung inflammation ( allergic inflammation ) , significant inhibition of tnf- , il-4 , il-13 , and rantes ( regulated on activation , normal t - cell expressed and secreted ) in lung homogenates was observed with jnk inhibitor , sp600125 . in addition , we also found that lta or il-1 could induce cpla2 , cox-2 , or mmp-9 in human airway smooth muscle cells or a549 cells [ 3 , 44 ] . therefore , mapks play an important role in mediating airway and lung inflammation ( figure 2 ) . nf-b is viewed as a master regulator of inflammatory responses because it plays an essential role in the evolution as well as the resolution phase of inflammation . nf-b controls a wide spectrum of biological effects ranging from immune and stress - induced responses to cell fate decisions such as proliferation , differentiation , tumorigenesis , apoptosis , and tissue remodeling . nf-b usually exists as a heterodimeric complex of p50 and p65/rela subunits . in unstimulated cells , nf-b is found in the cytoplasm as an inactive non - dna - binding form , associated with an inhibitor protein called inhibitory b ( ib ) which masks the nuclear translocation signal and so prevents nf-b from entering the nucleus . upon cell stimulation with various nf-b inducers , ib is rapidly phosphorylated on two serine residues , which targets the inhibitor protein for ubiquitination by the e3 ubiquitin - ligases ( e3rsib ) and subsequent degradation by the 26s proteasome . the released nf-b dimer can then be translocated into the nucleus and activate target genes by binding with high affinity to b elements in their promoters . nf-b is activated by numerous extracellular stimuli , including cytokines such as tnf- and il-1 , viruses and environmental particulates ( pm10s ) , and oxidative stress . exogenous h2o2 also activated nf-b in a murine model of ros - induced acute lung injury . administration of otc ( l-2-oxothiazolidine-4-carboxylate ) resulted in significant reduction of nf-b translocation into the nucleus and expression of adhesion molecules , chemokines , and cytokines . previous study demonstrated that nf-b activation occurred rapidly in the ovalbumin ( ova ) model of allergic airways disease and that nf-b activation predominantly occurred in the epithelial cells of the conducting airways , in association with enhanced mrna expression of nf-b - regulated chemokine genes , including mip-2 and eotaxin . a novel cyclin - dependent kinase inhibitor ( bai ) has been shown to downregulate tnf--induced expression of cell adhesion molecules by inhibition of nf-b activation in human pulmonary epithelial cells . recently , we also demonstrated that overexpression of ho-1 protects against tnf--mediated airway inflammation by downregulation of tnfr1-dependent oxidative stress and nf-b activation . taken together , these results show that nf-b plays a key role in mediating the expression of inflammatory proteins in airway and lung inflammation and injury ( figure 2 ) . ap-1 transcription factor typically consists of combinations of jun ( c - jun , jun b , jun d ) and fos proteins ( c - fos , fos b , fra-1 , fra-2 ) , which bind to the promoters of target genes . it was found to be responsible for the transcriptional activation of various genes that were activated by phorbol esters ( such as pma ) via activation of pkc . ap-1 may be activated via pkc and by various cytokines , including tnf- and il-1 , via several types of ptk and mapks , which themselves activate a cascade of intracellular kinases . certain signals rapidly increase the transcription of the fos gene , resulting in increased synthesis of fos protein . other signals lead to activation of kinases that phosphorylate c - jun , resulting in increased activation . specific jun and fos kinases are now recognized and may play a key role in the regulation of cellular responsiveness to cytokine signals . recent studies showed that sirtuin 1 ( sirt1 ) directly interacted with c - jun and repressed the transcriptional activity of ap-1 , thus decreasing mmp-9 expression . more recently , it was reported that sirt1 decreased c - fos / c - jun acetylation induced by p300 and inhibited the transcriptional activity of ap-1 and subsequent cox-2 expression and pge2 generation . thus , ap-1 may play a critical role in mediating expression of various inflammatory proteins . there is evidence for increased expression of c - fos in epithelial cells in asthmatic airways , and many of the stimuli relevant to asthma that activate nf-b will also activate ap-1 . thus , ap-1 is also a key factor in respiratory diseases ( figure 2 ) . kinase pathways have become recognized as key cellular signal transducers , and several protein kinase inhibitors are in development for the treatment of respiratory diseases . the pyridinylimidazole compounds , exemplified by sb203580 , were originally prepared as inflammatory cytokine synthesis inhibitors that subsequently were found to be selective inhibitors of p38 and mapk . sb203580 was shown to attenuate bal tnf- production in an ovalbumin challenged rat model of asthma and sb2439063 reduced neutrophilia and mediator expression in rat copd models . in addition , a recent study also indicated that in acute and chronic animal models of asthma , sp600125 ( a jnk inhibitor ) reduces bal accumulation of eosinophils and lymphocytes , cytokine release , serum ige production , and smooth muscle proliferation after repeated allergen exposure . intratracheal administration of ly294002 reduced ova - induced increases in total cell counts , eosinophil counts , and il-5 , il-13 , and ccl11 ( eotaxin ) levels in bal fluid and dramatically inhibited ova - induced tissue eosinophilia and airway mucus production . inhibition of sfks using specific inhibitors for src ptks ( either pp2 or su-6656 ) attenuated lps - induced lung injury and capillary permeability and reduced lps - dependent cytokine and chemokine levels in the lung and the serum . rnai is the process of sequence - specific , post - transcriptional / transcriptional gene silencing through small interfering rna ( sirna ) . rnai is a popular method of controlling gene expression and has a potential in the development of drugs for several diseases , such as various types of cancer and viral infections . gene therapy for asthma has already been developed and has demonstrated promising results in animal models . recent progress in delivering sirna to the respiratory system has also improved the therapeutic feasibility of rnai for asthma . in the context of allergic immune responses , activation of stat6 is pivotal for th2-mediated ige production and development of airway inflammation and hyperreactivity . moreover , stat6 sirna has been shown to inhibit allergic airway inflammation and hyperreactivity in mice . inflammatory airway and lung diseases are characterized by chronic inflammation and oxidant / antioxidant imbalance , a major cause of cell damage / injury . numerous studies have shown the effectiveness of polyphenols in limiting the progression of chronic diseases . this is likely to occur , at least in part , because of the antioxidant capacity of these molecules , which extends from the availability of hydroxyl groups and the presence of conjugated double bonds . resveratrol has been reported to increase antioxidant capacity and reduce various markers of oxidative stress . recently , lee et al . indicated that resveratrol inhibits the activation of nf-b ( p65 ) by tnf- or pma and reduces atp - induced mucin secretion from cultured primary rat tracheal surface epithelial ( rtse ) cells . on the other hand , edaravone ( 3-methyl-1-phenyl-2-pyrazolin-5-one ) , a novel radical scavenger protects neurons by reducing endothelial injury and by ameliorating neuronal damage caused by brain ischemia . treatment of edaravone decreases interstitial edema and inflammatory cell infiltration as well as prevents the process of pulmonary fibrosis . in addition , reports of clinical benefit in airway and lung diseases for increased vitamins c and e and other dietary antioxidants have been varied . indicated that vitamin e treatment prior to injury largely prevents the increase in pulmonary permeability index and moderates the increase in lung lymph flow , increases the pao2/fio2 ratio , ameliorates both peak and pause airway pressure increases , and decreases plasma conjugated dienes and nitrotyrosine . erdosteine is a thiol antioxidant having mucoactive properties and the ability to reduce bacterial adhesiveness . this compound was introduced as a mucolytic agent for the treatment of chronic airway and pulmonary diseases . erdosteine breaks the disulfide bonds of mucus glycoproteins , affecting the physical properties of the mucus , thus leading to increased cough clearance . in addition , erdosteine has been reported to have antioxidant , anti - inflammatory , and antibacterial activity . negro et al . showed that erdosteine at a dose of 600 mg / day proved effective in significantly reducing ros levels in peripheral blood of stable copd patients who are current smokers , together with reduction in levels of some chemotactic proinflammatory cytokines ( il-6 and il-8 ) in their bronchial secretions . recently , greene and gaughan represent new therapeutic targets and medicines that target specific micrornas ( mirnas ) and may have potential in the treatment of asthma . there have been a number of studies in the field of mirna that implicate specific mirnas in the pathophysiology of asthma . for example , studies using mouse models have identified mirnas that are altered in response to allergen challenge . certain mirnas that are involved in the regulation of il-13 and the th2 response , key components of the asthmatic response , have been shown to be amenable to modulation by pre - mirs and anti - mirs . other studies have identified mirnas that are implicated in bronchial smooth muscle hyperresponsiveness and proliferation . thus , developing mirna - based medicines to treat the pulmonary manifestations of asthma could yield therapeutics with new properties that have the potential to treat both the inflammation and hyperresponsiveness associated with this disease . there is an increasing evidence that inflammatory proteins , such as vcam-1 , icam-1 , cpla2 , cox-2 , and mmp-9 are involved in the pathogenesis of respiratory diseases , such as asthma and copd ( figure 3 ) . moreover , various inflammatory signaling pathways , including pkcs , nadph oxidase / ros , egfr , pdgfr , c - src , pi3k / akt , mapks , ap-1 , and nf-b , are involved in the regulation of these inflammatory proteins ( figure 3 ) . further exploration of the role of vcam-1 , icam-1 , cpla2 , cox-2 , and mmp-9 in these highly prevalent diseases is crucial to identify which are possible therapeutic targets . the development of new inhibitors that are highly specific but have no major adverse effects is essential . as targeted delivery of inflammatory proteins inhibitors directly to the airway and lung might result in fewer side effects , this option should also be explored . although the use of inhibitors of inflammatory signaling pathways in the treatment of respiratory diseases seems very attractive , further studies are needed to identify the exact role of inflammatory signaling molecules in these diseases and to develop highly specific inhibitors . polycystic liver disease is a condition characterized by the presence of multiple cysts in the liver , which may be inherited or sporadic , and most cases of adult polycystic liver disease ( apld ) are related to autosomal dominant polycystic kidney disease ( adpkd ) . in 1964 , there have been some reports , till date , that describe the association of malignant neoplasms with potter type iii cystic disease of the liver and kidney [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] ; however , most of these describe pancreatic and hepatobiliary neoplasms arising in patients with polycystic liver and kidney diseases including adpkd , and no report has described the association of gastric carcinoma with potter type iii cystic disease of the liver and kidney . this is the first case report of multiple gastric carcinomas associated with potter type iii cystic disease of the liver , mesenterium and kidney . although it is unclear whether the development of multiple gastric carcinomas is directly associated with the pathogenesis of multiple cysts in the liver , mesenterium and kidney , this case provides a perspective for etiology of gastric carcinoma with polycystic disease . he had been receiving hemodialysis treatment 3 times a week for 2 years because of chronic renal failure due to polycystic kidneys . he stated that his sister had suffered from polycystic kidney disease . physical examination on admission revealed distended abdomen due to polycystic disease . the patient had high serum levels of carcinoembryonic antigen ( 7.9 ng / ml ) and carbohydrate antigen 19 - 9 ( 202 mg / dl ) . an abdominal computed tomography scan showed multiple cysts in the liver and cystic lesions on both sides in the kidney and mesenterium ( fig . gastrointestinal fiberscopy showed the presence of two ulcerated lesions in the lesser curvature in the middle and lower portions of the stomach . histopathological analysis of the gastric tumor biopsy specimens revealed papillary adenocarcinoma in one tumor and poorly differentiated adenocarcinoma in another . distal gastrectomy with lymph node dissection was performed , and the gastric tumors were surgically diagnosed to be t2n1m0 , stage ii , according to the tnm classification for gastric carcinoma . macroscopic examination showed that two tumors of type ii were present in the middle and lower portions of the stomach ( fig . the distal lesion was diagnosed as a poorly differentiated adenocarcinoma ( 38 39 6 mm ) within the muscularis propria , with marked lymphatic invasion ( ly2 ) and vascular invasion ( v2 ) ( fig . 3b ) ; the lesion on the proximal side was diagnosed as a papillary adenocarcinoma ( 58 47 6 mm ) within the submucosal layer , with marked lymphatic invasion ( ly2 ) and vascular invasion ( v1 ) ( fig . adpkd is the most common of all inherited renal cystic diseases and has various external manifestations , including cysts in the pancreas , seminal vesicles , arachnoid membrane , and liver . the most accepted among them is the osathanondh and potter system that was established in 1964 , according to which polycystic kidneys are classified into three types . the cysts may be present in any portion of the nephron or tubule , but they are most commonly found in bowman 's space and the angle of the loop of henle . in the older classification , potter type iii cystic disease was considered to represent adult - type polycystic disease of the kidney . in 1972 , they also observed cysts in the liver and/or pancreas in 58 of these patients ; hence , they described this condition as potter type iii polycystic disease . in the present case , computed tomography and ultrasonography scans showed multiple renal and hepatic cysts in addition to multiple cysts in the mesenterium . therefore , according to hatfield and pfister 's criteria , we considered that this case of association of apld with multiple renal cysts could be classified as potter type iii cystic disease . the patient stated that his sister had suffered from polycystic kidney disease ; however , we could not confirm the presence of a family history of polycystic kidney disease because his sister had been out of contact for long . in the absence of a family history of adpkd , bilateral multiple renal cysts with hepatic cysts , together with the absence of other manifestations suggesting a different renal cystic disease , provide evidence for diagnosis . in these instances , a family history , however , may be absent in 2040% of new patients in whom the diagnosis of adpkd first is suspected from imaging studies , which can be due to a de novo mutation of pkd-1 or pkd-2 genes . although we could not confirm the mutation of pkd-1 or pkd-2 in this case , family history , clinical findings and imaging studies suggested that the patient suffered from adpkd . reports on the association of malignant neoplasm with apld and adpkd ( potter type iii cystic disease ) are extremely rare . to our knowledge , only 8 such cases have been reported in the english language literature ( table 1 ) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ] . till date , most cases of neoplasms associated with polycystic disease of the liver and kidney have been reported as either pancreatic or hepatobiliary neoplasms , namely 5 cases of pancreatic neoplasms and 3 cases of cholangiocarcinoma . since 5 of these cases were pancreatic neoplasms , we think that adpkd has some genetic influence on the development of pancreatic neoplasms . aziz et al . reported 2 patients from the same family ( a brother and a sister ) with adpkd who developed adenocarcinoma of the gastroesophageal junction ; however , these patients did not have cysts in the liver or other organs except for the kidney . in the present case , although a detailed family history could not be obtained , multiple cysts were observed in the liver , both kidneys , and mesenterium . these genes encode a member of the polycystin protein family , which is an integral membrane protein that functions as a regulator of calcium - permeable cation channels and intracellular calcium homoeostasis . some studies have described an association between the pkd genes and cancer cells ; polycystin-1 , encoded by pkd-1 , was reported to induce apoptosis and cell cycle arrest in the g0/g1 phase in cancer cells . moreover , pkd-1 inhibits cancer cell migration and invasion and may be considered a new member of the tumor suppressor family of genes . therefore , adpkd , which is caused by defective pkd-1 and pkd-2 , may be a carcinogenic condition . in addition , mutations in two genes , namely prksch ( which codes for hepatocystin ) and sec63 , have been found to cause apld . however , as mutations in these genes have not been found in all families , undiscovered genes responsible for causing apld probably still exist . although it is unclear whether the development of multiple gastric carcinomas is directly associated with the pathogenesis of cysts in the liver , mesenterium and kidney , the present case demonstrates that gastric carcinoma can be an external complication in patients with polycystic disease .

in respiratory diseases , there is an increased expression of multiple inflammatory proteins in the respiratory tract , including cytokines , chemokines , and adhesion molecules . chemokines have been shown to regulate inflammation and immune cell differentiation . moreover , many of the known inflammatory target proteins , such as matrix metalloproteinase-9 ( mmp-9 ) , intercellular adhesion molecule-1 ( icam-1 ) , vascular cell adhesion molecule-1 ( vcam-1 ) , cyclooxygenase-2 ( cox-2 ) , and cytosolic phospholipase a2 ( cpla2 ) , are associated with airway and lung inflammation in response to various stimuli . injuriously environmental stimuli can access the lung through either the airways or the pulmonary and systemic circulations . the time course and intensity of responses by resident and circulating cells may be regulated by various inflammatory signalings , including src family kinases ( sfks ) , protein kinase c ( pkc ) , growth factor tyrosine kinase receptors , nicotinamide adenine dinucleotide phosphate ( nadph)/reactive oxygen species ( ros ) , pi3k / akt , mapks , nuclear factor - kappa b ( nf-b ) , activator protein-1 ( ap-1 ) , and other signaling molecules . these signaling molecules regulate both key inflammatory signaling transduction pathways and target proteins involved in airway and lung inflammation . here , we discuss the mechanisms involved in the expression of inflammatory target proteins associated with the respiratory diseases . knowledge of the mechanisms of inflammation regulation could lead to the pharmacological manipulation of anti - inflammatory drugs in the respiratory diseases . we report a case of multiple gastric carcinomas associated with potter type iii cystic disease of the liver , mesenterium and kidney . a 65-year - old man with chronic renal failure due to polycystic kidneys and under hemodialysis treatment 3 times a week for 2 years was admitted to our hospital because of anemia . he stated that his sister had suffered from polycystic kidney disease . gastrointestinal fiberscopy showed two lesions in the lesser curvature in the lower portion of the stomach , and histopathological analysis of the gastric tumor biopsies revealed that one of the tumors was a papillary adenocarcinoma and the other a poorly differentiated adenocarcinoma . helicobacter pylori infection was not detected in the stomach mucosa . abdominal computed tomography scan revealed polycystic lesions in the liver , mesenterium and both kidneys . these imaging findings and family history suggested that the patient suffered from multiple gastric carcinomas associated with potter type iii cystic disease of the liver , mesenterium and kidney . reports on the association of malignant neoplasm with potter type iii cystic disease are extremely rare . especially , no case of the association of gastric carcinoma with potter type iii cystic disease of the liver and kidney has been described previously . this is a first report of the association of gastric carcinoma with potter type iii cystic disease . we also review reports of other malignant neoplasms associated with polycystic disease .

canaliculitis is a common encounter in ophthalmic practice but supernumerary puncta and canaliculi ( spc ) are rare congenital disorders . in a large series a 59-year - old gentleman presented with painful swelling of the left lower lid for a week , which was associated with epiphora . the swelling was confined to the nasal aspect of the left lower lid ( 0.50.5 mm ) with inflamed overlying skin ( figure 1a ) . eversion of the lower eyelid revealed two puncta , 0.5 mm apart ( figure 1b ) . the outer punctum was situated at the normal anatomical position ; whereas the inner punctum in the caruncle . gentle pressure did not result in any regurgitation from the both puncta . the patient was treated with oral cloxacillin 500 mg , 6 hourly for 5 days . the outer punctum had a soft stop with regurgitation of fluid from the same punctum . the outer punctum - canaliculus system was a cul - de - sac ( figure 1c ) . c ) dacryocystography showed pooling of dye in the cul - de - sac ( white arrow ) . c ) dacryocystography showed pooling of dye in the cul - de - sac ( white arrow ) . most spcs ( 78% ) present with epiphora . among the 23 patients reported by satchi et al . , none presented with canaliculitis . sequestration of tear and debris in the cul - de - sac served as nidus for infection . the resultant canaliculitis with its surrounding edema caused obstruction of the lacrimal drainage ; hence epiphora . epiphora however , may develop despite patent lacrimal drainage system . the 2-compartment model for lacrimal canalicular drainage of kakizaki et al . , suggested that the muscle of duverney - horner may deviate normal flow within the accessory canaliculus and thence transport tears back to the lacrimal tear lake , leading to epiphora . a solid epithelial cord forms in the region of the medial lower eyelid ( figure 2a ) and sends projections to form the canaliculi and the nasolacrimal duct ( figure 2b ) . spc is due to extra out - budding of the solid epithelial cord ( figure 2c ) . canalization begins at 4 months of gestation with disintegration of the central ectodermal core , forming lacrimal drainage outflow system . in this case , the extra inner canalicular epithelial bud ( nearer to the main epithelial cord ) underwent complete canalization and remained connected to the main epithelial cord . the outer canalicular epithelial bud , although its punctum is located at the normal anatomical position , was separated from the main epithelial cord ; forming a cul - de - sac ( figure 2d ) . c ) extra out - budding of the solid epithelial cord in supernumerary puncta and canaliculi . d ) the outer canalicular epithelial cord was separated from the main epithelial cord , forming a cul - de - sac . c ) extra out - budding of the solid epithelial cord in supernumerary puncta and canaliculi . d ) the outer canalicular epithelial cord was separated from the main epithelial cord , forming a cul - de - sac .

we report the first case of supernumerary puncta and canaliculi presented with canaliculitis . a-59 year - old gentleman presented with painful swelling of the left lower lid for a week , which was associated with epiphora . the swelling was confined to the nasal aspect of the left lower lid ( 0.50.5 mm ) with inflamed overlying skin . two puncta ( 0.5 mm apart ) were noted . the outer punctum at the normal anatomical position was a cul - de - sac while the inner punctum it the caruncle was patent . we described the embryology leading to supernumerary puncta and canaliculi to explain the paradoxical patency of the abnormally located punctum as well as the pathomechanism leading to canaliculitis . the patient was treated with oral cloxacillin 500 mg , 6 hourly for 5 days ; the cellulitis subsided after three days . mypro is a software pipeline for high - quality prokaryotic genome assembly and annotation . it was validated on 18 oral streptococcal strains to produce submission - ready , annotated draft genomes . mypro installed as a virtual machine and supported by updated databases will enable biologists to perform quality prokaryotic genome assembly and annotation with ease .

diabetes imposes a large economic burden on the individual , national healthcare systems , and countries.[15 ] healthcare expenditures due to diabetes account for 11% of the total healthcare expenditures in the world in 2011 . estimated global healthcare expenditures to treat diabetes and prevent complications totaled $ 465 billion in 2011 . by 2030 , this number is projected to exceed some $ 595 billion . on average , the estimated healthcare spending due to diabetes was $ 5,063 per person with diabetes in high - income countries compared with $ 271 in low- and middle - income countries . worldwide , the estimated number of adults living with diabetes has soared to 366 million , representing 8.3% of the global adult population . this number is projected to increase to 552 million people by 2030 , or 9.9% of adults , which equates to approximately three more people with diabetes every 10 or almost 10 million per year.[78 ] the kingdom of saudi arabia is not exception of this global pandemic . over the past four decades the ageing of populations , together with rapid socio - economic development ( progressive urbanization , decreasing infant mortality and increasing life expectancy ) and tremendous changes in lifestyle towards the westernized pattern reflected by changes in nutrition , less physical activity , tendency to increased obesity and more smoking , has resulted in the dramatic increase in the diabetes prevalence.[916 ] worldwide , the desire of many healthcare players ( governments , diabetes associations , health professionals , health economists , and people with diabetes themselves ) to reduce the economic burden of diabetes and its related complications costs makes the question of economic impact of diabetes an important component in : determining the present economic burden of diabetes and its related complications on government healthcare systempredicting the likely future economic burden of diabetes and its related complications , anddeveloping effective policies and strategies about treatment of diabetes and its related complications and the future public health spending related diabetes healthcare costs . determining the present economic burden of diabetes and its related complications on government healthcare system predicting the likely future economic burden of diabetes and its related complications , and developing effective policies and strategies about treatment of diabetes and its related complications and the future public health spending related diabetes healthcare costs . the purpose of this research study , therefore , is to determine the economic impact of diabetes mellitus ( dm ) on saudi arabia 's healthcare system , both now and in the future . this research study , however , focuses on the prevalence of diabetes and aims at estimating and predicting the total and per capita healthcare expenditure for people diagnosed with diabetes . to examine and predict the economic impact of diabetes costs on saudi arabia 's healthcare system , this study relied on data provided by ministry of health ( moh ) and ministry of finance ( mof ) for the years 1992 and 2010 databases.[1921 ] the 1992 and 2010 time period is used as the study time frame . population figures from the 1992 and 2010 moh databases for patients who reported that they had diabetes were selected for this study . data on government budget , total healthcare budget , and per capita health expenditure were obtained directly from the moh and mof databases . data on population ( by nationality , sex and age groups ) with undiagnosed diabetes obtained directly from central department of statistics and information databases . it should be noted that this study is based on single - source of diabetic data ( moh database ) only ; therefore , it does not account for the considerable number of people diagnosed with diabetes in other healthcare providers such as private and semi - private healthcare services ( military , universities , arabian american oil company ( aramco ) healthcare services , etc . ) . consequently , diabetes prevalence is probably much higher than figures presented in this study because a significant number of people diagnosed with diabetes are not included in the analysis . it should also be noted that the perspective adopted in this study is that of the moh . if other perspective were used such as payer , society or health care provider , the results would be different . in addition , data on diabetes was obtained from moh database is based on diabetic clinics visits in both moh hospitals and healthcare centers , and it combined in- and out - diabetic - patients which is categorized by nationality ( saudi / non - saudi ) ; sex ( male / female ) ; and age groups ( < 1 ; 1 - 4 ; 5 - 14 ; 15 - 44 ; 45 - 60 ; and 60 + ) . it should be noted that , it is not clear whether data provided by the moh database is based on first visits related to diabetes . therefore , it is not certain whether diabetic data is protected from double counting or not . this research study used prevalence - based approach that combines the demographics of the population with and without diagnosed diabetes in 1992 and 2010 . the general principle for estimating the healthcare costs of diabetes is straightforward.[2427 ] healthcare use attributable to diabetes is determined by a comparison of the healthcare use patterns of individuals with and without diabetes . dividing healthcare expenditures by the size of the population with and without diabetes provides an estimate of per capita healthcare expenditures.by multiplying diabetes population figures by the per capita healthcare expenditures , the total healthcare budget for people diagnosed with diabetes is derived.assuming diabetes prevalence rates remained constant over time , the exponential projection method ( i.e. , annualized change ) is used to reflect the growing prevalence of diabetes in the saudi arabia and the increasing costs of healthcare expenditures over the next 10 years ( i.e. , 2020 ) . healthcare use attributable to diabetes is determined by a comparison of the healthcare use patterns of individuals with and without diabetes . dividing healthcare expenditures by the size of the population with and without diabetes provides an estimate of per capita healthcare expenditures . by multiplying diabetes population figures by the per capita healthcare expenditures , the total healthcare budget for people diagnosed with diabetes is derived . assuming diabetes prevalence rates remained constant over time , the exponential projection method ( i.e. , annualized change ) is used to reflect the growing prevalence of diabetes in the saudi arabia and the increasing costs of healthcare expenditures over the next 10 years ( i.e. , 2020 ) . it should be mentioned that , the modern approach to conducting cost - of - illness studies is based on system of health account ( sha ) approach . sha is used to describe the health care system from an expenditure perspective . however , as there is no sha in saudi arabia , other approaches had to be used . knowledge of the costs of diabetes improves understanding of the importance of addressing healthcare and prevention issues associated with diabetes and help to inform and motivate decisions that can reduce the national burden of this disease . based on the moh database approximately 0.9 million people in 1992 and 2.5 million people in 2010 have been diagnosed with diabetes . analysis of the 1992 - 2010 moh database suggests that the number of people diagnosed with diabetes increased by 1.6 million ( or 183% ) during the last 18 years . this increases the proportion of the total population with diabetes from 5.3% in 1992 to 9.3% in 2010 . comparison of the 2010 diabetes figure to the 1992 figure suggests that the net number of people diagnosed with diabetes is growing by about 0.1 million per year . this increased prevalence of diabetes over the period of study is not only attributable to changing pattern of saudi lifestyle due to rapid socio - economic development ( together with increased obesity , smoking and less physical activities ) but may also be attributed to increased awareness programs related to diabetes and its health complications , community screening campaigns for diabetes , better diagnostic facilities specially in health centers and healthcare units , and better diabetes management systems and protocols . in fact , several community - based screening campaigns studies for dm and hypertension were conducted in different parts of saudi arabia revealed that quite a large percentage of these participants ( diabetic patients ) were not detected and remain for a long time without significant symptoms . these studies concluded that community - based screening campaigns are extremely efficient in identifying undiagnosed diabetic and hypertensive individuals in the society.[2933 ] table 1 summarizes the demographic component of population diagnosed with diabetes . the moh database for population diagnosed with diabetes by nationality , sex , and age - group , suggests that saudi citizens comprised 84% and 96% of total population diagnosed with diabetes in 1992 and 2010 , respectively , while , non - saudi population diagnosed with diabetes comprised 16% in 1992 and about 4% in 2010 . the demographic analysis also revealed that over half of total population diagnosed with diabetes , in 1992 and 2010 , are males while females represented about 47% and 48% of total population diagnosed with diabetes in 1992 and 2010 , respectively . the analysis revealed that about 1 in 11 males being diabetic in 2010 as compared with 1 in 19 males in 1992 . for females , about 1 in 9 were diabetic in 2010 compared with 1 in 17 in 1992 . population ( in millions ) and percent of population with and without diabetes , 1992 and 2010 the population between 45 and 60 years represented the highest among population diagnosed with diabetes during the period of analysis . this age group comprised about 52% and 45% of total population diagnosed with diabetes in 1992 and 2010 , respectively , with remaining population under age 15 , age 15 - 44 , and 60 years and above comprised about 3% , 21% , 15% in 1992 and about 4% , 27% , 24% in 2010 , respectively . it is important to mention that the population age 60 years and above represents the highest percentage increase among population diagnosed with diabetes during the last 18 years of analysis . the population of age 60 years and above diagnosed with diabetes is growing by more than 33.6 thousand people per year . the analysis revealed that the ratio of population of age 60 years and above diagnosed with diabetes to with undiagnosed diabetes rose from ( 1:4 ) in 1992 to almost ( 1:1 ) in 2010 . it is well documented that the population with the highest use of healthcare resources attributed to diabetes is the population aged 60 years and above.[3438 ] healthcare expenditure attributed to diabetes reflects the additional expenditures the nation incurs because of diabetes . this equals to the total healthcare expenditures for people with diabetes minus the projected level of expenditures that would have been incurred in the absence of diabetes . in effect , this is estimates the excess healthcare use that is theoretically due to ( or caused by ) diabetes and its related complications . the term attributed also means the difference in healthcare use for people with diabetes compared with what their healthcare use would be in the absence of diabetes . approximately $ 0.9 billion in healthcare expenditures is incurred by people with diabetes , reflecting $ 1 of every $ 11 moh healthcare dollars . healthcare expenditures incurred by people with diabetes increased by more than 500% during the last 18 years . in 2010 on average , people diagnosed with diabetes have medical healthcare expenditures that are ten times higher ( $ 3,686 vs. $ 380 ) than what expenditures would be in the absence of diabetes ( $ 1 us dollar = 3.75 saudi riyal ) . healthcare expenditures by diabetes status , nationality , sex , and age groups , 1992 and 2010 ( in usa dollars ) dividing total attributed healthcare expenditures by the number of people diagnosed with diabetes , gives an estimate of the average annual excess expenditures for the population under age 15 , age 15 - 44 , age 45 - 60 , and age 60 and above at $ 9,244 , $ 1,255 , $ 767 , and $ 1,442 , respectively . the population between age 45and 60 has the highest per capita healthcare expenditure during the last 18 years . per capita expenditure for population between age 45 and 60 increased by more than 145% between 1992 and 2010 , followed by age 15 - 44 ( 137% ) , under age 15 ( 45% ) , and age 60 years and above ( 36% ) . the analysis revealed that the per capita healthcare expenditure for the population under age 15 is almost nine times higher ( $ 9,244 vs. $ 1,048 ) than healthcare expenditures of otherwise similar age group without diabetes . the analysis also revealed that the per capita healthcare expenditure for the population between age 15 and 44 is two times higher ( $ 1,255 vs. $ 590 ) than healthcare expenditures of the otherwise similar age group without diabetes . per capita healthcare expenditure for male / female population diagnosed with diabetes is equal ( to some extent ) to the per capita healthcare expenditure of male / female population without diabetes . similarly , per capita for saudi citizens diagnosed with diabetes is to some extent equal to per capita for saudi citizens with undiagnosed diabetes . while , per capita healthcare expenditure for non - saudi population is eight times higher ( $ 8,589 vs. $ 1,018 ) than healthcare expenditures of non - saudi nationality without diabetes . as mentioned earlier , the rapid increase of socio - economic development in saudi arabia coupled with improved living standards and technological advancements during the last four decades has resulted in dramatic change of saudi lifestyle . over - nutrition , increasing obesity and smoking rate , and less physical activity and many other factors reflecting the substantial increase in the size of saudi population diagnosed with diabetes.[4045 ] these factors are likely to remain , and if diabetes prevalence rates remained constant over time , then the number of people diagnosed with diabetes is expected to rise by 4.2 million in 2015 and by another seven million by 2020 . the economic burden of diabetes on saudi arabia is expected to be approximately $ 2.4 billion in 2015 [ table 3 ] . this is an increase of $ 1.5 billion , or nearly three times the level in 2010 , this cost is also expected to rise by another $ 6.5 billion in 2020 . given the expected increase in the number of people diagnosed with diabetes in saudi arabia , the proportion of public healthcare spending is expected to escalate from 9.3% in 2010 to 13.1% in 2015 and to 18.3% by 2020 . projection of population with diagnosed diabetes and their healthcare expenditure ( in usa dollars ) the actual economic burden of diabetes in future years is expected to be higher if the cost of health care outpaces the overall cost of living , or if the growing problem of obesity increases the prevalence rate diabetes . even so , such estimates do not account for the lost productivity and losses attributable to pain and suffering incurred by people diagnosed with diabetes , as well as to families and friends of those with diabetes . based on the moh database approximately 0.9 million people in 1992 and 2.5 million people in 2010 have been diagnosed with diabetes . analysis of the 1992 - 2010 moh database suggests that the number of people diagnosed with diabetes increased by 1.6 million ( or 183% ) during the last 18 years . this increases the proportion of the total population with diabetes from 5.3% in 1992 to 9.3% in 2010 . comparison of the 2010 diabetes figure to the 1992 figure suggests that the net number of people diagnosed with diabetes is growing by about 0.1 million per year . this increased prevalence of diabetes over the period of study is not only attributable to changing pattern of saudi lifestyle due to rapid socio - economic development ( together with increased obesity , smoking and less physical activities ) but may also be attributed to increased awareness programs related to diabetes and its health complications , community screening campaigns for diabetes , better diagnostic facilities specially in health centers and healthcare units , and better diabetes management systems and protocols . in fact , several community - based screening campaigns studies for dm and hypertension were conducted in different parts of saudi arabia revealed that quite a large percentage of these participants ( diabetic patients ) were not detected and remain for a long time without significant symptoms . these studies concluded that community - based screening campaigns are extremely efficient in identifying undiagnosed diabetic and hypertensive individuals in the society.[2933 ] table 1 summarizes the demographic component of population diagnosed with diabetes . the moh database for population diagnosed with diabetes by nationality , sex , and age - group , suggests that saudi citizens comprised 84% and 96% of total population diagnosed with diabetes in 1992 and 2010 , respectively , while , non - saudi population diagnosed with diabetes comprised 16% in 1992 and about 4% in 2010 . the demographic analysis also revealed that over half of total population diagnosed with diabetes , in 1992 and 2010 , are males while females represented about 47% and 48% of total population diagnosed with diabetes in 1992 and 2010 , respectively . the analysis revealed that about 1 in 11 males being diabetic in 2010 as compared with 1 in 19 males in 1992 . for females , about 1 in 9 were diabetic in 2010 compared with 1 in 17 in 1992 . population ( in millions ) and percent of population with and without diabetes , 1992 and 2010 the population between 45 and 60 years represented the highest among population diagnosed with diabetes during the period of analysis . this age group comprised about 52% and 45% of total population diagnosed with diabetes in 1992 and 2010 , respectively , with remaining population under age 15 , age 15 - 44 , and 60 years and above comprised about 3% , 21% , 15% in 1992 and about 4% , 27% , 24% in 2010 , respectively . it is important to mention that the population age 60 years and above represents the highest percentage increase among population diagnosed with diabetes during the last 18 years of analysis . the population of age 60 years and above diagnosed with diabetes is growing by more than 33.6 thousand people per year . the analysis revealed that the ratio of population of age 60 years and above diagnosed with diabetes to with undiagnosed diabetes rose from ( 1:4 ) in 1992 to almost ( 1:1 ) in 2010 . it is well documented that the population with the highest use of healthcare resources attributed to diabetes is the population aged 60 years and above.[3438 ] healthcare expenditure attributed to diabetes reflects the additional expenditures the nation incurs because of diabetes . this equals to the total healthcare expenditures for people with diabetes minus the projected level of expenditures that would have been incurred in the absence of diabetes . in effect , this is estimates the excess healthcare use that is theoretically due to ( or caused by ) diabetes and its related complications . the term attributed also means the difference in healthcare use for people with diabetes compared with what their healthcare use would be in the absence of diabetes . approximately $ 0.9 billion in healthcare expenditures is incurred by people with diabetes , reflecting $ 1 of every $ 11 moh healthcare dollars . healthcare expenditures incurred by people with diabetes increased by more than 500% during the last 18 years . in 2010 on average , people diagnosed with diabetes have medical healthcare expenditures that are ten times higher ( $ 3,686 vs. $ 380 ) than what expenditures would be in the absence of diabetes ( $ 1 us dollar = 3.75 saudi riyal ) . healthcare expenditures by diabetes status , nationality , sex , and age groups , 1992 and 2010 ( in usa dollars ) dividing total attributed healthcare expenditures by the number of people diagnosed with diabetes , gives an estimate of the average annual excess expenditures for the population under age 15 , age 15 - 44 , age 45 - 60 , and age 60 and above at $ 9,244 , $ 1,255 , $ 767 , and $ 1,442 , respectively . the population between age 45and 60 has the highest per capita healthcare expenditure during the last 18 years . per capita expenditure for population between age 45 and 60 increased by more than 145% between 1992 and 2010 , followed by age 15 - 44 ( 137% ) , under age 15 ( 45% ) , and age 60 years and above ( 36% ) . the analysis revealed that the per capita healthcare expenditure for the population under age 15 is almost nine times higher ( $ 9,244 vs. $ 1,048 ) than healthcare expenditures of otherwise similar age group without diabetes . the analysis also revealed that the per capita healthcare expenditure for the population between age 15 and 44 is two times higher ( $ 1,255 vs. $ 590 ) than healthcare expenditures of the otherwise similar age group without diabetes . per capita healthcare expenditure for male / female population diagnosed with diabetes is equal ( to some extent ) to the per capita healthcare expenditure of male / female population without diabetes . similarly , per capita for saudi citizens diagnosed with diabetes is to some extent equal to per capita for saudi citizens with undiagnosed diabetes . while , per capita healthcare expenditure for non - saudi population is eight times higher ( $ 8,589 vs. $ 1,018 ) than healthcare expenditures of non - saudi nationality without diabetes . as mentioned earlier , the rapid increase of socio - economic development in saudi arabia coupled with improved living standards and technological advancements during the last four decades has resulted in dramatic change of saudi lifestyle . over - nutrition , increasing obesity and smoking rate , and less physical activity and many other factors reflecting the substantial increase in the size of saudi population diagnosed with diabetes.[4045 ] these factors are likely to remain , and if diabetes prevalence rates remained constant over time , then the number of people diagnosed with diabetes is expected to rise by 4.2 million in 2015 and by another seven million by 2020 . the economic burden of diabetes on saudi arabia is expected to be approximately $ 2.4 billion in 2015 [ table 3 ] . this is an increase of $ 1.5 billion , or nearly three times the level in 2010 , this cost is also expected to rise by another $ 6.5 billion in 2020 . given the expected increase in the number of people diagnosed with diabetes in saudi arabia , the proportion of public healthcare spending is expected to escalate from 9.3% in 2010 to 13.1% in 2015 and to 18.3% by 2020 . projection of population with diagnosed diabetes and their healthcare expenditure ( in usa dollars ) the actual economic burden of diabetes in future years is expected to be higher if the cost of health care outpaces the overall cost of living , or if the growing problem of obesity increases the prevalence rate diabetes . even so , such estimates do not account for the lost productivity and losses attributable to pain and suffering incurred by people diagnosed with diabetes , as well as to families and friends of those with diabetes . diabetes is a personal crisis for people living with the disease , and for their families . treatment of the disease and its related complications are consuming an ever - larger share of healthcare budgets , and will soon force a tremendous increase in those budgets . although the population diagnosed with diabetes comprises more than 9% of the saudi population in 2010 , almost 1 in 11 people in saudi arabia having diabetes , and if the present prevalence rate remains unchanged over time , the ratio of people with to without diabetes will increase by almost 1 in 5 people having the disease in 2020 . people diagnosed with diabetes have medical healthcare expenditures that are ten times higher than what expenditures would be in the absence of diabetes . this cost is estimated to be $ 6.5 billion by 2020 reflecting an increase of $ 5.6 billion ( or 7 times higher than the cost in 2010 ) within the next 10 years or so . although , the diabetes cost estimates presented in this research study might be conservative for several reasons : omitted from this analysis due to data limitations is the number of people who reported that they had diabetes in both private and other governmental ( e.g. universities hospitals , armed forces medical services , security forces hospital , national guard medical services , royal commission hospitals , youth welfare hospital , now saudi aramco ) healthcare service providers . at the time of writing in 2012 , there are 148 hospitals and about 2,360 health clinics not related to moh healthcare system . this research study relied only on data obtained directly from the moh healthcare database on people diagnosed with diabetes and ignored other sources of data due to difficulty in obtaining them . consequently , diabetes prevalence is probably much higher than figures presented in this study because a significant number of cases are not included in the analysis.omitted from the cost estimates is the indirect cost associated with diabetes such as lost productivity due to disease - related absenteeism , unemployment due to disease - related disability , lost productivity due to early mortality by disease as well as the social cost of intangibles such as pain and suffering and care provided by non - paid caregivers . the prevention programs targeted to people with diabetes , research activities , and administration costs associated with diabetes are also omitted for the diabetes cost estimates in this study . if a portion of these costs were attributed to diabetes , the national healthcare cost of diabetes would be billions of dollars higher than what the estimate suggest in this study.sensitivity analysis related diabetic mellitus and its related complications were not carried out in this study . omitted from this analysis due to data limitations is the number of people who reported that they had diabetes in both private and other governmental ( e.g. universities hospitals , armed forces medical services , security forces hospital , national guard medical services , royal commission hospitals , youth welfare hospital , now saudi aramco ) healthcare service providers . at the time of writing in 2012 , there are 148 hospitals and about 2,360 health clinics not related to moh healthcare system . this research study relied only on data obtained directly from the moh healthcare database on people diagnosed with diabetes and ignored other sources of data due to difficulty in obtaining them . consequently , diabetes prevalence is probably much higher than figures presented in this study because a significant number of cases are not included in the analysis . omitted from the cost estimates is the indirect cost associated with diabetes such as lost productivity due to disease - related absenteeism , unemployment due to disease - related disability , lost productivity due to early mortality by disease as well as the social cost of intangibles such as pain and suffering and care provided by non - paid caregivers . the prevention programs targeted to people with diabetes , research activities , and administration costs associated with diabetes are also omitted for the diabetes cost estimates in this study . if a portion of these costs were attributed to diabetes , the national healthcare cost of diabetes would be billions of dollars higher than what the estimate suggest in this study . sensitivity analysis related diabetic mellitus and its related complications were not carried out in this study . worldwide , resources allocated to diabetes prevention and treatment , are growing , but prevalence is also growing steadily . several international reports on preventing dm indicated that people with diabetes are at greater risk of neurological disease , peripheral vascular disease , cardiovascular disease , renal disease , endocrine / metabolic complications , ophthalmic disease , and other chronic complications compared with individuals without diabetes.[18 ] chronic complications are the main cause of death among diabetic patients and account for the higher costs in hospitalization and drugs , and the costs of drugs for these complications are 2.5 times higher than those for the general population . these international reports suggested that better access to preventive healthcare system , more widespread diagnosis , more intensive disease management , and the advent of new medical technologies could significantly eliminate or reduce the health problems caused by diabetes . these factors could also improve the quality of life for people with diabetes and their families , while at the same time potentially reduce national expenditures for healthcare services and increasing productivity in the national economy . to sum up , further research studies are needed to improve our understanding of economic costs of diabetes and its substantial burden to saudi healthcare system . special attention should also be given to the role of diabetic awareness programs , community - based screening campaigns and different health educational programs in reducing health problems caused by diabetes , which in the long run , helps to reduce the national burden of this disease . sha should also be adopted by saudi health providers to deal with these developments of financial health flows related to the consumptions of healthcare goods and services . the prospective study of pravastatin in the elderly at risk ( prosper ) was a prospective multicenter randomized placebo - controlled trial to assess whether treatment with pravastatin diminishes the risk of major cardiovascular events in the elderly ( 12,13 ) . between december 1997 and may 1999 , a total of 5,804 participants ( aged 7082 years ) with preexisting vascular disease or increased risk of such disease due to a history of smoking , hypertension , or diabetes were recruited in scotland , ireland , and the netherlands . the institutional ethics review boards of all centers approved the protocol , and all participants gave written informed consent . participants with very severe cognitive impairment ( mini - mental state examination [ mmse ] score < 24 ) were excluded for inclusion in the study . the rotterdam study is a large prospective population - based cohort study that is conducted among all inhabitants aged 55 years of ommoord , a district of rotterdam , the netherlands ( 14 ) . the medical ethics committee of the erasmus university of rotterdam approved the study , and written informed consent was obtained from all participants . of 10,275 eligible subjects , 7,983 individuals ( 78% ) participated in the baseline examinations between 1990 and 1993 ( mean age 71 25 years , range 55106 years ) . all participants were interviewed at home and visited the research center for further examinations . in prosper , fasting glucose levels were assessed at baseline in 5,599 of the 5,804 participants . of the 5,599 participants , 580 did not have all cognitive function tests available at baseline . all of the resulting 5,019 participants had full data available for other cardiovascular risk factors including bmi , systolic and diastolic blood pressure , and hdl cholesterol levels at baseline . this resulted in a study sample of 5,019 participants for prosper . in the rotterdam study , fasting glucose levels were assessed at the third survey in 3,795 participants . of these participants , 3,664 were free of dementia , and 3,550 of those had data available for other cardiovascular risk factors including bmi , systolic and diastolic blood pressure , and hdl cholesterol levels . of these 3,550 participants , 122 did not have all cognitive function tests available at the start of the third survey . additionally , in 3,342 of these 3,428 participants , fasting insulin levels were assessed at the third survey . in both the prosper and the rotterdam sample , there were 4,690 of the 5,019 participants in the prosper sample and 2,364 of the 3,428 participants in the rotterdam sample who underwent at least one additional measurement of fasting glucose level in addition to the initial examination . these data were used to study the variability of the fasting glucose levels over time and to assess the appropriateness of using a single baseline fasting glucose measurement to assess the relationship between fasting glucose and cognitive function and decline . at baseline , history of diabetes was defined by self - reported history of diabetes ( reporting the use of oral antidiabetes medication , the use of insulin , treatment by diet , or registration by a general practitioner as having diabetes ) in both study samples . in addition , a dedicated neuropsychological test battery was used to assess executive function and memory . executive function was assessed with the letter digit substitution task ( ldst ) ( 16 ) and the abbreviated stroop test part 3 ( 17 ) in both studies , as well as with the word fluency test ( wft ) ( 18 ) in the rotterdam study only . memory was assessed with the 12-picture learning test ( 12-plt ) immediate and delayed recall ( 19 ) , in prosper only . individual test scores were transformed into standardized z scores [ z score = ( individual score a compound cognitive test score for global cognitive function was calculated by averaging the z scores of the mmse , the ldst , and the abbreviated stroop test part 3 . a compound cognitive test score for the 12-plt was calculated by averaging the z scores of the 12-plt immediate and the 12-plt delayed recall test . in prosper , cognitive function was measured at six time points during the study : before randomization , at baseline , after 9 , 18 , and 30 months , and at the end of the study . the time point of the last measurement was different for the participants and ranged from 36 to 48 months after baseline . therefore , we performed the analyses with their individual varying time point but report the results for the mean of these time points ( at 42 months ) . change in cognitive function could be assessed in 4,767 participants for whom at least one follow - up examination of cognitive function was available after the initial measurement . in the rotterdam study , cognitive function was assessed at the third survey ( 19971999 ) and additionally at the fourth survey ( 20022004 ) . of the 3,428 participants of the rotterdam sample who were present at the third survey , 2,601 remained in the study until the end of follow - up ( fourth survey ) and were available for the assessment of change in cognitive function . in both samples , level of education , bmi , systolic and diastolic blood pressure , hdl cholesterol level , and apoe 4 carriership were assessed at baseline ( prosper ) or at the third survey ( rotterdam study ) . level of education was dichotomized into primary education or less ( low ) and more than primary education ( high ) ( rotterdam study ) and into age when leaving school < 13 years ( low ) and age when leaving school 13 years ( high ) ( prosper ) . the relationship between baseline ( prosper ) or third survey ( rotterdam study ) fasting glucose levels and cognitive function and decline was assessed by use of linear mixed models . data from the prosper sample and the rotterdam study sample were merged into one large sample of 8,447 participants . all analyses were adjusted for age , sex , level of education , study ( prosper or rotterdam study ) , bmi , systolic and diastolic blood pressure , hdl cholesterol level , apoe 4 carriership , country , use of pravastatin , and , where appropriate , test version . analyses were carried out using the spss statistical package ( release 12.0.1 ; spss , chicago , il ) . data on fasting glucose and fasting insulin levels from the rotterdam study sample were used to calculate the degree of insulin resistance according to homa ( 20 ) . the homa index is calculated by dividing the product of fasting levels of glucose and insulin by a constant and has been shown to correlate well ( r = 0.82 , p < 0.0001 ) with the euglycemic - hyperinsulinemic clamp method ( 21 ) . the prospective study of pravastatin in the elderly at risk ( prosper ) was a prospective multicenter randomized placebo - controlled trial to assess whether treatment with pravastatin diminishes the risk of major cardiovascular events in the elderly ( 12,13 ) . between december 1997 and may 1999 , a total of 5,804 participants ( aged 7082 years ) with preexisting vascular disease or increased risk of such disease due to a history of smoking , hypertension , or diabetes were recruited in scotland , ireland , and the netherlands . the institutional ethics review boards of all centers approved the protocol , and all participants gave written informed consent . participants with very severe cognitive impairment ( mini - mental state examination [ mmse ] score < 24 ) were excluded for inclusion in the study . the rotterdam study is a large prospective population - based cohort study that is conducted among all inhabitants aged 55 years of ommoord , a district of rotterdam , the netherlands ( 14 ) . the medical ethics committee of the erasmus university of rotterdam approved the study , and written informed consent was obtained from all participants . of 10,275 eligible subjects , 7,983 individuals ( 78% ) participated in the baseline examinations between 1990 and 1993 ( mean age 71 25 years , range 55106 years ) . in prosper , fasting glucose levels were assessed at baseline in 5,599 of the 5,804 participants . of the 5,599 participants , 580 all of the resulting 5,019 participants had full data available for other cardiovascular risk factors including bmi , systolic and diastolic blood pressure , and hdl cholesterol levels at baseline . this resulted in a study sample of 5,019 participants for prosper . in the rotterdam study , fasting glucose levels were assessed at the third survey in 3,795 participants . of these participants , 3,664 were free of dementia , and 3,550 of those had data available for other cardiovascular risk factors including bmi , systolic and diastolic blood pressure , and hdl cholesterol levels . of these 3,550 participants , 122 did not have all cognitive function tests available at the start of the third survey . additionally , in 3,342 of these 3,428 participants , fasting insulin levels were assessed at the third survey . in both the prosper and the rotterdam sample , fasting glucose levels were additionally measured during follow - up . there were 4,690 of the 5,019 participants in the prosper sample and 2,364 of the 3,428 participants in the rotterdam sample who underwent at least one additional measurement of fasting glucose level in addition to the initial examination . these data were used to study the variability of the fasting glucose levels over time and to assess the appropriateness of using a single baseline fasting glucose measurement to assess the relationship between fasting glucose and cognitive function and decline . at baseline , history of diabetes was defined by self - reported history of diabetes ( reporting the use of oral antidiabetes medication , the use of insulin , treatment by diet , or registration by a general practitioner as having diabetes ) in both study samples . global cognitive function was measured with the mmse ( 15 ) in both studies . in addition , a dedicated neuropsychological test battery was used to assess executive function and memory . executive function was assessed with the letter digit substitution task ( ldst ) ( 16 ) and the abbreviated stroop test part 3 ( 17 ) in both studies , as well as with the word fluency test ( wft ) ( 18 ) in the rotterdam study only . memory was assessed with the 12-picture learning test ( 12-plt ) immediate and delayed recall ( 19 ) , in prosper only . individual test scores were transformed into standardized z scores [ z score = ( individual score a compound cognitive test score for global cognitive function was calculated by averaging the z scores of the mmse , the ldst , and the abbreviated stroop test part 3 . a compound cognitive test score for the 12-plt was calculated by averaging the z scores of the 12-plt immediate and the 12-plt delayed recall test . in prosper , cognitive function was measured at six time points during the study : before randomization , at baseline , after 9 , 18 , and 30 months , and at the end of the study . the time point of the last measurement was different for the participants and ranged from 36 to 48 months after baseline . therefore , we performed the analyses with their individual varying time point but report the results for the mean of these time points ( at 42 months ) . change in cognitive function could be assessed in 4,767 participants for whom at least one follow - up examination of cognitive function was available after the initial measurement . in the rotterdam study , cognitive function was assessed at the third survey ( 19971999 ) and additionally at the fourth survey ( 20022004 ) . of the 3,428 participants of the rotterdam sample who were present at the third survey , 2,601 remained in the study until the end of follow - up ( fourth survey ) and were available for the assessment of change in cognitive function . in both samples , level of education , bmi , systolic and diastolic blood pressure , hdl cholesterol level , and apoe 4 carriership were assessed at baseline ( prosper ) or at the third survey ( rotterdam study ) . level of education was dichotomized into primary education or less ( low ) and more than primary education ( high ) ( rotterdam study ) and into age when leaving school < 13 years ( low ) and age when leaving school 13 years ( high ) ( prosper ) . the relationship between baseline ( prosper ) or third survey ( rotterdam study ) fasting glucose levels and cognitive function and decline was assessed by use of linear mixed models . data from the prosper sample and the rotterdam study sample were merged into one large sample of 8,447 participants . all analyses were adjusted for age , sex , level of education , study ( prosper or rotterdam study ) , bmi , systolic and diastolic blood pressure , hdl cholesterol level , apoe 4 carriership , country , use of pravastatin , and , where appropriate , test version . analyses were carried out using the spss statistical package ( release 12.0.1 ; spss , chicago , il ) . data on fasting glucose and fasting insulin levels from the rotterdam study sample were used to calculate the degree of insulin resistance according to homa ( 20 ) . the homa index is calculated by dividing the product of fasting levels of glucose and insulin by a constant and has been shown to correlate well ( r = 0.82 , p < 0.0001 ) with the euglycemic - hyperinsulinemic clamp method ( 21 ) . table 1 shows the baseline characteristics of the total sample and for participants with a history of diabetes at baseline ( prosper ) or at the third survey ( rotterdam study ) . in prosper , the fasting glucose levels sd differed among the three countries from which the participants were enrolled : scotland 5.62 1.27 , ireland 5.09 1.34 ) , and the netherlands 5.76 1.64 mmol / l . this resulted in a lower mean fasting glucose level for the prosper study sample compared with that of the rotterdam study sample . participants with a history of diabetes had a higher fasting glucose level , bmi , and systolic blood pressure and lower levels of hdl cholesterol compared with participants without a history of diabetes . * fasting insulin levels were available for 3,342 participants in the rotterdam study . in prosper , fasting glucose levels were assessed during follow - up after 3 , 6 , 12 , 24 , and 36 months , in addition to the baseline assessment . in 3,491 participants without a history of diabetes , fasting glucose levels were available at baseline and after 36 months of follow - up . the quintiles of the mean fasting glucose sd at baseline and after 36 months were 4.30 0.20 and 4.77 0.67 mmol / l for the lowest quintile ( quintile 1 ) , 4.70 0.08 and 4.95 0.47 mmol / l for quintile 2 , 5.00 0.08 and 5.21 0.67 mmol / l for quintile 4 , and 6.25 0.83 and 6.30 1.40 mmol / l for the highest quintile ( quintile 5 ) . in the rotterdam sample , fasting glucose levels were assessed at the third survey as well as at the end of follow - up ( fourth survey ) in 2,209 participants without a history of diabetes . the quintiles of fasting glucose levels at the third survey and at the end of follow - up were 4.82 0.24 and 5.07 0.39 mmol / l for the lowest quintile ( quintile 1 ) , 5.20 0.08 and 5.36 0.42 mmol / l for quintile 2 , 5.50 0.08 and 5.55 0.50 mmol / l for the highest quintile ( quintile 5 ) . of the 8,447 participants who were present at baseline , 6,641 remained in the study sample until the end of follow - up , 985 withdrew from the study , and 821 died during follow - up . the 1,806 participants without a final examination comprised 20.8% of the participants without a history of diabetes and 26.3% of the participants with a history of diabetes at baseline ( prosper ) or at the third survey ( rotterdam study ) . figure 1a shows the relationship between fasting glucose levels and cognitive function at baseline for the 8,447 participants of the merged study sample . study - specific quintiles of the distribution of fasting glucose levels in participants without a history of diabetes were constructed to account for the differences in fasting glucose levels between the studies . cognitive test scores are shown for quintiles of the distribution of fasting glucose levels in participants without a history of diabetes and for participants with a history of diabetes . in participants without a history of diabetes , a rise in fasting glucose levels in the nondiabetes range was not associated with impairment in cognitive function , for any of the cognitive tests . additionally , we compared the cognitive test scores at baseline for participants with and without a history of diabetes ( fig . 1a ) and showed that participants with a history of diabetes had worse cognitive function across the majority of tests at baseline when compared with participants without a history of diabetes ( p < 0.05 for all tests except for the wft in the rotterdam study sample ) . z scores ( sem ) for different cognitive test scores are plotted for study - specific quintiles of fasting glucose levels in nondiabetic participants ( from lowest [ quintile 1 ] to the highest [ quintile 5 ] levels of fasting glucose ) and for participants with a history of diabetes ( dm ) . p values reflect the trend over the quintiles of fasting glucose levels , as well as the difference between participants without a history of diabetes ( n = 7,592 ) and participants with a history of diabetes ( n = 855 ) . estimates are based on the maximum number of participants available per cognitive test : global cognitive function ( n = 8,447 ) , wft ( n = 3,518 ) , and 12-plt ( n = 5,223 ) . z scores ( sem ) represent annual change in cognitive test scores for study - specific quintiles of fasting glucose levels in nondiabetic participants ( from lowest [ quintile 1 ] to the highest [ quintile 5 ] levels of fasting glucose ) and for participants with a history of diabetes . p values reflect the trend over the quintiles of fasting glucose levels , as well as the difference between participants without a history of diabetes ( n = 6,649 ) and participants with a history of diabetes ( n = 719 ) . estimates are based on the maximum number of participants available per cognitive test : global cognitive function ( n = 7,368 ) , wft ( n = 2,639 ) , and 12-plt ( n = 4,960 ) . linear mixed models were used , adjusted for age , sex , level of education , study ( prosper or rotterdam study ) , bmi , hdl level , systolic blood pressure , diastolic blood pressure , country , treatment group , and test version where applicable . in the longitudinal analyses of the study population , there was no clear association between baseline fasting glucose levels and change in cognitive function during follow - up in participants without a history of diabetes ( fig . higher levels of fasting glucose were associated with a decreased rate of decline on the 12-plt ( ptrend = 0.039 ) , but this was not seen for any of the other cognitive tests . furthermore , participants with a history of diabetes did not show an increased rate of decline for any of the cognitive tests . additionally , we assessed the relationship between insulin resistance and cognitive function and decline for the 3,342 participants of the rotterdam study sample for whom fasting insulin levels were available ( fig . the homa that was calculated for these participants was correlated with the fasting glucose levels ( r = 0.54 , p < 0.001 ) , although the overlap between quintiles of the homa index and quintiles of fasting glucose levels was limited : only 35% of the participants without a history of diabetes were in the same quintile of the distribution for both fasting glucose and homa index . the relationship between insulin resistance and cognitive function was in accordance with findings on fasting glucose levels and cognitive function : in participants without a history of diabetes , rising insulin resistance was not associated with cognitive function . similarly , there was no clear relationship between levels of insulin resistance and change in cognitive function during follow - up ( fig . when the data from the prosper sample and the rotterdam study sample were analyzed separately , the findings were consistent with the results of the merged sample of 8,447 participants . a : insulin resistance ( homa ) and cognitive function in the rotterdam study . z scores ( sem ) for different cognitive test scores are plotted for quintiles of insulin resistance ( homa ) in nondiabetic participants ( from lowest [ quintile 1 ] to the highest [ quintile 5 ] levels of insulin resistance [ homa ] ) and for participants with a history of diabetes ( dm ) . p values reflect the trend over the quintiles of fasting glucose levels , as well as the difference between participants without a history of diabetes ( n = 3,039 ) and participants with a history of diabetes ( n = 303 ) . estimates are based on the maximum number of participants available per cognitive test : global cognitive function ( n = 3,342 ) and wft ( n = 3,424 ) . z scores ( sem ) represent annual change in cognitive test scores for quintiles of insulin resistance ( homa ) in nondiabetic participants ( from lowest [ quintile 1 ] to the highest [ quintile 5 ] levels of insulin resistance [ homa ] ) and for participants with a history of diabetes . p values reflect the trend over the quintiles of fasting glucose levels , as well as the difference between participants without a history of diabetes ( n = 2,331 ) and participants with a history of diabetes ( n = 203 ) . estimates are based on the maximum number of participants available per cognitive test : global cognitive function ( n = 2,534 ) and wft ( n = 2,567 ) . linear mixed models were used , adjusted for age , sex , level of education , bmi , hdl level , systolic blood pressure , and diastolic blood pressure . our study shows that , in an unprecedented large number of individuals from two independent prospective studies , higher levels of fasting glucose in the absence of a history of diabetes are not associated with cognitive function or cognitive decline . furthermore , there was no association between insulin resistance ( homa index ) and cognitive function and decline in people without a history of diabetes . however , participants with a history of diabetes did have worse cognitive function at baseline than those without diabetes , although the magnitude of the observed effects was relatively small . the results of our analyses do not fully correspond with previous findings in the rotterdam study , in which diabetes was found to be related to an increased risk of developing dementia ( 22 ) . although our data show that those with a history of diabetes have worse cognitive function at baseline than those without , one could argue that the effect sizes are lower than expected based on the previous report of an almost twofold increased risk of dementia for people with diabetes ( 22 ) . it is possible that the measurement of cognitive function compared with the assessment of participants with dementia in the rotterdam study underlies this discrepancy . in the rotterdam study , we are able to continuously monitor the total cohort for incident dementia through computerized linkage between the study database and digitalized medical records from the general practitioners and the regional institute for outpatient mental health care . in contrast , in the rotterdam sample , cognitive function assessments were performed with a 4.6-year interval , which could have led to selective nonparticipation of those in whom dementia was diagnosed between cognitive examinations ( n = 85 ) and did not visit the research center for the follow - up assessment . furthermore , a previous report from the nurses ' health study showed that higher insulin levels in nondiabetic participants were related to faster cognitive decline ( 23 ) . these results are not in agreement with our results on the relationship between insulin resistance and cognitive decline in the rotterdam study sample . the prolonged length of follow - up ( > 10 years compared with 4.6 years ) in the nurses ' health study could have affected these results , as the effect of insulin resistance on cognitive function might be long - term . on the other hand , results from the ongoing memory in diabetes ( mind ) substudy of the action to control cardiovascular risk in diabetes ( accord ) trial showed that higher a1c but not fasting plasma glucose levels was associated with worse cognitive function in a sample of almost 3,000 participants ( 24 ) , which is in line with our results . in individual participants , diabetes and impaired fasting glucose levels often co - occur with other cardiovascular risk factors . the metabolic syndrome is seen as a clustering of a number of these risk factors ( abdominal obesity , hypertriglyceridemia , low hdl cholesterol , hypertension , and hyperglycemia ) and is the subject of an ongoing discussion on the clinical use of the syndrome and its individual components in establishing the risk of cardiovascular disease and diabetes ( 25 ) . other studies have suggested a relationship between the metabolic syndrome and the risk of cognitive impairment and dementia ( 2628 ) . in our study , the clustering of these factors of the metabolic syndrome with the fasting glucose levels could have influenced our results . however , adjustment for bmi , systolic and diastolic blood pressure , and hdl cholesterol did not differ from the analyses that were unadjusted for these covariates . furthermore , the participants in prosper were included based on their increased cardiovascular risk profile : having either preexisting vascular disease or increased risk of such disease due to a history of smoking , hypertension , or diabetes . the known association between these cardiovascular risk factors and cognitive function and decline might have interfered with our investigation of the relationship between fasting glucose levels and cognitive function and decline . however , when we excluded the 2,823 participants in prosper who had a history of vascular disease from the sample of 5,019 participants , the relationship between fasting glucose levels and cognitive function at baseline did not markedly differ from our findings in the total sample : there was no clear relationship between fasting glucose levels and cognitive function in participants without a history of vascular disease . moreover , after exclusion of the 2,823 participants , there was no significant relationship anymore between history of diabetes and baseline global cognitive function . the relationship between fasting glucose levels and change in cognitive function did not markedly change after exclusion of the participants with a history of vascular disease . the same was seen for the analyses on the relationship between history of diabetes and change in cognitive function . therefore , we do not think that the inclusion of participants with preexisting vascular disease in prosper has masked a possible association between fasting glucose and insulin resistance on cognitive function . we used fasting insulin levels that were available for almost the entire sample of the rotterdam study to calculate the homa index as a measure of insulin resistance to further investigate the relationship between glucose metabolism and cognitive function . however , the relationship between insulin resistance and cognitive function and decline showed similarities with the association between fasting glucose levels and cognitive function and decline : in participants without a history of diabetes , insulin resistance was not associated with cognitive function or decline . previous population - based studies that investigated the relationship between glucose metabolism and cognitive functions suggested a number of possible biological mechanisms that could be involved , ranging from accumulation of advanced glycation end products ( 2 ) and accelerated cerebrovascular disease ( 1 ) to the role of the insulin - degrading enzyme on amyloid metabolism ( 3 ) . although it is difficult to address the role of these suggested mechanisms , our study of > 8,000 participants shows that the effect of increased fasting glucose levels on cognitive function seems to be long - term and independent of other cardiovascular risk factors like bmi , blood pressure , and hdl cholesterol levels . the observed differences in cognitive test scores between people with and without a history of diabetes were relatively moderate and may therefore lack clinical significance for individuals . however , small effect sizes do not automatically imply irrelevance of the observed effect , as small effects on the group level can indeed represent large effects for a number of participants . in the analyses of the annual decline in cognitive function , the prosper sample failed to show a clear decline in mmse score over time , although this was seen in the rotterdam sample with participants of comparable age . it is possible that a potential learning effect of the mmse had a higher impact on the prosper sample compared with the rotterdam study sample because of the shorter time span between cognitive measurements ( 19 ) . additionally , the selection criteria for participants in prosper ( baseline mmse score 24 ) may have resulted in a sample of participants with slightly better cognitive function , which is also represented in the difference in mmse scores of both samples at baseline and might have had an effect on the annual decline of mmse score that was measured in prosper . the strengths of this study consist of the prospective design , the large number of participants in both studies , and the dedicated neuropsychological test battery that was used in both samples . furthermore , we had the possibility of studying the variability of fasting glucose levels during follow - up and of examining the appropriateness of using a single measurement of fasting glucose level to assess the association between fasting glucose levels and cognitive function and decline . a large variation in fasting glucose levels over time could have disturbed our analyses through the phenomenon of regression - to - the - mean . however , the levels of fasting glucose during follow - up did not materially differ from the baseline or third survey in both study samples . therefore , we decided to use the baseline or third survey fasting glucose measurement in our analyses . participants who were present at baseline but did not undergo follow - up examinations were predominantly present in the group with a history of diabetes . they had worse cognitive function at baseline compared with the participants who stayed in the study until the end of follow - up . this selective attrition of participants with relatively high levels of fasting glucose and concurrent low levels of cognitive function could have resulted in an underestimation of our estimates of cognitive decline for participants with a history of diabetes . we also recognize that some individuals with diabetes would have been missed because of lack of oral glucose tolerance testing . more importantly , undiagnosed diabetes would be more prevalent in those in the higher quintiles for fasting glucose and would have biased the study toward an association of higher quintiles and cognitive decline , not the other way around . thus , lack of oral glucose tolerance testing does not negate our findings ; rather it gives us added confidence that our observations are valid . in conclusion , elevations in fasting glucose levels are not clearly associated with impaired cognitive function or with an accelerated rate of cognitive decline in participants without a history of diabetes . furthermore , there was no clear relationship between insulin resistance ( homa index ) and cognitive function and decline in participants without a history of diabetes . these data suggest that cognitive decline accelerates strongly once a person is diabetic but not with lesser degrees of dysglycemia . as a result , preventing individuals at risk from developing diabetes through lifestyle changes may also lead to large societal gains by preventing such individuals from undergoing accelerated cognitive decline .

background : diabetes imposes a large economic burden on the individual , national healthcare systems , and countries.objective:to determine the economic impact of diabetes mellitus on saudi healthcare system , both now and in the future.materials and methods : this research study uses a prevalence - based approach that combines the demographics of the population ( classified by nationality , sex and age group ) with and without diagnosed diabetes in 1992 and 2010 . the economic impact of diabetes is estimated in this study , using secondary sources of information provided by ministry of health , ministry of finance and central department of statistics and information databases.results:people diagnosed with diabetes , on average , have medical healthcare expenditures that are ten times higher ( $ 3,686 vs. $ 380 ) than what expenditures would be in the absence of diabetes . over 96% of all medical healthcare expenditures attributed to diabetes are incurred by persons of saudi nationality , with the remaining 4% incurred by persons of non - saudi nationality . the population age 45 - 60 incurs 45% of diabetes - attributed costs , with the remaining population under age 15 incurs 3.8% , age 15 - 44 incurs 27.5% , and age 60 and above incurs 23.8%.conclusion : the actual national healthcare burden because of diabetes is likely to exceed the $ 0.87 billion estimated in this study , because it omits the indirect costs associated with diabetes , such as absenteeism , lost productivity from disease - related absenteeism , unemployment from disease - related disability , lost productivity due to early mortality by disease . the social cost of intangibles such as pain and suffering and care provided by non - paid caregivers as well as healthcare system administrative costs , cost of medications , clinician training programs , and research and infrastructure development is also omitted from this research study . further studies are needed to confirm the present findings and to improve our understanding of economic costs of diabetes and its related complications . objectiveto investigate the relationship between fasting glucose levels , insulin resistance , and cognitive impairment in old age . diabetes is associated with cognitive impairment in older people . however , the link between elevated fasting glucose levels and insulin resistance in nondiabetic individuals , and the risk of cognitive impairment is unclear.research design and methodswe analyzed data from , in total , 8,447 participants in two independent prospective studies : the prospective study of pravastatin in the elderly at risk ( prosper ) , 5,019 participants , aged 6984 years , and the rotterdam study , 3,428 participants , aged 6197 years . fasting glucose levels were assessed at baseline in both studies ; fasting insulin levels were assessed in the rotterdam study only . cognitive function was assessed in both studies at baseline and during follow-up.resultssubjects with diabetes had impaired cognitive function at baseline . in contrast , in people without a history of diabetes , there was no clear association between baseline fasting glucose levels and executive function and memory , nor was there a consistent relationship between elevated baseline fasting glucose levels and the rate of cognitive decline in either cohort . insulin resistance ( homeostasis model assessment index ) was also unrelated to cognitive function and decline.conclusionselevated fasting glucose levels and insulin resistance are not associated with worse cognitive function in older people without a history of diabetes . these data suggest either that there is a threshold for effects of dysglycemia on cognitive function or that factors other than hyperglycemia contribute to cognitive impairment in individuals with frank diabetes .

as mentioned in the report of the national eye institute , dry eye is a disorder of the tear film due to tear deficiency or excessive tear evaporation , which causes damage to the interpalpebral ocular surface , and is associated with symptoms of ocular discomfort . since contact lenses disrupt the integrity of the tear film and thin out the lipid layer , thus increasing tear evaporation , they can potentially cause symptoms of eye dryness in wearers . . found that ocular dryness affects 20% of wearers of new - generation silicone hydrogel contact lenses , as well as 24% of wearers of older - generation , low - oxygen permeable ( phema ) lenses . brennan and efron found that 75% of low - oxygen permeable lens wearers report a feeling of discomfort at the end of the day related to dry eye . in general , it is believed that more than 50% of contact lens wearers are symptomatic of dry eye . they may also present with clinical signs of dryness , such as damage to the surface of the eye , as well as instability and/or hyperosmolarity of the tear film ; however , the symptoms do not always correlate with the objective signs . in all of these cases , the subject develops discomfort in the absence of any ocular pathology that could trigger the dryness . all other factors being discarded , the contact lens wear must therefore be considered as the most likely etiology of the subject 's symptoms . subjects with induced ocular dryness by contact lens wear usually tend to drop out from this modality when the benefits of the lenses are outweighed by the discomfort they feel , especially during the last hours of wear , day after day . this is the main reason why 20% of low - oxygen permeable lens wearers and 13% of silicone hydrogel lens wearers stop wearing contact lenses every year . this discontinuation rate represents a continuous loss for the industry and for eyecare professionals , and the quest to find new methods to prevent induced dryness of the ocular surface is an ongoing concern . manufacturers first tried to deal with the materials used for contact lenses . among other things , comfort depends on a given material 's rate of on - eye dehydration [ 711 ] , wettability [ 12 , 13 ] , and the presence of surface deposits [ 810 ] . silicone hydrogel lenses are currently considered to be the material of choice compared with hydrogels . clinically , while many benefits [ 14 , 15 ] are recognized , studies have shown that silicone hydrogel lenses have not altered end - of - day discomfort , as described above , at least not as expected , with the exception of senofilcon a , which was found to improve comfort in adverse and challenging environments . when materials proved not to be a solution to the problem of dry eye , researchers turned their attention to the lens design ( spherical versus aspherical ) , the edge profile ( sharp and thin versus rounded and thicker ) , the fit of the lens on the eye ( flat or steep ) , and the replacement rate ( 2 weeks versus 1 month ) . on many of these aspects , care products were also considered as triggering ocular dryness symptoms because they are chemical products that are known to cause short- and medium - term toxic and allergic reactions , leading to patient discomfort . preservatives have been identified as the main components of these products that can trigger these reactions among contact lens wearers [ 1822 ] . it is also known that specific biocides found in contact lens solutions can be related to toxic and allergic reactions that produce more severe symptoms in wearers . accordingly , solutions containing polyhexanides , specifically polyaminopropyl biguanide ( papb ) and polyhexamethylene biguanide ( phmb ) , are known to increase the potential for adverse reactions . polyhexanides are polycationic agents with a relatively high molecular weight , which , in theory , should limit their absorption by the contact lens . however , studies have shown that certain materials can easily absorb a large quantity of polycationic agents during the lens soaking phase ( at night ) . other factors that can explain this absorption are the number and the nature of buffers in the solution and the presence of sodium chloride which determines the ionic strength of the lens - solution interaction . on insertion , the lens balances out with the surrounding ocular environment , and the biocides absorbed by the lens matrix are released into the eye , possibly contributing to a temporary alteration of the ocular surface , especially during the first four hours of wear . absorption and release rates vary depending on the agents and materials used , and not their surface treatment , which determines compatibility between certain solutions and certain materials . for example , aldox penetrates more than phmb and is released more rapidly ( between 0 and 60 min ) into the eye after insertion of the contact lens . this has prompted certain authors to suggest that wearers neutralize the polycationic agents ( such as phmb ) absorbed during soaking by adding a drop of anionic solution to the lenses before insertion . the anionic - cationic reaction creates a chemically neutral environment , sparing the cornea from temporary structural changes during wear . this has been proven to contribute to increased patient comfort , even at the end of the day . the simple use of a lubricant in the morning would therefore significantly improve end - of - day symptoms by creating a chemically neutral environment in the cornea from the outset , thus eliminating the potential for toxic or allergic reactions . the only agent that has been tested in this regard and that has confirmed this hypothesis is carboxymethyl cellulose ( cmc ) , an anionic polymer found in certain lubricants ( refresh , allergan , irivne , ca ) . cmc is a water - soluble synthetic cellulose derivative with marked hydrophilic and bioadhesive properties that promote faster repair of the eye surface . this study aims to evaluate the performance of sodium hyaluronate ( sh ) in the same way . in fact , it is believed that the use of this product , instilled in the lens , before insertion , would chemically neutralize the biocide effects on the ocular surface , helping to alleviate initial and long - term discomfort associated to contact lens wear . sh is a viscoelastic agent used in several eye care products , both topically and in surgery , to protect and maintain the integrity of ocular tissues . sixty - four patients were recruited among 4 optometric offices in the greater montreal area , each of them being selected as representative of an average optometric practice in north america , based on the number and type of patients seen . one optometrist per office was designated and appropriately trained to conduct the study , in order to maximize the interobserver agreement rate . training and coordination of the study were the responsibility of l'cole d'optomtrie de l'universit de montral . regular contacts by email or phone were maintained during the study between the university coordinator ( lm ) and the associate researchers . this study received approval from the ethics committee for health sciences of universit de montral . inclusion and exclusion criteria of subjects are listed in table 1 . after being enrolled , subjects were asked to observe a wash - out period of 72 hours without contact lenses before the beginning of the study . subjects had to be established wearers , for at least 3 months , of the silicone hydrogel lenses ( comfilcon a - coopervision , fairport , ny ) . subjects were then randomly assigned to four groups ( a d ) , based on the care regimen to be used for the length of the study a : complete ( amo - abbott , irvine , ca ) ; b , opti - free replenish ( alcon , forth worth , tx ) ; c : renu fresh ( b&lomb , rochester , ny ) ; d : clear care ( ciba vision , duluth , ga ) . once recruited , the subject was provided with a new pair of contact lens and the care regimen steps were reviewed , according to the manufacturer 's recommendations . there was only one base curve available for the lens , limiting the customization of the fit . however , every lens fitted had to be centered , to offer a movement of 0.2 mm maximum upon blinking , and did not expose the limbus in any manner , in any gaze . for half of the subjects , they were told to wear the lens for a month , on a daily wear basis , and to not use any drops prior to insertion of the lenses . for the other half , they were told to instill a sodium hyaluronate based comfort drop ( blink contacts , abbott - amo , irvine , ca ) in the lens ( not in the eye ) , before insertion . nothing was said to subjects about potential clinical effects of this product not to justify its use before insertion . , subjects were seen back for a control exam and assessed . at the 1-month visit , a case history was performed to identify subjects ' level of compliance with the protocol and to answer any questions that might be raised by them . visual acuity was measured using an etdrs high - contrast chart under dim standard illumination . a slit lamp exam was performed to evaluate conjunctival and corneal staining , conjunctival hyperemia , and tear break - up time ( tbut ) . efron 's grading scale was used to evaluate the presence and severity of clinical signs , with grades varying from 0 ( absence ) to 4 ( severe ) . fluorescein was observed using a cobalt blue filter and a yellow filter ( kodak wratten filter number 12 ) to reveal any contrasts . the same filters were also used to evaluate tbut , which is measured 2 times and averaged . the first questionnaire used in this study is the contact lens dry eye questionnaire ( cldeq ) . it consists of 36 questions specific to symptoms of ocular dryness related to contact lens wear . there are nine symptom subscales : discomfort , dryness , visual changes , soreness and irritation , grittiness and scratchiness , foreign body sensation , burning , photophobia , and finally itching . for each of these subscales , any occurrence of the symptom was also evaluated in term of intensity at different times ( 2 hours after insertion , middle and end of the wearing period ) . this questionnaire had already been validated and is sensitive and specific at predicting a diagnosis of dry eye associated with the wearing of contact lenses . in addition to the cldeq questionnaire , subjects were asked to complete a brief survey about their contact lens experience over the last month . this is made through a forced - choice questionnaire where the subject had to click a box to select the best answer . questions of this questionnaire are related to the subjective evaluation of comfort , with the lenses on , at different times of the day ( morning , afternoon , evening ) . subjects have to select from 5 options , from very comfortable ( score of 5 ) to very uncomfortable ( score of 1 ) . the 1-month visit ended with a review of the care regimen procedures with instructions to the subject to wear the lenses for at least 8 hours / day , 5 days / week , and not to sleep or take a nap while wearing contact lenses . a new pair of lenses was then provided and subjects were asked to observe a 48 h00 wash - out period of time , without lenses worn . for the next month , the subjects that did not use sh were asked to put a drop of a sodium - hyaluronate- ( sh- ) based product ( blink contacts , amo - abbott , irvine , ca ) on the lens before insertion . the other ones had to cease to use such a drop . subjects were seen a month later for the final visit , and the same clinical procedures were repeated . the results provided with and without the use of sh drops were considered for comparison . at the end of the second visit , the second questionnaire was slightly different from the first one , with an additional question about which care regimen they would prefer to use in the future ( with or without sh drops ) . one file was rejected due to missing clinical data at one visit ; another one was discarded because the subject admitted to have worn his lenses on an extended - wear basis most of the month . a total of 61 patients completed the study successfully ( 18 males ( 30% ) and 43 females ( 70% ) ) . the mean subject age was 25.0 ( 7,3 ) years old ( range 1835 ) . at the beginning of the study , subjects reported spending 5 hours / day , on average ( 3 hours at work and 2 hours outside ) on computer work . only 5/61 ( 8.1% ) had received a positive diagnosis of dry eye in the past , while 19 of them ( 31.1% ) self - reported suffering from ocular dryness periodically . some others ( 37 subjects60.6% ) were uncertain on this issue , having had some symptoms in the past . fifteen subjects were assigned to group a , thirteen to group b , seventeen to group c , and sixteen to group d. based on the population sample , characteristics of each group were found to be similar . for the following , visit 1 ( v1 ) is related to the clinical results obtained when the subject was free of sh drops while visit 2 ( v2 ) represents the results obtained after its usage . on average , a total of 76 ( 22.00 ) hours of wear / week was estimated at v1 which is slightly less than 81 ( 29.50 ) hours / week for v2 . however , this difference is not considered statistically significant ( t - test , p = 0.12 ) . table 2 shows the results for corneal and conjunctival staining , conjunctival hyperemia , and tear break - up time ( tbut ) . more subjects ( 41 versus 38 ) showed no staining with the use of drops . also , 50% fewer subjects showed clinically significant staining ( grades 2 - 3 ) with this usage . the differences on the presence of the corneal staining show a tendency for statistical significance ( f(2,122 ) = 0.6323 ; p = 0.05 ) . no care regimen was associated with a significant increase in corneal staining . conjunctival staining ( grade 1 ) was noticed in both temporal and nasal quadrants for 24% of the subjects on visit 1 and 17% on visit 2 . this ocular sign was markedly reduced with the use of sh drop before insertion of the lens . there was an increase of 50% of subjects ( 23 versus 14 ) showing no hyperemia at v2 compared to v1 . clinically significant hyperemia ( grade 2 and over ) was also markedly reduced ( 10 subjects at v1 versus 3 at v2 ) . moreover , a 2/3 reduction in the severity of the hyperemia is observed . this difference is statistically significant ( pearson , f(2,122 ) = 3.1217 ; p = 0.04 ; r = 0.0281 ) . overall , the average tear break - up time increased slightly at the second visit from 5.8 ( 3.1 ) sec to 6.3 ( 3.3 ) sec od and from 5.8 ( 3.2 ) sec to 6.5 ( 3.5 ) sec os . this difference is not considered statistically significant ( od p = 0,2372 ; os p = 0,1598 ) . on the other hand , when we paired results for the same individual ( subject compared to himself from v1 to v2 ) , there was an increase in the tbut value that is considered statistically significant ( tukey - kramer , q = 1,9801 , p = 0,05 od and q = 1,9765 , p = 0,05 os ) . finally , visual acuity remained stable during the study ( v1/v2 : od 1.1 ( 0.2)/1.0 ( 0.2 ) ; os 1.1 ( 0.1)/1.0 ( 0.2 ) ) . from this questionnaire , we kept for analysis each symptom that was reported by at least 10% of the subjects , at one visit , intensity of the symptoms was analyzed ( subquestions ) . accordingly , discomfort ( q4 ) , dryness ( q5 ) , visual changes ( q6 ) , soreness and irritation ( q7 ) , grittiness and scratchiness ( q8 ) , foreign body sensation ( q9 ) , burning ( q10 ) , photophobia ( q11 ) , and itching ( q12 ) were analyzed . grittiness and scratchiness ( q8 ) , foreign body sensation ( q9 ) , burning ( q10 ) , photophobia ( q11 ) , and itching ( q12 ) were not reported sufficiently to be considered for further analysis . table 3 shows the results . on average , there was a 10% increase in subjects reporting comfortable or very comfortable wear at the second visit . this difference is considered significant ( chi square , f(4,61 ) = 5.16 ; p = 0.0353 ; r = 0.118 ) . this result applies regardless of the care regimen used ( f(6,61 ) = 4.675 ; p = 0.15 ) . looking at the intensity of this symptom ( table 4 ) , a greater difference is found at the beginning of the day , after 2 hours of wear ( p = 0.00 ) . this difference decreases by midday ( p = 0.01 ) and at the end of the day ( p = 0.03 ) but remains statistically significant at every time point . the difference is highly significant ( chi square , f(4,61 ) = 20.2312 , p < 0.00 ) with a strong correlation ( r = 0.3191 ) , showing a positive effect from the use of the sh drop , reducing the presence or the intensity of this symptom . in fact , there is a decrease of 20% of subjects reporting this symptom as constant at visit 2 compared to visit 1 . the severity of the symptoms was also analyzed ( cldeq questions 5b , c , and d ) . overall , there is a marked difference in the reduction of the severity of eye dryness felt by the patient ( q5b - chi square f4,61 : 9.538 ; p = 0.0008 ; r = 0.1847 ) , after 2 hours of wear ( q5c - f2,61 : 2.8366 ; p = 0,05 ; r = 0.1439 ) , and at the end of the wearing time ( q5d - f4,61 : 11.6696 ; p = 0.00 ; r = 0.1999 ) . there is a respective increase of 13% at 2 hours , 8% ( mid - day ) and 14% ( end of the day ) in subjects reporting reduced symptoms on v2 compared to v1 . with the use of sh drop , 8 out of 10 patients report this symptom as not intense at all . considering that astigmatism is not in play among study 's subjects ( exclusion criteria ) , blurry vision is considered to be related to tear film instability and contact lens dehydration . the use of sh drop helps to reduce almost by half the number of subjects experiencing blurry vision during their contact lens wear . sixty percent of subjects in this study reported not feeling that symptom at all , after using sh drop before insertion ( chi square f4,61 : 13.964 ; p < 0.00 ) . this result shows a strong correlation ( r = 0.2154 ) and is verified the same way in all care system groups . questions 6 b , c , and d provide more details on this issue by asking how noticeable the blurry vision was at different times : after 2 hours ( q6b ) , in the middle of the day ( q6c ) , and at the end of the wearing period ( q6d ) . there is no statistical difference between subjects at the first two time points ( 6d ) . at the end of the day , 15% more patients report no symptom with the use of sh drop ( v2 ) ( f4,61 = 5.4858 ; p = 0.03 ) , even after many hours of wear . again , this result is the same with all the care system used , with no significant differences among groups . at visit 1 , 84.7% of the wearers report this symptom as never or infrequently present while 15.3% report it as frequent or constant . at visit 2 , only 5.1% of the wearers still experienced this symptom . this is considered a significant difference in favor of the use of the sh drop ( chi square f1,61 = 2.1899 ; p = 0.04 ; r = 0.0869 ) . in other words , 10% of the subjects experiencing eye soreness and irritation became asymptomatic with the use of a comfort drop on the lens before insertion . this difference was not present after 2 hours of wear ( q7b , p = 0.90 ) but significant in the middle of the day ( q7c , p = 0.00 ) and highly significant at the end of the wearing time ( q7d , p = 0.00 ) . this was again validated across all groups ( a , b , c , d ) . a highly significant difference exists between visit 1 and 2 ( chi square f4,61 = 14.0198 , p < 0.00 ) , with a strong correlation ( r = 0,2463 ) . at v1 , 21% of the subjects admit removing their lenses many times / week because of discomfort , while only 11% report the same at v2 . this is a 50% reduction of symptomatic patients with the use of a sh - based drop on the lens before insertion . ocular discomfort and ocular dryness were , respectively , the number one and two reasons identified by subjects to explain this need to withdraw their lenses . looking at intensity of these two symptoms , a positive effect of the comfort drop applied before insertion is found . in fact , there is a highly significant difference in the grading of the severity of ocular discomfort and ocular dryness ( q13b , item ( a ) and ( b ) ) between v1 and v2 ( p < 0.00 for each symptom ) . table 9 shows the results . there is a significant difference at every time point , but the most obvious difference is during the evening ( f4,61 = 10.943 ; p = 0,00 ; r = 0.1817 ) . we find again here the 10% positive difference in favor of the use of sh drops , confirming the cldeq results . over 60% of subjects reported a very comfortable / comfortable lens during the evening , after more than 10 hours of wear , compared with 52% without the use of the drops . this positive effect is already measured in the afternoon with a 7% increase in the number of wearers reporting comfortable instead of average wear . these results indicate that the subjective positive effect from the use of sh drops starts early during the wear and increases over time . at the last visit , subjects rated the ease of use of the 2 care regimens they used in the past 2 months ( with and without sh drops ) . ninety percent ( 90% ) of the subjects rated as very easy or easy both care regimen , without statistically significant differences . the care regimen used at v1 was rated as easy ( 20% ) or very easy ( 70% ) , while the second one , including drops before insertion , was rated easy ( 25% ) or very easy ( 65% ) . when asked , near 9 out of 10 subjects would recommend the care regimen with sh drops ( 58.3% strongly and 30% certainly ) . none of the subjects would not recommend the system ( item 4 and 5 ) . finally , 75% of subjects would definitely opt for the system with sh drops in the future ( see figures 2 and 3 ) . this study aimed to evaluate the clinical outcome from the instillation of sh drop , as an additional step in the wearing regimen of contact lens wearers , on contact lens related comfort and visual performance over the day . the cross - over design of the study was established to alleviate hawthorne - like effects as well as sequencing effects . one could argue that the lack of a control group , using another type of lubricating drop , would have been beneficial . even if this is true , we believe that the cross - over design eliminates any bias that can be raised and helps to determine the real effect from the usage of sh drop instilled in the lens before insertion . the results of the present study prove that sodium - hyaluronate - based drops can be added to the list of products that can help symptomatic contact lens wearers to reduce their overall discomfort ( cldeq - q4 ) , but especially at the end of the day ( q4 subquestions ) , when it matters most . even if the symptoms remain , symptom severity is greatly reduced with the use of sh drops ( cldeq - q5 ) . sh drops also have a positive impact on the subjective visual acuity ( cldeq - q6 ) . there is a significant reduction of blurry vision reported by the subjects on visit 2 , which means that the use of sh drops probably helps to maintain tear film stability and also contributes to reduce lens dehydration . we have to consider that blurry vision represents the number 2 reason ( and the first one for astigmatic patients ) to drop out from contact lens wear . in this study , the use of a sh drop initiated a switch for a significant percentage of the wearers from symptomatic to asymptomatic ( eye soreness and irritation ( cldeq - q7 ) ) . for these subjects , discomfort was no longer a reason to remove their contact lenses prematurely ( cldeq - q13 ) . more than 60% of wearers reported remaining comfortable in their lenses during the evening , compared with 50% when they did not use the sh drop before insertion ( comfort questionnaire ) . clinical findings observed through slit lamp were also improved with the use of sh drops on the lens before insertion . it was observed a reduction in the level of corneal staining , whereas clinically significant hyperemia levels were reduced by more than 50% . pairing of the results indicates an improvement of tbut , when subjects are compared to themselves , after the use of the sh drop . these objective findings correlate with subjective reports of increased comfort with the use of sh drops as part of the regular care regimen . they are not surprising considering the nature of the sodium hyaluronate and its known beneficial effects on the ocular surface restoration , namely , on dry eye patients . consequently , we can say that the use of sh drops before insertion helps to address the two major reasons why patients cease to wear contact lens wear ( discomfort and vision ) and contributes to a reduction in the physiological impact from contact lens wear . this is not anecdotal and should represent a significant step forward to keep patients in the contact lens market . it is easy to realize the impact on wearers , practitioners , and the contact lens industry if , with just one easy additional step before lens insertion , it is possible to keep 10% more of the wearers in contact lenses on an ongoing basis and to improve symptoms of others by 50 to 60% . a single drop put on the lens before insertion can considerably change the contact lens experience for subjects who report ocular dryness . noncompliance to this new step is certainly not a big issue : instilling one drop before lens insertion was rated easy by a large majority of subjects and 3 out of 4 of them would recommend this to other wearers or their relatives . it is a well - accepted and easy way to improve contact lens wear . another important aspect of this study is that the positive outcome from the use of sh drops before insertion was verified with multiple types of care systems habitually recommended to wearers . even subjects using a hydrogen peroxide system found a positive effect from the use of sh drop before lens insertion . practitioners , therefore , should not hesitate to recommend this step to their symptomatic wearers in order to improve their overall contact lens experience . a sodium hyaluronate drop before insertion of contact lenses improves the overall comfort but its clinical effect is highly significant at the end of the wearing period , during the evening . its use helps to eliminate or to significantly reduce discomfort symptoms related to contact lens wear and to improve contact lens experience . sh drops also help to maintain more stable vision throughout the day . because of that , it is possible to say that the use of sh - based drops ( blink contacts ) before insertion addresses the two major reasons why contact lens wearers drop out from the market . the use of sh drops before insertion should be considered to improve overall contact lens performance for symptomatic contact lens wearers . we are grateful for financial support from the industrial source technology development program of the ministry of knowledge economy ( 10044909 ) , the next - generation biogreen 21 program ( ssac grant # pj01111803 ) , rda , and the kribb initiative program of south korea .

purpose . this study aimed to evaluate outcomes from the use of a sodium hyaluronate ( sh)-based comfort drop , instilled before the insertion of contact lenses , in a population of symptomatic contact lens wearers . methods . this was a cross - over , open - label , multi - sites study . subjects were fitted with silicone hydrogel lenses and followed for two months . before insertion of the lenses , sh drops was instilled in the lens for half of the group . the other half did the same on the second month . objectives and subjective outcomes were measured and compared before from baseline with the ones collected after usage of sh drops . abstractbacterial volatiles protect plants either by directly inhibiting a pathogenic fungus or by improving the defense capabilities of plants . the effect of bacterial volatiles on fungal growth was dose - dependent . a low dosage did not have a noticeable effect on botrytis cinerea growth and development , but was sufficient to elicit induced resistance in arabidopsis thaliana . bacterial volatiles displayed negative effects on biofilm formation on a polystyrene surface and in in planta leaf colonization of b. cinerea . however , bacterial volatile - mediated induced resistance was the major mechanism mediating protection of plants from b. cinerea . it was responsible for more than 90% of plant protection in comparison with direct fungal inhibition . our results broaden our knowledge of the role of bacterial volatiles in plant protection .

surgical intervention for corneal perforation is indicated when the anterior chamber does not reform within a short period of time . herein , we report the successful management of a small paracentral corneal perforation using autologous iris incarceration and tissue adhesive . a 41-year - old man developed a small paracentral corneal perforation ( 0.5 mm in size ) in the right eye , while the treating physician attempted to remove the residual rust ring after removal of a piece of metallic foreign body . the eye was initially managed with a bandage soft contact lens to ameliorate the aqueous leakage ; however , without success . iatrogenic iris incarceration of the wound was first induced , followed by application of cyanoacrylate tissue adhesive to the perforated site . as a result , the anterior chamber was immediately reformed and maintained . cyanoacrylate tissue adhesive with iatrogenic incarceration of the autologous iris was effective in treating this type of small corneal perforation . this technique is simple and potentially useful for small paracentral corneal perforations outside the visual axis and without good apposition . corneal perforation caused by small corneal punctures can sometimes be managed with a bandage soft contact lens , patching , and/or tissue adhesive [ 2 , 3 , 4 ] . however , surgical intervention is usually indicated when the anterior chamber does not reform within a short period of time . herein , we report the successful experience of managing a small paracentral corneal perforation without good apposition , which could not be tamponaded by a soft bandage lens only , with iatrogenic iris incarceration followed by cyanoacrylate tissue adhesive . a 41-year - old man noted irritation in the right eye and was diagnosed to have a piece of metallic foreign body in his right cornea . iatrogenic corneal perforation was encountered , while the attending physician attempted to remove the residual rust ring with a corneal burr . slit - lamp examination revealed a paracentral corneal penetration with a diameter of approximately 0.5 mm ( fig . the anterior chamber was flat , and active aqueous leakage was confirmed by a positive seidel test with fluorescein . intravenous antibiotics with cefozopran hydrochloride were initiated , in conjunction with prophylactic topical antibiotic with 0.5% levofloxacin three times daily . after 48 h of treatment , there was no significant improvement in the right eye . concerns were raised about possible complications such as secondary angle closure glaucoma and prolonged hypotony . since the puncture was located paracentrally near the visual axis , simple primary closure of the wound or corneal patch graft was deemed undesirable . as the wound was completely punched out , injected viscoelastic materials in the anterior chamber came out from the wound , which made it quite difficult to dry the surface of the cornea for application of tissue adhesive . therefore , to provide a necessary support for the gluing , amniotic membrane plugging was tried , but it was too slippery to be fixed on the wound . a paracentesis port was created . a blunt 27-gauge needle ( with a 2-mm bent tip ) attached to a viscoelastic injecter was gently inserted into the anterior chamber through the paracentesis . using the bent tip , a small tug of iris care was taken not to touch or injure the corneal endothelium or anterior capsule of the lens . meticulous drying of the wound and incarcerated iris was performed and a tiny amount of ethyl-2-cyanoacrylate adhesive ( aron alpha a ; sankyo co. ltd . , attention was paid to ensure that the glue did not spread on to the surrounding cornea . the corneal puncture was thus successfully closed without any sutures , and the anterior chamber remained well formed . postoperatively , the patient was treated with topical 0.1% fluorometholone and 0.5% levofloxacin three times daily . pupillary distortion was observed when the iris was dilated ; however , the patient felt no visual disturbance . herein , we reported a case of iatrogenic paracentral corneal perforation without good apposition successfully treated with autologous iris incarceration and cyanoacrylate adhesive . conventionally , small corneal perforations were treated with stitches with or without corneal patch graft , tissue adhesives [ 2 , 3 , 4 ] , and recently with amniotic membrane and fibrin glue . the indications for the use of a bandage soft contact lens to treat a corneal laceration include small wounds with good apposition , good alignment of the wound edges , and no incarceration or prolapse of the uvea or lens . corneal puncture wounds of less than 2.0 mm in size can usually heal without being sutured . in this case , the wound was relatively small ( less than 1 mm ) , but the apposition of the wound was not optimal due to a sharply demarcated punch , which made restoring the anterior chamber extremely difficult using a bandage soft contact lens only . in cases with good apposition , a small amount of viscoelastic materials through the perforation site is often enough to allow safe application of tissue adhesive without gluing intraocular contents . the technique described in this case appears useful secondary to the minimal residual scarring and related astigmatism . a second advantage of this technique is that the iris is introduced to the perforation site from inside the eye in a controlled manner to prevent excessive iris prolapse and provide a stable support for the tissue adhesive . this is more adequate than the use of an amniotic membrane , which is very slippery and difficult to be fixed on the wound . furthermore , this technique requires no foreign tissue , thereby eliminating the risk of immunological tissue reactions . the tissue adhesive may also theoretically circumvent the potential risks of epithelial down - growth by preventing corneal epithelium from growing directly over the iris . this technique is safe and effective for paracentrally located corneal perforation ; however , significant synechiae involving the angle could prove to be problematic when the perforation is more peripheral . very small descemet 's membrane transplantation instead of iris incarceration may be the other option for this situation . in conclusion , application of cyanoacrylate adhesive following iatrogenic incarceration of the iris seems to be simple and effective in managing small paracentral corneal perforations without good apposition . it has long been recognized that psychiatric disorders and symptoms aggregate in families and the evidence for a substantial role for genetic factors is incontrovertible . genetic epidemiological studies of autism , bipolar disorder and schizophrenia show that the risk of developing one of these specific psychiatric illnesses is proportional to the amount of genetic material shared with an affected individual . heritability has been estimated as being at least 80% for all these disorders [ 2 - 4 ] , which , to put it in context , is equivalent to that for type i diabetes ( about 80% ) but greater than that for breast cancer or parkinson 's disease . the majority of psychiatric disorders , like other common conditions , are genetically complex . in psychiatry , genetic complexity has been compounded by phenotypic complexity . psychiatric diagnosis can not be made on the basis of biological investigation or validated against a common pathogenesis . psychiatric ' disorders ' such as autism , schizophrenia and bipolar disorder are therefore effectively groups of symptoms making up syndromes that define groups of patients who show broadly similar outcomes and who respond similarly to treatment . such diagnostic categories are therefore likely to be heterogeneous and the boundaries between them somewhat arbitrary . autism , schizophrenia and bipolar disorder have traditionally been considered as separate disease entities , although they do share some common behavioral characteristics and cognitive deficits . the distinction between schizophrenia and bipolar disorder has been justified for many years by reference to family studies showing that these disorders seem to ' breed true ' . however , this view has been challenged , and a recent large - scale study has shown that relatives of individuals affected with schizophrenia have increased risks of bipolar disorder , and vice versa . definitive genetic epidemiological studies of the genetic relationship between autism and these disorders are lacking , although there is some evidence for shared genetic factors . in recent years new molecular genetic findings , particularly from the application of genome - wide association studies ( gwass ) and other genomic technologies [ 11 - 14 ] , have implicated risk factors for these disorders , and this has allowed the possibility of a genetic relationship between them to be explored directly and current orthodoxies to be challenged [ 8 - 10 ] . autism spectrum disorders ( asds ) such as autism , asperger 's syndrome and rett 's syndrome , are developmental psychiatric disorders with high heritability . over the past few years , genetic studies of asds have consistently identified rare and de novo point mutations and large structural variants present in genes encoding interacting synaptic proteins . such studies have reported co - segregation of putative high - risk alleles ( such as deletions or point mutations ) with asds or performed so - called ' burden analysis ' , in which different alleles of a particular gene are aggregated and the frequency in cases compared with that in controls . initial studies of asds using small samples found rare missense point and structural mutations ( such as copy number variants , cnvs ) in the x - linked neuroligin-3 and neuroligin-4 ( nlgn4 ) genes [ 17 - 19 ] . neuroligins are a family of post - synaptic proteins that bind trans - synaptically to a family of pre - synaptic proteins called neurexins . although these findings were interesting , incomplete penetrance of the mutations and lack of power made the results equivocal . however , missense mutations were subsequently identified in the neurexin-1 ( nrxn1 ) gene at a high frequency in individuals with autism . these suggestive findings have been augmented by the results of recent genomic studies discussed below . traditional karyotyping , gwass and comparative genome hybridization ( cgh ) analyses have been used to identify large chromosomal structural losses ( deletions ) and gains ( duplications ) in individuals with asds [ 21 - 24 ] . a burden analysis study of approximately 200 affected individuals identified two translocation events at nrxn1 in separate samples , one disrupting the coding sequence and the other lying 5 ' to the gene . a further study of over 1,000 pedigrees using approximately 10,000 single - nucleotide polymorphisms ( snps ) identified a 300 kb deletion of coding exons of nrxn1 co - segregating with autism . genome - wide analyses have also implicated further related and interacting synaptic protein - coding genes in the etiology of asds . first , a study of 427 asd cases using approximately 500,000 snps identified a 6 mb de novo deletion encompassing nlgn4 and a 270 kb deletion at shank3 . second , burden analyses revealed a high frequency of point mutations of shank3 in asd cases . third , cntnap2 , which encodes a member of the neurexin family that resides in the juxtaparanodal region of myelinated neurons , shows evidence for common - allele association with asds as well as an increased burden of rare protein - coding mutations and large de novo deletions . the evidence implicating synaptic cell adhesion molecules and their related proteins in asds is strong . data implicating them in schizophrenia is now arguably even stronger . a whole - genome screen for large chromosomal abnormalities using array - cgh performed in 93 individuals with schizophrenia identified a hemizygous loss at nrxn1 in one case . the deletion of exon 1 was also present in an affected sibling and no deletions of nrxn1 were observed in 372 controls , suggesting that the allele may be pathogenic . the same study also found a large ( 1.4 mb ) de novo duplication event in an individual with an asd that spanned apba2 ; this is an intriguing result given that the gene encodes a protein ( mint2 ) that binds to intracellular domains of neurexins . further to this , an independent cgh study identified rare deletions of nrxn1 in monozygotic twins both diagnosed with early - onset schizophrenia . higher - density , lower - cost genome - wide screens using gwas technologies have made it feasible to screen many thousands of individuals for smaller copy number variations . using over 300,000 probes across the genome assayed in approximately 3,000 european cases and 10 times as many european controls , burden analysis revealed many deletions at the nrxn1 locus . the authors reported a significant excess of protein - coding deletions present in cases , a finding that is replicated in a similar analysis performed on an independent sample of approximately 3,000 cases of european descent and 3,000 controls and also in two further studies using smaller sample sizes . so far , no data implicating neuroligins and shanks in schizophrenia have been reported . however , hemizygosity of the cntnap2 gene , which encodes a member of the neurexin family , contactin - associated protein - like 2 ( caspr2 ) , has been reported in schizophrenia and also in individuals with mental retardation . although the available data provide relatively strong evidence that disruption of the neurexin-1 locus ( nrxn1 ) is a risk factor for schizophrenia and asds , evidence in relation to bipolar disorder is lacking . this might reflect the relative paucity of studies addressing this hypothesis , but it could be the result of a discontinuity between bipolar disorder and schizophrenia in relation to the role of cnvs ( see below ) . the recent application of genome - wide technologies has shown that the burden of large , rare cnvs is increased in schizophrenia when compared with controls , and that this implicates specific loci . studies of such magnitude have not yet been performed for asds , although there is evidence for the involvement of specific cnvs . in contrast , there is evidence that the global burden of duplications or deletions in bipolar disorder is substantially less than for schizophrenia and asds . specific deletions associated with schizophrenia include those at 22q11.2 , 1q21.1 and 15q13.3 , and these have also been found in association with mental retardation , autism and attention deficit hyperactivity disorder [ 36,41,46 - 50 ] while that at 15q13.3 has also been implicated in idiopathic generalized epilepsy . therefore , just as for nrxn1 deletions , it is apparent that these large cnvs confer risk of a range of neurodevelopmental phenotypes , including autism , mental retardation and schizophrenia . however , similar evidence is lacking for bipolar disorder and there is a suggestion that cnvs might have a less prominent role in this phenotype . the advent of the gwas has allowed most of the common snp variation in the human genome to be tested for association and the first wave of such studies has been reported for schizophrenia , bipolar disorder [ 54 - 56 ] and autism . several loci have been implicated at genome - wide levels of statistical significance for schizophrenia , including znf804a ( encoding a protein with zinc finger and nucleic acid binding domains ) and the major histocompatibility complex ( mhc ) region . these studies have also provided strong evidence for genetic overlap between schizophrenia and bipolar disorder . however , these associations implicate common alleles with small effects , and findings from gwass do not yet clearly suggest a specific biological process . so far there have been no systematic comparisons of gwas data for asds with those from schizophrenia or bipolar disorder . however , intriguing associations have been reported at voltage - gated calcium channel genes across all these phenotypes [ 56,58 - 60 ] . furthermore , there have been recent reports of association for common alleles at several gaba receptor genes in a subtype of bipolar disorder and schizophrenia , which implicate loci also reported as associated with asds [ 23,63 - 65 ] . the evidence for involvement of neurexins ( nrxn1 ) , neuroligins ( nlgn4 ) and related proteins such as shanks ( shank3 ) , mint2 ( a2bp1 ) and caspr2 ( cntnap2 ) in asds is substantial and growing . there is also strong evidence implicating some of these genes in schizophrenia , although not all of them have been examined . given this overlap , it is possible that the genes may be exerting their effects through a biological pathway common to both disorders . the neurexins are a family of transmembrane proteins that have extracellular , membranous and intracellular domains . neurexins can be divided into two groups , and neurexins ; both are encoded by three genes . the neurexins are primarily expressed in neurons , where they are known as pre - synaptic heterophilic adhesion molecules , and they typically bind across the synapse to neuroligins . the neuroligins represent a similar class of proteins to neurexins and the binding of the two types of molecule to each other is controlled by alternative splicing . the intracellular domains of neurexins ( and neuroligins ) bind scaffolding proteins and assemble large molecular complexes that are known to link to synaptic systems such as receptors , ion channels and vesicle release machinery . neurexins are best known for their ability to promote cell adhesion and synaptogenesis when neuroligins are present on the neighboring cell . even in non - neuronal cell lines , it seems that neurexins and neuroligins are necessary for both excitatory and inhibitory synaptogenesis , and possibly in functional synapse maturation . it has been hypothesized that neurexins and neuroligins are involved in the promiscuous generation of many synapses , before their activity - dependent pruning . however , multiple neurexin gene knockout studies in mice seem to contradict this and suggest that the neurexin - neuroligin complex is not essential for synapse formation but for synapse function . deletions of -neurexin result in increased lethality , normal synapse number and gross anatomy but severely impaired synaptic functioning , a pattern strikingly similar to neuroligin gene knockouts . such biological roles fit with hypotheses of the etiology of autism and schizophrenia in which a neurodevelopmental insult and adult imbalance in excitatory and inhibitory neurotransmission occur in the absence of overt macro - pathology . shank3 is implicated in autism by several lines of evidence [ 23,25,26,68 - 70 ] and functions as a post - synaptic scaffolding protein that binds indirectly to neuroligins , forming a potentially functional circuit of neurexin - neuroligin - shank that is dysregulated in asds . the involvement of -neurexins in pre - synaptic neurotransmission suggests a functional link with voltage - gated calcium channels , which are integral to pre - synaptic function and plasticity and have been implicated to be involved in autism , schizophrenia and bipolar disorder [ 56,58 - 60,73 ] . therefore , the evidence from asds , schizophrenia and bipolar disorder suggests a convergence on specific processes involved in the development and regulation of synaptic transmission . further work on the biology of neurexins , neuroligins and related proteins is certainly required and it seems likely that the pathogenic roles of these proteins will be illuminated by further human genetic studies . whole - genome studies of many thousands of affected individuals are uncovering evidence for genetic overlap between autism , schizophrenia and bipolar disorder . studies of cnvs and other rare alleles have found overlap between autism and schizophrenia , whereas those of common snp variants have shown overlap between schizophrenia and bipolar disorder . these findings suggest that schizophrenia , autism and other neurodevelopmental disorders may share underlying pathogenic mechanisms and challenges the view that these are completely unrelated diagnostic entities . the findings also support the view that schizophrenia has a stronger neurodevelopmental component than bipolar disorder and suggest that it lies on a gradient of decreasing neurodevelopmental impairment between syndromes such as mental retardation and autism , on one hand , and bipolar disorder on the other . the identification of rare and common alleles predisposing to prototypically distinct psychiatric disorders provides challenges for the ways in which such disorders are diagnosed and researched . we have argued on the basis of recent genetic data that these findings point to common pathophysiological mechanisms , and this is now an important area for future research . we have based this conclusion on the fact that several rare cnvs , including deletions of nrxn1 , are associated with mental retardation , autism and schizophrenia , and on the overlap in common risk alleles seen between schizophrenia and bipolar disorder [ 11 - 13 ] . we do not propose that the disorders are the same phenomenologically , and we accept that there may be many genetic and environmental risk factors not shared between the phenotypes . it is clear that much future work is required and equally clear that this should not be constrained by current categorical diagnostic systems . such studies should explore the relationship of genes and other biological variables to dimensional measures of key domains of psychopathology across current diagnostic categories . we have previously argued the need to undertake such endeavors across the functional psychoses of schizophrenia and bipolar disorder . however , recent data point to the need to consider a broader clinical spectrum that includes also autism and mental retardation / cognitive impairment . asd : autism spectrum disorder ; cnv : copy number variant ; cgh : comparative genome hybridization ; gwas : genome - wide association study ; nlgn4 : neuroligin-4 ; nrxn1 : neurexin-1 ; snp : single - nucleotide polymorphism .

backgroundsurgical intervention for corneal perforation is indicated when the anterior chamber does not reform within a short period of time . herein , we report the successful management of a small paracentral corneal perforation using autologous iris incarceration and tissue adhesive.casea 41-year - old man developed a small paracentral corneal perforation ( 0.5 mm in size ) in the right eye , while the treating physician attempted to remove the residual rust ring after removal of a piece of metallic foreign body.observationsthe eye was initially managed with a bandage soft contact lens to ameliorate the aqueous leakage ; however , without success . iatrogenic iris incarceration of the wound was first induced , followed by application of cyanoacrylate tissue adhesive to the perforated site . as a result , the anterior chamber was immediately reformed and maintained . complete corneal epithelialization of the perforation was achieved in 2 months without visual compromises.conclusionscyanoacrylate tissue adhesive with iatrogenic incarceration of the autologous iris was effective in treating this type of small corneal perforation . this technique is simple and potentially useful for small paracentral corneal perforations outside the visual axis and without good apposition . there is strong evidence that genetic factors make substantial contributions to the etiology of autism , schizophrenia and bipolar disorders , with heritability estimates being at least 80% for each . these illnesses have complex inheritance , with multiple genetic and environmental factors influencing disease risk ; however , in psychiatry , complex genetics is further compounded by phenotypic complexity . autism , schizophrenia and bipolar disorder are effectively syndromic constellations of symptoms that define groups of patients with broadly similar outcomes and responses to treatment . as such the diagnostic categories are likely to be heterogeneous and the boundaries between them somewhat arbitrary . recent applications of whole - genome technologies have discovered rare copy number variants and common single - nucleotide polymorphisms that are associated with risk of developing these disorders . furthermore , these studies have shown an overlap between the genetic loci and even alleles that predispose to the different phenotypes . the findings have several implications . first , they show that copy number variations are likely to be important risk factors for autism and schizophrenia , whereas common single - nucleotide polymorphism alleles have a role in all disorders . second , they imply that there are specific genetic loci and alleles that increase an individual 's risk of developing any of these disorders . finally , the findings suggest that some of the specific genetic loci implicated so far encode proteins , such as neurexins and neuroligins , that function in synaptic development and plasticity , and therefore may represent a common biological pathway for these disorders .

fever is a common reason for seeking medical attention and accounts for more than 20% of emergency department visits.1 many mothers and caregivers have perceived phobia associated with their child 's fever for reasons that include serious infection , seizure , brain damage and death.2 paediatricians have regularly advised parents to seek immediate medical attention if their children developed a fever because fever is of great diagnostic importance especially in developing countries where infectious diseases such as malaria and pneumonia are very prevalent.3 in developing countries , because of the relatively low level of literacy and financial constraint of procuring reliable thermometers , many parents rely on palpation to assess if their children have fever.4 the objective use of the thermometer to detect the presence of fever is often limited to health care settings in most resource - poor settings . an accurate determination of the absence of fever in a child assures parents and saves cost by preventing unnecessary investigations and medication . ownership of thermometer seems poor , although no study in our environment was found to support that . in a study in new york , usa , of the 78% of caregivers that owned thermometers , nearly half ( 48% ) still utilised tactile method to assess fever.4 in another study , in india in contrast , only 15% owned thermometer with 23.8% of them not knowing how to use it.5 this underscores the fact that the use of palpation as a means of assessing fever by mothers is common and universal . majority of mothers and care givers in our environment still utilise tactile assessment to determine if their children have fever . controversies over the reliability of tactile assessment of fever have been variously reported ; while a study found it accurate enough , at least in younger infants,4 others reported that it overestimates the presence of fever.67 the reliability of the use of palpation to detect fever still remains uncertain . this study was carried out to determine the reliability of tactile assessment of fever by nigerian mothers in under-5 children and also to determine if the surface of the hand used influence the accuracy . the study further ascertained if palpation of a single or multiple anatomical sites influence the accuracy of tactile assessment of fever . children under the age of 5 years presenting for care with their mothers were recruited for the study . ethical approval was obtained from the research and ethics committee of federal medical centre owerri , imo state nigeria before commencement of the study . the subjects were recruited from the children outpatient clinic and children emergency room of the hospital . information such as age and sex of the child , mother 's age and highest educational qualification ( heq ) were captured using a profoma . thereafter , each eligible child had a tactile assessment of fever by the mother . the response of the mother as regard presence or absence of fever was documented following which the child 's axillary temperature was measured and documented . the axillary temperature was measured using mercury in glass thermometer , which was left in the axilla for 5 minutes before reading . frequencies , percentages and mean were analysed where appropriate . the sensitivity , specificity , positive predictive values ( ppvs ) and negative predictive values ( npvs ) of tactile assessment was calculated using axillary temperature of 37.5c as cut - off for fever . the different hand surfaces used for palpation , as well single versus multiple sites of palpation were also compared . there were 60 males and 53 females giving a male : female ratio of 1.1:1 . seven ( 6.2% ) of the mothers had only primary school education , while 38 ( 33.6% ) and 68 ( 60.2% ) had secondary and tertiary education , respectively . fever detected by touch was reported in 81 ( 71.6% ) children by mothers while , only 65 ( 57.5% ) mothers were able to correctly predict the presence of fever in their children . there was no statistical significant difference in the ability of the mothers to correctly predict fever with respect to their heq ( = 2.66 , p = 0.265 ) . comparison of fever detection by palpation with the mothers highest educational qualification ( heq ) irrespective of the part of hand used for palpation , the sensitivity of detection of fever by palpation was 82.4% , while the specificity was 37.1% . the use of the palmer surface of the hand had a better sensitivity ( 95.2% ) than the dorsum of the hand ( 69.2% ) . table 2 shows the sensitivity , specificity , ppv and npv of the part of hand used for palpation . sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) of the part of hand used for palpation a total of 71 ( 62.8% ) of the mothers palpated at more than one site while the remaining 42 ( 37.2% ) used only single site . table 3 shows the different sites used by the mothers in palpation , while table 4 compares the sensitivity , specificity , ppv and npv of use of single site versus multiple sites in palpation . the use of multiple sites had better sensitivity ( 86.7% ) than the use of single site ( 76.2% ) . sites used by the mothers to palpate for fever palpation of single site versus multiple sites the present study shows that mothers can be trusted to a good extent to assess for the presence of fever in children by palpation . a sensitivity of 82.4% obtained in this study is similar to those reported by okposio and abhulimhen - iyoha in benin , nigeria as well as graneto and soglin in chicago , usa , who documented sensitivity of 89.2% and 84% , respectively.89 some studies in nigeria have reported a higher sensitivity value of 96.3% and 94.6% , respectively.610 a specificity of 37.1% obtained in the present study is quite poor and is lower than the specificities reported by other workers ranging from 64.3% to 82%.489 it is , however , higher than 23% reported by akinbami et al.6 the heterogeneity in the methodology of these studies may have been responsible for the differences obtained between the present study and some of the earlier studies . for instance , akinbami et al.,6 used rectal temperature of 38c as the cut - off of fever , wammanda and onazi10 adopted axillary temperature of 37.2c as the cut - off for fever , while the present study and that of okposio and abhulimhen - iyoha8 used axillary temperature of 37.5c as the definition of fever . although the specificity in the present study was poor , the relatively high sensitivities obtained in all the studies still make maternal palpation for the presence of fever a considerably useful method of assessment of childhood fever . although , palpation overestimates fever , the elicitation of fever by mothers would encourage early presentation as it has been documented that the concern for fever is a strong motivational factor for seeking medical care.2 the educational qualification of a mother did not influence the accuracy of palpation for the presence of fever . it is therefore expected that any mother , irrespective of her educational background should be able to appreciate fever by palpation . with a npv of 71.9% , if a mother says her child does not have fever , her history of subjective assessment of fever without the use of a thermometer is about 72% reliable . the likelihood of a child adjudged to be febrile by a mother to be truly febrile measured by the ppv in the present study is 51.9% . this is higher than 39% reported by wheybrew and co - workers.7 this higher ppv reported in this study may have been influenced by the prevalence of fever in the present study . in the present study , 57.5% of the study subjects had fever , while in the study of wheybrew et al.,7 27% of children had fever . the palmer surface of the hand was the most commonly used hand surface by mothers to palpate for fever . the sensitivity , specificity , npv and ppv of palmer surface was higher than that of the dorsal surface or the use of both surfaces . this may be attributed to the fact that receptors for detection of heat and temperature are more concentrated on the palmer surface and finger tips.11 the use of both surfaces may confuse the mother as the sensation obtained from both are likely to vary , hence the reduced sensitivity obtained in this study when compared with palmer surface alone . the forehead was the most common site used for tactile assessment of fever in this study . this corroborates reports from previous studies , which also found the forehead to be the most common anatomical site used for tactile assessment of fever.4 this may possibly be explained by the fact that the head is easily accessible compared with the other parts of the body . it was observed that palpation of multiple sites was more sensitive than use of single site . this is in agreement with an earlier report by okposio and abhulimhen - iyoha as well as singhi and sood.812 this implies that if fever is to be assessed by palpation without the use of a thermometer , palpation of multiple body sites should be utilised . palpation with the palmer surface of the hand using multiple sites improves the reliability of tactile assessment of fever . clinicians should not discountenance mothers complaint of fever in their children detected by palpation without the use of a thermometer . mothers should be taught to palpate at multiple sites with the palmer surface when palpating for the presence of fever in their children to improve the accuracy . pancreatic ductal adenocarcinoma ( pdac ) represents the fourth leading cause of cancer mortality worldwide , with an incidence of approximately 217,000 new cases each year nearly matched by 213,000 deaths ( parkin et al . , 2001 ) . several of the most frequent genetic events underlying the initiation and progression of human pancreatic cancer have been identified ( hezel et al . , 2006 ; maitra and hruban , 2008 ) . these include activating mutations in the kras proto - oncogene , which occur in > 90% of pdac ( caldas and kern , 1995 ) and are considered as a key driver for pancreatic carcinogenesis , and mutations inactivating the tp53 gene , which occur in 5075% of patients ( redston et al . , 1994 ) . moreover , several lines of evidence implicate mutations inactivating the brca2 tumour suppressor in an estimated 520% of familial pdac ( hahn et al . , 2003 ; couch et al . , 2007 ) . germline carriers of deleterious brca2 mutations that commonly truncate the encoded protein exhibit an increased lifetime risk of developing pdac , in addition to their well - known predisposition to cancers of the breast and ovary ( breast cancer linkage consortium , 1999 ) . within high - risk pancreatic cancer kindreds , inherited mutations in brca2 represent the most frequently encountered germline genetic alteration ( hahn et al . , 2003 ) . the incidence of germline brca2 mutations in apparently sporadic pancreatic cancers may be as high as in breast or ovarian cancer ( goggins et al . , 1996 ) . more recently , palb2 , which encodes a brca2-interacting protein also essential for homology - directed dna repair , has emerged as a pancreatic cancer susceptibility allele ( jones et al . , 2009 ) . three new transgenic models for pancreatic adenocarcinoma associated with brca2 inactivation have recently been described ( skoulidis et al . one of these models does not incorporate activation of the kras oncogene ( feldmann et al . , 2011 ) . in contrast , the other two models ( skoulidis et al . , 2010 ; rowley et al . , 2011 ) use a conditional gene - targeted allele developed by tuveson , jacks and colleagues ( jackson et al . , 2001 ; johnson et al . , 2001 ) , in which tissue - specific activation of oncogenic krasg12d is driven on a single allele by loxp - cre mediated recombination , mimicking a genetic event that frequently triggers kras activation in human cancers . cre recombinase expression is controlled by the pdx1 promoter , which is expressed at e8.5 and required for organogenesis of the pancreas , whereby loss of the gene is associated with an absence of pancreatic formation ( jonsson et al . , 1994 ; offield et al . , the expression of the pdx1-cre transgene therefore occurs throughout the pancreatic cellular compartment , albeit in a stochastic manner , to trigger kras activation ( hingorani et al . , patients who carry germline mutations affecting brca2 harbour the germline mutant allele in all somatic tissues , whereas the second brca2 allele is wildtype ( wooster et al . , 1994 ) . it has been widely believed that loss of the second , wild - type brca2 allele in nascent cancer cells ( termed loss of heterozygosity or loh ) is necessary for the emergence of tumours in germline mutation carriers . the pancreatic cancer model developed in our laboratory ( skoulidis et al . , 2010 ) it carries in all somatic tissues a truncated allele of murine brca2 ( brca2 ) , which truncates the gene in an evolutionarily conserved and functionally critical region encoded by exon 11 , resembling deleterious germline mutations found in human carriers ( friedman et al . , 1998 ) . the second brca2 allele , brca2 ( jonkers et al . , 2001 ) can be conditionally disrupted to remove exon 11 by loxp - cre recombination in the pancreas . this event is driven by pdx1-cre , and therefore occurs in the same tissues which undergo kras activation . there is evidence that both the brca2 and brca2 alleles can express a truncated protein product ( patel et al . , 1998 ; notably , the models developed by rowley et al . and feldmann et al . both exclusively use a strain homozygous for the conditional brca2 allele ; thus , germline heterozygosity for brca2 is not modelled in their experiments . all three of the new pancreatic cancer models incorporate conditional alleles that inactivate tp53 in the pancreas , to mimic the frequent loss of this tumour suppressor in human pancreatic cancers . somewhat different tp53 alleles are used by each group , an important distinction given that the nature of tp53 mutations is thought to affect pancreatic cancer development ( olive et al . , 2004 ) . two of the studies employ gain - of - function mutants affecting a single allele . these are either the structural mutant tp53 ( feldmann et al . , 2011 ) or the contact mutant tp53 ( skoulidis et al . , 2010 ) . both these tp53 mutants are associated with the development of carcinomas ( olive et al . , 2004 ) . in contrast , the third study uses a null allele of tp53 , wherein exons 210 are deleted by pdx1-cre activation ( rowley et al . , 2011 ) , in a manner that less faithfully represents human cancer - associated mutations . thus , it should be clear from the foregoing that these three recently - published models for pancreatic carcinogenesis associated with brca2 inactivation harbour important differences not only in the tissue - specificity , nature and timing of mutant tp53 and brca2 alleles , but also in the presence of mutant kras . we believe that these distinctions are vital to understanding the marked differences in pancreatic carcinogenesis observed in the studies , the key findings from which are highlighted in table 1 . brca2 has been believed to follow the classical two - hit paradigm for tumour suppression ( smith et al . , 1992 ; collins et al . initial studies soon after the discovery of brca2 reported consistent inactivation of the wild - type brca2 allele through loss - of - heterozygosity ( loh ) in breast or ovarian cancer cells from mutation carriers ( collins et al . , 1995 ; gudmundsson et al . , 1995 ) , engendering the widely accepted view that brca2 loh is an essential event in carcinogenesis . a few notes of dissent have emerged in later studies ( king et al . , 2007 ; willems et al . , 2008 ) , but they have not gained widespread attention . in this context unexpectedly reveal that brca2 heterozygosity promotes pancreatic cancer development in mice and men . in both the tp53 wildtype and tp53 cohorts from the murine model , heterozygosity for brca2 ( through the brca2 genotype ) acts with kras to accelerate the progression and development of pdac . a similar conclusion is reached from studies on a small number of human pancreatic cancer samples from carriers of the icelandic founder mutation in brca2 , the allele brca2 , which is 5 bp deletion in exon 9 that causes a frame - shift leading to the expression of a very short and unstable protein product ( mikaelsdottir et al . , 2004 ) . one possibility is that this genotype causes a mutator phenotype , owing to defects in dna repair arising from a known role of brca2 in homologous dna recombination ( patel et al . , 1998 ; however , previous studies on murine embryo fibroblasts ( mefs ) heterozygous for brca2 reveal no statistically significant effects on sensitivity to genotoxic agents ( patel et al . neither brca2 mice ( friedman et al . , 1998 ) , nor strains heterozygous for other brca2 truncation mutants ( connor et al . , 2001 ; yan et al . , 2004 ) , exhibit cancer predisposition . notably , a lacz mutation reporter gene ( boerrigter et al . , 1995 ) incorporated into the germline of mice heterozygous for a brca2 truncation similar but not identical to brca2 ( tutt et al . , 2002 ) reveals no evident mutator phenotype . on the other hand , mefs from this strain showed a mild alteration in dna repair kinetics during recovery from 4gy of ionizing radiation . thus , there is little convincing evidence that heterozygosity for these brca2 mutant alleles creates a dna repair defect that could explain heightened cancer predisposition , although the possibility has not yet been conclusively excluded . in this connection , it is important to note that these cellular approaches do not yet account for the cooperative effect of mutant kras on pancreatic carcinogenesis associated with brca2 heterozygosity , as suggested by the murine model developed by skoulidis et al . even a subtle increase in mutational load induced by brca2 heterozygosity in mutant kras expressing cells which might be undetectable in cellular experiments , but significant in vivo could plausibly accelerate the progression of pre - malignant pancreatic intra - epithelial ( panin ) lesions ( which occur frequently even in apparently normal pancreatic parenchyma ( hruban et al . , 2008 ) ) to overt malignancy . whether different brca2 alleles behave in a manner similar to brca2 is not clear . like brca2 , however , the instability of the truncated protein encoded by brca2 ( mikaelsdottir et al . , 2004 ) suggests that haploinsufficiency for brca2 ( as opposed to any trans - dominant effect of a mutant brca2 protein ) accounts for the phenotypic effects of heterozygosity in patients who carry this icelandic founder mutation . describe no heterozygous effect in any of their brca2 cohorts despite the presence of mutant kras . interpretation of this difference is not straightforward , since the brca2 allele engenders brca2 loss only in the cells expressing pdx1-cre , unlike brca2 , which is expressed in all somatic cells . this raises the possibility that non - cell autonomous effects of brca2 heterozygosity for example on stromal cells rather than the nascent cancer cells may account for the cancer - predisposing effect of the brca2 allele . mitotic functions have also been ascribed to brca2 , and interestingly , defects in g2 checkpoint function ( menzel et al . , 2011 ) , mitotic checkpoint enforcement ( choi et al . , 2012 ) and the completion of cell division by cytokinesis ( daniels et al . , 2004 ; whether or not these roles for brca2 may explain the effect of heterozygosity in tumour development is yet to be explored . heterozygosity for the brca2 allele is enough to trigger cytokinetic defects in mefs ( daniels et al . , 2004 ) , but it is unclear whether the other mitotic functions are perturbed by brca2 heterozygosity . importantly , recent data from human studies further support that brca2 heterozygosity is enough to promote carcinogenesis . in breast cancers , incomplete loss of the remaining wild - type allele has been observed using techniques more sensitive than those applied in the original studies ( king et al . , 2007 ) . importantly large - scale , unbiased genomic sequencing of high - grade serous ovarian carcinomas highlighted the retention of the wild - type allele in end stage disease from 25% of germline brca2 carriers ( atlas , 2011 ) . furthermore , a detailed study of prostate tumour progression in brca2 germline mutation carriers uncovered no loh in high - grade prostatic intraepithelial neoplasias , considered precursor lesions to the development of prostate adenocarcinoma , and up to 55% of the malignant tumours analysed ( willems - jones et al . , 2012 ) . collectively , these data suggest that cancers arising in germline brca2 mutation carriers frequently fail to exhibit loss of the wildtype allele , and that failure to exhibit loh occurs in brca2-mutant cancers from several different tissues . interestingly , these conclusions can be set against the emerging backdrop of ongoing studies on tissue samples from patients with familial forms of pancreatic cancer . a study of 58 pancreatic intra - epithelial neoplasms and intraductal papillary mucinous neoplasms reveals that somatic losses in brca2 copy number are infrequent ( hong et al . , 2012 ) . however , definitive evidence addressing the extent to which the lessons from gemms of brca2-deficient pancreatic cancers can be applied to human neoplasia awaits the results of more extensive genome sequencing studies on pancreatic cancer samples from patients harbouring germline brca2 mutations . murine pancreatic cancers emerging in brca2 strains in which both brca2 alleles are inactivated in pdx1-cre expressing cells exhibit a preponderance of acinar cell carcinoma histology . correspondingly , 3 of the four human pancreatic cancers from brca2 mutation carriers that exhibited loh were also of the acinar type ( skoulidis et al . , 2010 ) , which normally accounts for only 12%% of human pancreatic cancers ( hruban , 2007 ) . this raises the possibility that these genotypes promote the evolution of acinar cell carcinomas rather than pdac . ( 2011 ) also observe differences in the histopathological spectrum of pancreatic malignancies from mice in which brca2 as well as tp53 had been inactivated , when compared to tp53 deficiency alone . these observations raise the possibility that the nature of brca2 mutations , their timing , or their coincidence with alterations with tp53 may alter the histopathological evolution of pancreatic cancers in mice . however , these observations remain too limited to allow firm conclusions to be drawn , and we draw attention to them here simply to emphasize the need for further studies . we and others have shown ( patel et al . , 1998 ; lee et al . , 1999 ; tutt et al . , 1999 ) that the genome - wide dna damage that follows homozygous inactivation of brca2 leads to checkpoint activation and cell cycle arrest , rather than the unrestrained cellular proliferation typical of cancer . we have previously proposed ( venkitaraman , 2009 ) that checkpoint inactivation may therefore be an essential pre - requisite for homozygous brca2 inactivation through loh during carcinogenesis . the work of skoulidis et al . provides strong in vivo evidence for this hypothesis , supported by the observations of rowley et al . in both murine models , bi - allelic brca2 inactivation by itself leads to a loss of exocrine pancreatic parenchyma , a concomitant increase in adipose tissue , and progressive loss of organ functionality . further demonstrate that pancreatic insufficiency is preceded by the widespread occurrence of dna double - strand breakage marked by h2ax staining in cells lacking both copies of brca2 . moreover , both skoulidis et al . and rowley et al . find that the concomitant inactivation of tp53 function prevents pancreatic insufficiency , and allows rapid pdac development , in the pancreas of mice carrying bi - allelic mutations inactivating brca2 . when the observations from these studies are synthesized , a picture emerges wherein brca2 heterozygosity in germline mutation carriers may suffice to allow the development of kras - driven pdac . later inactivation of tp53 or other checkpoint genes may then allow eventual loss of the second brca2 allele : although loh is not an obligate step , it may promote the emergence of advanced cancers . indeed , inferences from a very small study of just 5 samples from human pancreatic cancer patients support such a scenario , although it remains to be firmly established . , our results suggest that brca2 heterozygosity suffices for pdac formation driven by mutant kras in mice and men . however , the rationale for the use of targeted agents such as parp1 inhibitors ( parp1i ) in brca2-deficient cancers is contingent upon bi - allelic brca2 inactivation in the tumour cells ( bryant et al . , therefore , as confirmed in our work ( skoulidis et al . , 2010 ) , pdac cells that retain a functional brca2 allele are resistant to parpi such as the astrazeneca compound olaparib . thus , parp1 inhibitors should be reserved for clinical use when brca2 loh can be verified in the tumour , assessment of which emerges as a critical requirement in the design of human clinical trials for the treatment of brca2-deficient cancers . these findings exemplify how the new generation of gemms for pdac may represent valuable surrogate models for preclinical tests of therapeutic efficacy in patients . importantly , such models not only allow in vivo proof of new therapeutic concepts , but may also provide a platform to assess the pharmacodynamic and pharmacokinetic properties of new agents , although species - specific differences may limit such interpretations . the models also provide a flexible method to assess the impact of therapy on tumour progression using adapted multimodal imaging and drug bioavailability ( including tissue drug penetrance ) analyses . an important feature that determines if a particular gemm is useful as a preclinical platform is if the model recapitulates a similar clinical response to standard therapy agents in clinical use in human . for instance , the kpc mouse model is relatively unaffected by gemcitabine similar to the small clinical benefit from this agent in the advanced pancreatic cancer setting in humans ( olive et al . , 2009 ) . each gemm can be likened to a patient with a particular tumour type , and hence , can be enrolled into preclinical trial of novel agents ( eklund et al . , 2013 ; guerra and barbacid , 2013 ) . such trials are facilitated by the use of adapted imaging techniques to monitor for tumour development and progression . such utility is beginning to have an impact in the clinical setting . in humans , early phase clinical trials have shown promise for the combination of nanoparticle albumin - linked paclitaxel ( nab - paclitaxel ) and gemcitabine in advanced pdac . frese and colleagues have used the kpc mouse model of pancreatic cancer to provide a mechanistic understanding of the synergistic effect of this combination . paclitaxel appears to inhibit the breakdown of gemcitabine through modulation of a degradative enzyme , cytidine deaminase through a reactive oxygen species - dependent pathway ( frese et al . , 2012 ) . such mechanistic analyses may help to rationalize our clinical strategy of using such drug combination . for instance , the nab - paclitaxel can be used as an inducting agent followed by gemcitabine to enhance the tumouricidal effect of the latter . rational clinical trials in man are likely to benefit from the incorporation of an in vivo component that provides relatively rapid feedback of the predicted response to new agents . the preclinical assessment using gemms can either be used to screen potentially useful therapeutic agents before progressing to clinical trials , or alternatively , to critically assess the mechanisms of action in vivo once an agent has been found to be effective in a small - scale trial , before progressing to larger phase iii clinical trial . one potential advantage of such gemms is that unlike human trials , they will allow sequential sampling of appropriate tumour tissues to assess the pharmacodynamic impact of a particular agent . in pancreatic cancer , several novel agents targeting a diverse range of molecular pathways have been tested in gemms to complement early phase clinical trials ( olive et al . , 2009 ; plentz et al . , 2009 ; cook et al . the results of these trials will in due course affirm or refute the value of pdac gemms as a predictive tool for clinical efficacy in human cancers . the potential value of pdac gemms as surrogates for the preclinical testing of new therapies is critically dependent on how closely these models mimic human pdac . several studies ( hingorani et al . , 2003 ; tuveson and hingorani , 2005 ; olive et al . , 2009 ; plentz et al . , 2009 ; cook et al . , 2012 ) have emphasized the similarities in histopathology , cancer progression , clinical behaviour and even drug pharmacodynamics between pdac gemms and human pdacs . initial observations suggest that murine kpc pdacs bear resemblance to the human disease insofar as they exhibit a high degree of genomic instability , evident from multiple non - reciprocal chromosomal translocations ( hingorani et al . , 2005 ) . however , with emerging data from large - scale sequencing of human pdac tumours ( biankin et al . , 2012 ) , it is now imperative that we further validate the gemms at the genomic level to compare the genomic landscapes of murine and human tumours . because pdac gemms incorporate high - penetrance genetic events such as initiating oncogenes or inactivated tumour suppressor genes from an early stage in a large number of susceptible cells , the resulting stereotypy of the malignancies arising therein may not reflect the heterogeneity likely to be present in human cancers . importantly , the genetic heterogeneity of human cancers may give rise to differing therapeutic responses to any particular agent due to the differing genetic and epigenetic signatures of the constituent cells . it is conceivable that individual tumours can take differing genetic routes to achieve tumoural progression , depending on the type of initiating genetic lesions and secondary genetic hits that occur stochastically . it is presumed that through inactivation of genes involved in maintaining genomic stability ( e.g. brca2 in models of pancreatic cancer ) may promote the stochastic acquisition of genetic and consequent morphologic heterogeneity due to the expected increase in mutation rate . however , this point remains to be established in future studies , and also has important implications for the potential value of gemm models in testing new therapeutic approaches against pdac .

background : many mothers still rely on palpation to determine if their children have fever at home before deciding to seek medical attention or administer self - medications . this study was carried out to determine the accuracy of subjective assessment of fever by nigerian mothers in under-5 children.patients and methods : each eligible child had a tactile assessment of fever by the mother after which the axillary temperature was measured . statistical analysis was done using spss version 19 ( ibm inc . chicago illinois , usa , 2010).result : a total of 113 mother / child pairs participated in the study . palpation overestimates fever by 24.6% . irrespective of the surface of the hand used for palpation , the sensitivity , specificity , positive predictive value ( ppv ) and negative predictive value ( npv ) of tactile assessment were 82.4% , 37.1% , 51.9% and 71.9% , respectively . the use of the palmer surface of the hand had a better sensitivity ( 95.2% ) than the dorsum of the hand ( 69.2% ) . the use of multiple sites had better sensitivity ( 86.7% ) than the use of single site ( 76.2%).conclusion : tactile assessment of childhood fevers by mothers is still a relevant screening tool for the presence or absence fever . palpation with the palmer surface of the hand using multiple sites improves the reliability of tactile assessment of fever . chromosomal instability is a hallmark of human cancer cells , but its role in carcinogenesis remains poorly resolved . insights into this role have emerged from studies on the tumour suppressor brca2 , whose inactivation in human cancers causes chromosomal instability through the loss of essential functions of the brca2 protein in the normal mechanisms responsible for the replication , repair and segregation of dna during cell division . humans who carry heterozygous germline mutations in the brca2 gene are highly predisposed to cancers of the breast , ovary , pancreas , prostate and other tissues . here , we review recent studies that describe genetically engineered mouse models ( gemms ) for pancreatic cancer associated with brca2 mutations . these studies not only surprisingly show that brca2 does not follow the classical knudson two hit paradigm for tumour suppression , but also highlight features of the interplay between tp53 inactivation and carcinogenesis in the context of brca2 deficiency . thus , the models reveal novel aspects of cancer evolution in carriers of germline brca2 mutations , provide new insights into the tumour suppressive role of brca2 , and establish valuable new preclinical settings for testing approaches to pancreatic cancer therapy ; together , these features emphasize the value of gemms in cancer research .

central venous catheters ( cvc ) are often used in the intensive care unit ( icu ) for monitoring and treatment of critically ill patients to provide long - term venous access . however , their use may result in nosocomial catheter - related bloodstream infection ( crbsi ) . the estimated number of crbsi is approximately 250,000 annually in the united states [ 24 ] , with an incidence of approximately 1.65 infections per 1000 central line days , and it was reported to be of 6.8 infections per 1000 central line days in asia . indeed , crbsi is associated with a 2.27-fold increased risk of mortality in the icu . host factors such as chronic illness , bone marrow transplantation , immune deficiency , malnutrition , total parenteral nutrition , previous history of crbsi , old age , and skin trauma increase the risk of crbsi . in addition , catheter factors also play a role in crbsi onset , such as duration of catheterization , type of catheter , conditions of insertion , insertion site care , and skill of the person who inserted the catheter [ 1013 ] . in cases of crbsi , empiric antibiotic therapy must be started as soon as possible , and must be based on the clinical characteristics of the patients . catheter removal is necessary in cases of severe sepsis , hemodynamic instability , endocarditis , erythema , or bacteremia persisting after 72 hours of antibiotic therapy . once the responsible organism is identified , tailored antibiotic therapy may be started , and usually lead to better outcomes . however , little is known about the risk factors for early crbsi onset , and few data are available about the clinical manifestations and mortality of crbsi in china . therefore , the aim of the present study was to assess the risk factors and catheter characteristics of 73 cases of nosocomial crbsi in the icu between january 2010 and december 2012 . we analyzed the clinical features and treatment outcomes , and provide evidence for early diagnosis and treatment of crbsi , as well as risk factors for early crbsi onset . this study was performed at the taizhou hospital of integrated traditional chinese and western medicine in zhejiang ( zhejiang wenlin , china ) in patients hospitalized in the icu between january 2010 and december 2012 . the ethics committee of the hospital approved the study , and waived the requirement for individual consent . patients were included if : 1 ) they developed bacteremia or fungemia while having a cvc or within 48 h of cvc removal ; 2 ) fever ( > 38c ) , shivering or hypotension ; and 3 ) no other apparent source of infection . in addition , at least one of the following criteria had to be met : 1 ) the same pathogenic agent was isolated from the catheter tip and from the peripheral venous blood ( at least once ) , detected with a semi - quantitative method ( each catheter fragment contained > 15 bacteria / cfu ) or quantitative method ( each catheter fragment contained > 10 bacteria / cfu ) ; 2 ) intra - catheter and peripheral blood culture was positive , and the bacterial colony count in the intra - catheter blood was at least 3 times greater compared with the peripheral blood ; or 3 ) the time to report the positive intra - catheter blood culture was at least 2 h earlier than that of the positive peripheral blood culture . patients were excluded if : 1 ) there was no clinical manifestation of bloodstream infection ; or 2 ) other causes of bloodstream infection could not be excluded . the clinical data of patients with nosocomial crbsi were collected and analyzed in accordance with the 2009 clinical practice guidelines for the diagnosis and management of intravascular catheter - related infection from the infectious diseases society of america ( idsa ) . patients age , sex , reason of icu admission , underlying diseases , time and position of indwelling cvc , type of cvc , pathogenic identification , treatment , prognosis , and mortality attributable to crbsi were reviewed . timely removal of the catheter was defined as cvc removal within 12 h of fever onset . appropriate antibiotic use was defined as the empirical use of antibiotics , which were confirmed to have covered the pathogenic bacteria in the drug sensitive test later . patients with timely cvc removal and appropriate antibiotic use were assigned to group a. patients with timely cvc removal and inappropriate antibiotic use were assigned to group b. patients with untimely cvc removal and appropriate antibiotic use were assigned to group c. finally , patients with untimely cvc removal and inappropriate antibiotic use were assigned to group d. all continuous data are expressed as means standard deviation ( sd ) . inter - group comparison of the measured data was performed using analysis of variance ( anova ) , and t - tests were used for post hoc analysis . categorical data are presented as proportions , and were compared using chi - square tests , using a segmentation approach to compare group pairs . the risk factors of early crbsi were evaluated using univariate and multivariate logistic regression analyses . this study was performed at the taizhou hospital of integrated traditional chinese and western medicine in zhejiang ( zhejiang wenlin , china ) in patients hospitalized in the icu between january 2010 and december 2012 . the ethics committee of the hospital approved the study , and waived the requirement for individual consent . patients were included if : 1 ) they developed bacteremia or fungemia while having a cvc or within 48 h of cvc removal ; 2 ) fever ( > 38c ) , shivering or hypotension ; and 3 ) no other apparent source of infection . in addition , at least one of the following criteria had to be met : 1 ) the same pathogenic agent was isolated from the catheter tip and from the peripheral venous blood ( at least once ) , detected with a semi - quantitative method ( each catheter fragment contained > 15 bacteria / cfu ) or quantitative method ( each catheter fragment contained > 10 bacteria / cfu ) ; 2 ) intra - catheter and peripheral blood culture was positive , and the bacterial colony count in the intra - catheter blood was at least 3 times greater compared with the peripheral blood ; or 3 ) the time to report the positive intra - catheter blood culture was at least 2 h earlier than that of the positive peripheral blood culture . patients were excluded if : 1 ) there was no clinical manifestation of bloodstream infection ; or 2 ) other causes of bloodstream infection could not be excluded . the clinical data of patients with nosocomial crbsi were collected and analyzed in accordance with the 2009 clinical practice guidelines for the diagnosis and management of intravascular catheter - related infection from the infectious diseases society of america ( idsa ) . patients age , sex , reason of icu admission , underlying diseases , time and position of indwelling cvc , type of cvc , pathogenic identification , treatment , prognosis , and mortality attributable to crbsi were reviewed . timely removal of the catheter was defined as cvc removal within 12 h of fever onset . appropriate antibiotic use was defined as the empirical use of antibiotics , which were confirmed to have covered the pathogenic bacteria in the drug sensitive test later . patients with timely cvc removal and appropriate antibiotic use were assigned to group a. patients with timely cvc removal and inappropriate antibiotic use were assigned to group b. patients with untimely cvc removal and appropriate antibiotic use were assigned to group c. finally , patients with untimely cvc removal and inappropriate antibiotic use were assigned to group d. inter - group comparison of the measured data was performed using analysis of variance ( anova ) , and t - tests were used for post hoc analysis . categorical data are presented as proportions , and were compared using chi - square tests , using a segmentation approach to compare group pairs . the risk factors of early crbsi were evaluated using univariate and multivariate logistic regression analyses . the 73 crbsi patients included 42 males and 31 females , with a mean age of 63.221.3 years ( range : 4387 years ) . the mean time of cvc indwelling was 20.89.2 days ( range : 739 days ) , and 64.3% of the participants had cvc indwelling for 14 days . there were 27 , 20 , 15 , and 11 patients assigned to groups a , b , c , and d , respectively . there were 21 patients ( 28.8% ) with 3 types of underlying diseases , 43 patients with 4 types of underlying diseases , and 9 patients ( 12.3% ) with 5 types of underlying diseases . there were 23 cases ( 31.5% ) with 3 high - risk factors of crbsi and 50 cases ( 68.5% ) with 4 high - risk factors . univariate logistic regression analysis revealed that an acute physiology and chronic health evaluation ii ( apache ii ) score > 23 , parenteral nutrition or transfusion of blood products , age > 65 years , diabetes , > 3 types of underlying diseases , renal insufficiency or multiple organ dysfunction syndrome ( mods ) , and immunosuppression or chemoradiotherapy were correlated with the occurrence of crbsi within 14 days of cvc indwelling ( table 2 ) . the factors that were statistically significant ( p<0.05 ) in univariate analyses were included in the multivariate analysis . apache ii score > 20 and > 3 types of underlying diseases were independent risk factors of crbsi occurring within 14 days of cvc indwelling ( table 3 ) . the types of cvc and the position of cvc indwelling are listed in table 1 . the duration of cvc indwelling until crbsi occurrence is shown in table 4 . femoral cvc showed the shortest time to crbsi onset ( 14.25.1 days ) , followed by internal jugular cvc ( 20.98.0 ) , and subclavian cvc ( 33.83.9 ) ( all p<0.05 ) . time to crbsi onset was longer with single - lumen cvc compared with dual - lumen cvc ( 33.73.9 vs. 16.96.3 days , p<0.05 ) . the most common pathogens were gram - positive bacteria , followed by gram - negative bacteria and fungi ( all of which were candida strains ) . infection associated with methicillin - resistant staphylococcus aureus ( mrsa ) and methicillin - resistant coagulase - negative staphylococci ( mrcons ) were observed in 32 cases . infection with extended - spectrum -lactamase ( esbl)-producing bacteria was observed in 15 cases . vancomycin - resistant enterococci ( vre ) infection was seen in 5 cases . carbapenem - resistant gram - negative bacteria ( crgnb ) were detected in 13 cases ( table 5 ) . all 73 crbsi patients manifested fever , with a body temperature ranging from 38.1 to 40.3c , including 20 cases ( 27.3% ) with a body temperature of 38.139c , and 53 cases ( 77.7% ) with a body temperature of > 39c . routine blood test showed a mean white blood cell count of 19.69.310/l ( range : 12.130.210/l ) , and a mean neutrophile count of 87.38.5% ( range : 8291.2% ) . / l in all subjects , and 39 cases ( 53.4% ) had crp levels of > 165 mg / l . abnormal liver function ( mild to moderate elevation of alt and ast levels ) was detected in 17 cases ( 23.2% ) . abnormal renal function ( except 6 cases with renal failure before enrollment in the study ) was found in 27 cases ( 36.9% ) , with the highest serum creatinine level being 671 mol / l . circulation failure was observed in 23 cases ( 31.5% ) and 27 cases ( 50.9% ) were found to be complicated by mods . redness and swelling at the site of indwelling catheter was found in 14 cases ( 19.1% ) , including 7 cases ( 9.7% ) with local purulent secretions . the major treatment for patients with crbsi was cvc removal and the use of effective antibiotics . we observed 47 cases with cvc being removed within 12 h after blood sampling , and all of them showed a rapid drop in body temperature following cvc removal . seventeen cases had their cvc removed after a report of positive blood culture , while cvc was not removed in 9 cases . empirical antibiotic therapy was administered to all subjects before confirmation of a positive blood culture , and was continued after positive blood culture was confirmed , or replaced by another antibiotic . the survivors showed a mean time to defervescence of 6.92.1 days , and a mean time to negative conversion of blood culture of 6.42.4 days ( table 6 ) . the 73 crbsi patients included 42 males and 31 females , with a mean age of 63.221.3 years ( range : 4387 years ) . the mean time of cvc indwelling was 20.89.2 days ( range : 739 days ) , and 64.3% of the participants had cvc indwelling for 14 days . there were 27 , 20 , 15 , and 11 patients assigned to groups a , b , c , and d , respectively . there were 21 patients ( 28.8% ) with 3 types of underlying diseases , 43 patients with 4 types of underlying diseases , and 9 patients ( 12.3% ) with 5 types of underlying diseases . there were 23 cases ( 31.5% ) with 3 high - risk factors of crbsi and 50 cases ( 68.5% ) with 4 high - risk factors . univariate logistic regression analysis revealed that an acute physiology and chronic health evaluation ii ( apache ii ) score > 23 , parenteral nutrition or transfusion of blood products , age > 65 years , diabetes , > 3 types of underlying diseases , renal insufficiency or multiple organ dysfunction syndrome ( mods ) , and immunosuppression or chemoradiotherapy were correlated with the occurrence of crbsi within 14 days of cvc indwelling ( table 2 ) . the factors that were statistically significant ( p<0.05 ) in univariate analyses were included in the multivariate analysis . apache ii score > 20 and > 3 types of underlying diseases were independent risk factors of crbsi occurring within 14 days of cvc indwelling ( table 3 ) . the types of cvc and the position of cvc indwelling are listed in table 1 . the duration of cvc indwelling until crbsi occurrence is shown in table 4 . femoral cvc showed the shortest time to crbsi onset ( 14.25.1 days ) , followed by internal jugular cvc ( 20.98.0 ) , and subclavian cvc ( 33.83.9 ) ( all p<0.05 ) . time to crbsi onset was longer with single - lumen cvc compared with dual - lumen cvc ( 33.73.9 vs. 16.96.3 days , p<0.05 ) . the most common pathogens were gram - positive bacteria , followed by gram - negative bacteria and fungi ( all of which were candida strains ) . infection associated with methicillin - resistant staphylococcus aureus ( mrsa ) and methicillin - resistant coagulase - negative staphylococci ( mrcons ) infection with extended - spectrum -lactamase ( esbl)-producing bacteria was observed in 15 cases . vancomycin - resistant enterococci ( vre ) infection was seen in 5 cases . carbapenem - resistant gram - negative bacteria ( crgnb ) were detected in 13 cases ( table 5 ) . all 73 crbsi patients manifested fever , with a body temperature ranging from 38.1 to 40.3c , including 20 cases ( 27.3% ) with a body temperature of 38.139c , and 53 cases ( 77.7% ) with a body temperature of > 39c . routine blood test showed a mean white blood cell count of 19.69.310/l ( range : 12.130.210/l ) , and a mean neutrophile count of 87.38.5% ( range : 8291.2% ) . / l in all subjects , and 39 cases ( 53.4% ) had crp levels of > 165 mg / l . abnormal liver function ( mild to moderate elevation of alt and ast levels ) was detected in 17 cases ( 23.2% ) . abnormal renal function ( except 6 cases with renal failure before enrollment in the study ) was found in 27 cases ( 36.9% ) , with the highest serum creatinine level being 671 mol / l . circulation failure was observed in 23 cases ( 31.5% ) and 27 cases ( 50.9% ) were found to be complicated by mods . redness and swelling at the site of indwelling catheter was found in 14 cases ( 19.1% ) , including 7 cases ( 9.7% ) with local purulent secretions . the major treatment for patients with crbsi was cvc removal and the use of effective antibiotics . we observed 47 cases with cvc being removed within 12 h after blood sampling , and all of them showed a rapid drop in body temperature following cvc removal . seventeen cases had their cvc removed after a report of positive blood culture , while cvc was not removed in 9 cases . empirical antibiotic therapy was administered to all subjects before confirmation of a positive blood culture , and was continued after positive blood culture was confirmed , or replaced by another antibiotic . the survivors showed a mean time to defervescence of 6.92.1 days , and a mean time to negative conversion of blood culture of 6.42.4 days ( table 6 ) . the aim of the present study was to investigate the clinical characteristics , treatment , and prognosis of crbsi in the intensive care unit ( icu ) in a chinese center , as well as the risk factors for early crbsi . among the 73 crbsi patients enrolled in the present study , the major risk factors included advanced age , long - term catheter indwelling , parenteral nutrition , diabetes , and apache ii score > 23 , and > 3 types of underlying diseases . multivariate analysis showed that an acute physiology and chronic health evaluation ii ( apache ii ) score > 20 and > 3 types of underlying diseases were independent factors associated with crbsi occurring within 14 days of cvc indwelling . advanced age , long - term catheter indwelling , parenteral nutrition , diabetes , apache ii score > 23 , and > 3 types of underlying diseases are well known risk factors of crbsi . patients with advanced age and multiple underlying diseases and diabetes usually show decreased immune function . our findings showed that significantly more subjects had catheter indwelling for 14 days compared with catheters indwelling for < 14 days . the prolongation of catheterization by one day was reported to be associated with a 1.8-fold increased risk of crbsi . early crbsi may have serious implications for the patients since it can interfere with patient treatment . the results of the present study showed that poor patient condition , indicated by the apache ii score , and high number of disease types were independently associated with an early crbsi onset . therefore , the prevention of crbsi should be for a priority in critically ill patients with complicated diseases . indeed , the mortality rate from crbsi is reported to be 8.8% in subjects aged > 65 years , but only 3.8% in those aged < 65 years . indeed , a large proportion of our patients had high apache ii scores and a large number of concomitant diseases . accordingly , a previous study showed that the mortality rate due to crbsi in the icu was 28.1% in critically ill patients . the present study demonstrated that the position of cvc indwelling was closely associated with the time of crbsi onset . the longest mean duration of catheterization was found in the subclavian vein at crbsi onset , followed by the internal jugular vein . the shortest duration was observed in the femoral vein , as observed by previous studies . the skin at the subclavian vein is flat , which is easier to clean and disinfect , resulting in late crbsi onset . accordingly , the subclavian vein is recommended as the first choice of the site for catheterization in the idsa guidelines , followed by the internal jugular vein and the femoral vein . in addition , crbsi occurred later when using the single - lumen catheter than with double - lumen catheter . indeed , a significantly higher occurrence of crbsi was observed with double - lumen catheters compared with single - lumen catheters . in the present study , the most common pathogens causing crbsi were gram - positive bacteria , followed by gram - negative bacteria and fungi . cons were predominant in the gram - positive bacteria , followed by mrsa , with 85.7% of the staphylococci resistant to methicillin , and 41.7% of the enterococci resistant to vancomycin . the most common gram - negative bacteria were acinetobacter baumannii , followed by pseudomonas aeruginosa , in which esbls - producing bacteria were 80.0% and 68.4% , respectively , resistant to carbapenem antibiotics . the high prevalence of antibiotic - resistant bacteria may be associated with the wide use of broad - spectrum antibiotics in the icu . candida albicans constituted only 42.9% of candida infections . in a systematic review of eligible studies related to crbsi associated with parenteral nutrition published between 1970 and march 2012 , the major pathogens were staphylococcus spp . , followed by gram - negative bacillus and candida spp . , which was consistent with the current study findings . however , the distribution of the causal agents in the present study was slightly different from that observed in the united states . therefore , the choice of empiric treatment should be made according to these possible geographical differences . empirical selection of antibiotics should be based on patient clinical profile , such as recent history of antibiotic use , drug resistance or allergy , underlying diseases , clinical epidemiology , and health facility . in addition , adequate anti - infective therapy for crbsi should be administered to prevent complications such as inflammatory deep venous thrombosis and infectious endocarditis . the number of patients was small , and they all were from a single center . no control group was included , preventing us from studying the risk factors for crbsi in china . however , the present study is the first to identify some risk factors for early crbsi onset . apache ii score > 20 and the presence of > 3 types of diseases were associated with earlier crbsi onset . in this chinese center , critically ill patients in the icu should be particularly monitored for the appearance of crbsi . there is a critical need to develop potent and selective therapeutic agents capable of targeting malignant tissue without compromising normal cell viability . while chemotherapeutic agents ( e.g. , doxorubicin and docetaxel ) remain widely used in the clinic , they lack inherent selectivity desired to limit toxicity to normal cells . in addition , administration of chemotherapeutic agents can induce drug resistance , resulting in disease progression . thus , the development of more targeted therapies could circumvent nonspecific interactions and potentially overcome drug resistance in cancer therapy . intriguing studies are currently exploring new methods to engage biomolecular targets with high affinity and specificity , including the generation of multivalent and heterobifunctional constructs . advances in chemical synthesis techniques , such as cross - coupling and conjugation strategies , have enabled chemists to decorate a plethora of molecular species with targeting moieties , providing access to elaborate molecular architectures that can be tailored to occupy distinct binding sites within one or multiple biomacromolecules . although these types of compounds fall outside the molecular weight range of typical drug compounds ( 5003000 da ) , increasing interest in developing new chemical entities that can modulate biomolecular targets in novel ways and address selectivity requirements are emerging . to date , there have been only limited examples evaluating the potential for targeting the androgen receptor ( ar ) with steroidal conjugates . the ar is an important drug target for treatment of prostate cancer and has been the subject of research for several decades . a large number of bioactive compounds targeting ar have been identified via screening efforts . in this review , we begin by providing a rationale for continued studies in prostate cancer pharmacology targeting the ar . particular focus is placed on examining current approaches to specifically engage and modulate ar activity with steroid conjugates utilizing rational design principles . androgens are a class of steroid hormones that consist of 19-carbon derivatives of cholesterol and are synthesized by the testis and adrenal glands . they are also precursors for estrogens , the female sex hormones , produced by hydroxylation , elimination , and aromatization of androgens through the enzyme aromatase . functioning primarily through the ar , which is a ligand - dependent transcription factor , androgens play a fundamental role in the development and survival of male reproductive tissues , such as the prostate , by influencing gene expression levels . the body maintains control of testosterone ( the most abundant androgen in men ) levels within a normal reference range of 240800 ng / dl . low levels of testosterone resulting from zinc deficiency or aging can lead to fatigue and erectile dysfunction . by contrast , high levels of testosterone have been linked to a variety of diseases , including prostate cancer . prostate cancer remains the most common cancer among men and is globally estimated to affect 900 000 patients every year . as the second leading cause of cancer - related deaths in men ( 258 000/year ) , approximately one out of every six men will be diagnosed with prostate cancer in the u.s . if detected early , an arsenal of therapeutic options currently provide a promising chance for long - term survival . however , 40% of patients will develop castration - resistant prostate cancer ( crpc ) , arising from drug resistance ( vida infra ) , which is associated with poor survival rates . the ar is a 110 kda protein that shares sequence homology with other nuclear hormone receptors in the superfamily , including the progesterone receptor ( pr ) , glucocorticoid receptor ( gr ) , and estrogen receptor ( er ) . the ar consists of four basic elements : n - terminal domain , dna binding domain , hinge region , and the ligand binding domain ( lbd ) . the first domain is the 559 amino acid long intrinsically disordered n - terminal domain , which contains the ligand - independent activation function 1 ( af-1 ) . activation function sites encode signature motifs containing lxxll or fxxlf sequences to recruit co - regulatory proteins that are essential for transcription . the most highly conserved region within all nuclear hormone receptors , including ar , is the centrally located dna binding domain , consisting of two zinc finger domains that recognize specific dna consensus sequences known as the androgen response elements ( figure 1a ) . the third domain , dubbed the hinge region , connects the dna binding domain to the ligand - binding domain ( figure 1b ) . the ligand - binding domain ( lbd ) contains ligand - dependent activation function 2 ( af-2 ) , forms the ligand - binding pocket , and mediates interactions between the ar and heat shock proteins ( figure 1b ) . importantly , af-2 can interact with an fxxlf binding motif located within the n - terminal domain , a feature unique to ar . x - ray crystal structure of ( a ) androgen receptor ( ar ) dna binding domain ( ribbon , red ) in complex with the androgen response elements ( sticks , pdb code 1r4i ) and ( b ) ar ligand binding domain ( ribbon , gray ) and portion of hinge region ( ribbon , blue ) in complex with native ligand ( sticks , green , pdb code 1i37 ) . ( c ) amino acids residues that establish high affinity binding with native ligand dht ( pdb code 2ama ) . the crystal structure of the ar lbd bound to native ligand ( dht ) reveals the amino acid residues critical for maintaining high binding affinity ( figure 1c ) . although van der waals forces contribute to binding affinity , hydrogen bonds establish stronger interactions with the native ligand . arg752 forms a hydrogen bond with the o3 atom ( ketone ) of the steroid ligand . mutagenesis of arg752 has been shown to compromise binding affinity , suggesting the importance of this interaction for achieving high affinity . in addition , asn705 and thr877 form hydrogen bonds with the 17- hydroxyl group of the steroid ligand . mutagenesis of asn705 and thr877 have also resulted in reduced binding affinity and specificity , establishing their importance to maintaining high affinity . it is important to note that modifications to the 17- hydroxyl group can result in diminished binding affinity , while even large substituent modifications at the 17- position often retain strong binding interactions . the ar is a ligand - dependent transcription factor that is stabilized in the cytoplasm by chaperone proteins ( figure 2 ) . competitive displacement of the chaperones by dihydrotestosterone ( dht ) , an androgen biosynthesized from testosterone through the enzyme 5-reductase , activates the ar . upon activation , a conformational change brings the n- and c - termini into proximity and facilitates ar dimerization . upon translocation into the nucleus , ar binds to palindromic 5-tgttct-3 consensus sequences ( androgen response elements ) in the promoter regions of target genes . this event stimulates the recruitment of necessary cofactors , including lxxll or fxxlf motif - containing proteins , and other components of the transcriptional machinery to regulate gene expression . abbreviations : dht , dihydrotestosterone ; hsp , heat shock protein ; p , phosphorylation site ; fxxlf , coactivator protein . the ar mediates a variety of androgen - dependent diseases including benign prostatic hypertrophy ( bph ) , prostatic intraepithelial neoplasia ( pin ) , and prostate cancer . it has been proposed that prostate cancer often originates from high - grade prostatic intraepithelial neoplasia ( hgpin ) , a process in which subtle alterations in the shape and size of prostate cells occur . more importantly , progression of prostate cancer has been linked to elevated expression of ar in malignant tissue , suggesting that ar plays a central role in prostate cancer cell biology . although many hypotheses regarding the involvement of ar in prostate cancer progression have been postulated , the precise molecular mechanisms are not fully understood . patients diagnosed with localized or metastatic prostate cancer usually undergo androgen deprivation therapy ( reduction of circulating androgen levels ) , through chemical castration ( gonadotropin - releasing hormone agonists ) or surgical castration . unfortunately , these methods do not completely eliminate circulating levels of androgens , as the tumor itself is capable of local androgen synthesis , due to the expression of androgen biosynthetic enzymes . this has led to numerous research efforts focusing on the development of inhibitors that interfere with key enzymes , such as cytochrome p450 17a1 ( cyp17a1 ) , in androgen biosynthesis as exemplified by the recent fda approval of abiraterone ( zytiga ) . the standard treatment approach for prostate cancer involves androgen deprivation therapy in conjunction with small molecule anti - androgens that block ar signaling ( figure 3a ) . anti - androgens compete with dht for binding to ar , thus inhibiting ar transactivation through a variety of mechanisms , including disruption of nuclear localization , interruption of dna binding , and interference with coactivator recruitment . unfortunately , most patients receiving anti - androgen therapy eventually develop drug resistance as indicated by rising levels of serum prostate - specific antigen ( psa ) , a gene regulated by ar , leading to the lethal disease state termed castration - resistant prostate cancer or crpc . small molecule inhibitors targeting the ar : ( a ) anti - androgens ; ( b ) activation function 2 inhibitors ; ( c ) allosteric ( bf3 site ) regulators ; ( d ) n - terminal domain inhibitors . purple denotes approved therapies for androgen - dependent prostate cancer , and orange represents approved therapies for castration - resistant prostate cancer . current mechanisms proposed for advancement to crpc include the following:(1)alterations in ar co - regulatory protein balance;(2)somatic gain of function mutations within ar , with the majority in the lbd , resulting in activation by other steroid hormones and anti - androgens;(3)generation of new fusion gene products;(4)ar ligand - independent activation via cross - talk with other signaling pathways.these mechanisms have garnered significant attention because of their ability to reactivate ar and disease progression , and provide a conceptual underpinning to guide development of new therapeutic interventions . nevertheless , currently crpc is primarily treated with chemotherapeutic agents , immunotherapy , or abiraterone ( vida supra ) . alterations in ar co - regulatory protein balance ; somatic gain of function mutations within ar , with the majority in the lbd , resulting in activation by other steroid hormones and anti - androgens ; generation of new fusion gene products ; ar ligand - independent activation via cross - talk with other signaling pathways . ar have been identified via chemical screening efforts and include compounds that act on the af-2 ( figure 3b ) or bf3 site ( figure 3c ) on ar to regulate its activity . the bf3 site is a hydrophobic binding pocket located adjacent to af-2 on the surface of ar that can allosterically regulate binding interactions between ar and coactivator proteins . the development of noncompetitive modulators ( that do not compete against dht for ligand binding ) could circumvent drug resistance in ar pharmacology . while promising , these noncompetitive approaches have yet to yield candidates for clinical implementation , likely because of the high concentrations required to suppress ar activity . in the future , it may be important to utilize structure - based design to generate more potent af-2 or bf3 inhibitors . in contrast , continuing interest in anti - androgen drug development has led to the fda approval of enzalutamide , which targets the ar ligand binding domain for the treatment of crpc ( figure 3a ) . unfortunately , recent evidence suggests that drug resistance to enzalutamide can emerge from point mutations within the ar lbd , such as f876l . additionally , drug resistance has been proposed to arise from constitutively active ar splice variants lacking the ar ligand binding domain . this has led researchers to focus on innovative ways to antagonize ar splice variants and the development of n - terminal domain inhibitors ( figure 3d ) . it is important to note , however , that no structural information exists for the ar n - terminal domain , complicating the design of n - terminal domain antagonists . although it is tempting to speculate that ar splice variants are mainly responsible for drug resistance to enzalutamide , the precise molecular mechanisms remain unknown . evidence suggests that full - length ar is required for signaling , although different sets of studies demonstrate that er splice variants can be constitutively active in the absence of ligand . also , an intriguing report has similarly suggested that the gr can become constitutively active in the absence of its lbd . future research may illuminate whether other nuclear hormone receptors can exhibit similar modes of action . bioactive hit compounds , typically identified from screening efforts , often lack the potency and selectivity required for translation to a clinical setting . for this reason compounds through iterative rounds of synthesis and rigorous bioassays . while this strategy remains widely utilized in both academic and industrial research programs , rational design of therapeutic agents aims to streamline these issues by initially identifying more potent and selective compounds . below , we describe different strategies that have been used to target ar with steroid conjugates , along with preliminary evaluation of their potential applications in ar pharmacology . the atp - dependent ubiquitin - proteasome pathway is a quality control mechanism that conducts the programmed metabolic degradation of proteins . ubiquitin - protein ligase ( e3 ) associates with ubiquitin - conjugating enzyme ( e2 ) , providing subsequent tagging of ubiquitin chains to protein substrates that results in degradation by the proteasome . rational design strategies aimed toward selectively targeting proteins for degradation through e3 could establish an approach to diminish the levels of aberrantly functioning proteins . the crews lab has pioneered a general strategy to modulate levels of selective proteins by engagement of the ubiquitin system . by use of conjugates dubbed proteolysis targeting chimeric molecules ( protacs ) , the first steroid conjugate to selectively induce ar degradation was developed . protacs consist of three components : a targeting moiety ( dht ) , a linker , and a recognition element for e3 . the modular synthesis of protacs establishes a significant pharmacological advantage because protacs are particularly amenable to chemical modification , permitting control over the physicochemical features of the products . initial ex vivo studies aimed toward degrading ar yielded protac-5 ( figure 4a ) . protac-5 was outfitted with a peptide sequence ( alapyip ) as an e3 recognition domain and to induce ubiquitination upon hydroxylation of the central proline residue . to assess biological activity , protac-5 was administered to human embryonic kidney cells ( hek293 ) that stably expressed an ar fluorescent fusion protein . protein degradation was quantified by a reduction in the fluorescence signal . at a concentration of 25 m , protac-5 successfully degraded ar without compromising normal cell viability . in control studies , vehicle treated cells maintain fluorescence , suggesting that protac-5 engages ar in the cell and induces degradation . to confirm these results , cells were treated with protac-5 and immunoblotted for ar . a significant decrease in ar protein level proteolysis targeting chimeric molecules ( protacs ) for ar : ( a ) synthesis of protac-5 ; ( b ) chemical structure of protac - aa ; ( c ) chemical structure of small molecule e3 recognition element ( left ) and cocrystal structure of small molecule e3 recognition element ( blue sticks ) and e3 ( orange surface rendered , right ; pdb code 3zrc ) . figure is adapted from refs ( 53 ) , ( 55 ) , and ( 56 ) . more recently , a derivative of protac-5 , dubbed protac - aa ( figure 4b ) , was administered to an ar - expressing prostate cancer cell line ( lncap ) to evaluate effects on cell proliferation . protac - aa contains a shorter hydroxylated recognition element for e3 and a slightly modified arginine tail to enhance cell permeability . the arginine tail enhances cell permeability through an uptake mechanism mimicking the antennapedia and hiv tat proteins . protac - aa inhibited cell growth with an inhibitory concentration ( ic50 ) value of 3.8 m at 72 h and 0.217 m at 144 h. a control protac lacking the arginine tail displayed ic50 values 12.5 m at 72 h and 1.5 m at 144 h. western blot analysis was performed to establish that ar protein levels were reduced . taken together , these results suggest that the arginine tail enhances biological activity while maintaining specificity . importantly , in prostate cancer cell lines that do not express ar ( pc-3 and du-145 cells ) , protac - aa had no significant effect on cell viability , establishing selective activity . while protacs remain promising candidates for applications in ar pharmacology , difficulties in large - scale production may impede rapid translation into the clinic . current efforts have focused on developing more druglike protac molecules and the recent discovery of the first small molecule targeting e3 ( figure 4c ) with an ic50 value of 4.1 m . competitive fluorescence polarization data indicated that the small molecule binds to e3 , which was confirmed by a cocrystal structure . subsequent optimization led to the first submicromolar small molecule targeting e3 ( ic50 = 0.90 m ) . in the future , we may begin to see small molecule protacs targeting ar , which may include , for example , enzalutamide tethered to similar small molecules that are capable of recruiting e3 . apoptosis , or programmed cell death , is a physiological cell suicide mechanism critical to cellular homeostasis . inadequate activation of the apoptotic pathway can play a role in the development of cancer and autoimmune diseases . inhibitors of apoptosis proteins ( iaps ) play a fundamental role in regulating apoptosis and other cellular processes . iaps contain a ring domain that possesses e3 activity , establishing the ability to induce proteasomal degradation by tagging proteins with ubiquitin chains . the hashimoto lab has developed specific and nongenetic iaps - dependent protein erasers ( snipers ) . relative to protacs , snipers consist of a targeting moiety ( dht ) , linker , and a recognition element for iaps . the targeted ubiquitination of proteins by snipers relies on small molecule iap recognition elements ( figure 5 ) . the biological activity of an ar targeting compound , sniper-13 , was evaluated by western blot . in human mammary tumor ( mcf-7 ) cells that express ar , sniper-13 decreased ar protein levels at a concentration of 30 m . the high concentration required to induce degradation may be attributed to the hydrolytically unstable ester and oxime linkages . chemical structure of specific and nongenetic iaps dependent protein eraser 13 ( sniper-13 ) . the modular assembly of snipers allows for the incorporation of virtually any targeting moiety . this characteristic , and the ability to recruit e3 with a small molecule , establishes a versatile molecular platform to address many protein targets . in the future , research efforts may focus on generating stable linkages between the targeting moiety and the iap recognition element or altering linker lengths to optimize activity of sniper conjugates against various protein targets . metallo - based cytotoxic agents , such as cisplatin , remain a viable option for the treatment of cancer . from a mechanistic standpoint , these compounds exert their biological activities by binding to nucleobases in dna and inducing damage to dna that ultimately triggers apoptosis . although widely used in the clinic , these agents are generally nonspecific and exhibit shortcomings that include severe side effects resulting from compromised normal cell viability and drug resistance . this has led to the exploration of metallo - based chemotherapeutic agents that target specific organs or tumors to minimize adverse side effects . conjugation of a targeting moiety to metallo - based cytotoxic agents could potentially circumvent nonspecific interactions by selectivity targeting cells that overexpress particular proteins , establishing a delivery vector to localize the effects of new therapeutic agents . recent studies from the hannon group have discovered the first metallo - based chemotherapeutic conjugates to target ar . the authors developed an efficient protocol to readily synthesize an array of steroid conjugates to act as delivery vehicles . ethisterone , the 17-ethynyl homologue of testosterone , was conjugated to pyridines , quinolines , and isoquinolines utilizing sonogashira cross - coupling conditions . subsequent coordination to platinum(ii ) complexes yielded metallo - based bifunctional agents ( figure 6 ) . initial evaluation of the cytotoxic effects for the two most promising metallo - based bifunctional agents in cell lines that express ar ( t47d cells ) revealed promising biological activity for a cis conjugate ( ic50 = 15.9 m ) but not the trans conjugate ( ic50 = 63 m ) , suggesting that the geometry of the platinum(ii ) complexes plays a critical role . additionally , the cis conjugate exhibited greater potency than cisplatin itself ( ic50 = 32 m ) . cell uptake studies reveal that the targeting moiety enhances drug delivery , suggesting that the hydrophobic character of ethisterone facilitates molecular transport across the cellular membrane . importantly , the presence of the targeting moiety in the cis conjugate ( relative to control compounds lacking steroid moieties ) led to significant structural effects on dna . the distortion of dna upon binding the cis conjugate was greater than that observed for cisplatin , suggesting that the steric bulk of ethisterone promotes greater unwinding of dna to accommodate binding of the complex . metallo - based conjugates have significant utility as a platform for targeted drug delivery . the work outlined above suggests that metallo - based conjugates can be crafted to exert toxic effects preferentially on cell types that overexpress ar . , demonstration that coadministration of a competitive ligand , such as dht , abrogates the activity of the metallo - conjugate would further support the hypothesis that targeting is mediated specifically through binding to ar . ultimately , similar strategies could potentially be elaborated for targeting additional metallo - conjugates to a range of malignant cell types , while mitigating cytotoxic effects on other tissues . alkylating agents act through dna damaging mechanisms and are commonly used in cancer therapy . crafting alkylating therapies to specifically target malignant cells could minimize cytotoxic effects to normal cells and lead to the development of potent anticancer agents . in an effort to block dna repair enzymes , the essigmann group has developed heterobifunctional dna - damaging agents to specifically target prostate cancer cells ( figure 7 ) . the alkylating agent n , n - bis-2-chloroethylaniline was linked to a steroid hormone that targets ar , allowing the conjugate to simultaneously bind ar and dna . this strategy results in the blockade of dna repair enzymes in prostate cancer cells that overexpress ar , subsequently leading to the disruption of ar - mediated transcription and signaling . using radiometric competitive binding assays , the relative binding affinity of n , n - bis-2-chloroethylaniline was determined to be 20% for ar and 4.2% for pr . this result establishes that the conjugate is more selective for ar than pr . in addition , only n , n - bis-2-chloroethylaniline , and not the negative control ( n , n - bis-2-methoxyethylaniline ) , covalently modified dna . administration of the alkylating agent at a concentration of 10 m induced apoptosis , as determined by flow cytometry and cleavage of poly adp - ribose polymerase ( parp ) in western blot analysis . as expected , the negative control did not induce apoptosis at an equivalent concentration . more importantly , xenograft studies in immunocompromised mice revealed 90% inhibition of tumor growth through intraperitoneal injection ( daily dose of 30 mg / kg ) . these results demonstrate the effectiveness of using targeted alkylating agents to selectively suppress prostate cancer tumor growth . an emerging avenue in molecular pharmacology is the development of multivalent therapeutic agents . multivalency can be used to establish enhanced binding affinity , termed avidity , and specificity for corresponding biomolecular targets through multisite binding contacts . displaying ligands or targeting moieties on modular oligomer frameworks allows chemists to precisely craft architectures capable of inhibiting highly specific protein in addition , the ability to create monodisperse molecular scaffolds enables control over important physicochemical features of the products , including solubility and cellular uptake . peptoids are a class of peptidomimetics composed of n - substituted glycine units joined through tertiary amide linkages . peptoids have recently been exploited as multivalent platforms to design conjugates capable of targeting different nucleic acids and protein receptors . peptoids are stable against proteases and display enhanced cell permeability profiles . the incorporation of over 200 different peptoid side chains has enabled numerous applications in chemistry and biology , including enantioselective catalysis , molecular recognition , antimicrobial activity , intracellular delivery , and antitumor activity in vivo . peptoids are compatible with solid - phase synthesis techniques and can be assembled in a sequence - specific manner to afford monodisperse products . additionally , the conformation of peptoid oligomers can be controlled though macrocyclic constraints and side chain interactions . utilizing peptoids as a versatile chemical platform , the kirshenbaum lab designed multivalent ethisterone conjugates to specifically target the ar ldb and modulate ar activity via different mechanisms of action . ethisterone was conjugated at the 17- position to the peptoid scaffold via highly stable triazole linakges . initial efforts evaluated effects of valency , spacing , and conformational ordering on ar activity ( figure 8) . fluorescence polarization assays were conducted to determine if the conjugates compete against dht for binding to the ar ligand binding domain . results from these studies revealed that hexavalent ( 4 ) and spaced divalent conjugates ( 5 and 6 ) compete for binding . mono- , di- , and trivalent conjugates ( 13 ) and a cyclic divalent conjugate ( 7 ) did not compete for ar binding . a control peptoid conjugate outfitted with pr ligands did not activate ar in a luciferase reporter assay , suggesting the ethisterone conjugates are selective for ar . in cell proliferation studies that model castration - resistant prostate cancer ( lncap - abl cells ) , conjugates 6 and 7 exhibited potent antiproliferative properties . as expected , the anti - androgen bicalutamide ( vide supra ) failed to suppress proliferation in this resistant cell line . importantly , cytotoxic effects of conjugates 6 and 7 were not observed in cell lines that do not express ar ( pc-3 and hek293 cells ) , establishing that conjugates selectively target ar . figure is adapted from ref ( 90 ) . in a follow - up investigation , the authors used confocal microscopy , time - resolved fluorescence resonance energy transfer , chromatin immunoprecipitation , flow cytometry , and microarray analysis to gain insight into the mechanism of action for conjugates 6 and 7 . upon administration of conjugates 6 and 7 to hek293 cells transfected with an ar fluorescent fusion protein , conjugate 6 did not promote ar nuclear localization while conjugate 7 did , suggesting competitive and noncompetitive mechanisms of action , respectively . ar coactivator recruitment assays revealed that conjugate 6 did not promote binding between ar and coactivator proteins while conjugate 7 partially recruited coactivator proteins . in dna binding experiments , both conjugates 6 and 7 reduced the occupancy of ar to the psa enhancer ( vida supra ) . conjugate 7 , but not conjugate 6 , induced arrest in the g0/g1 phase of the cell - cycle and displayed contrasting patterns in global gene expression . intriguingly , conjugate 6 and 7 share extensive chemical similarities , indicating that the disposition of the ligand presentation on the scaffolds can exert a significant influence on the mechanism of action . conjugate 6 did not promote ar nuclear localization or coactivator binding and inhibited dna binding . in contrast , conjugate 7 promoted ar nuclear localization and induced cell - cycle arrest through a noncompetitive mode of action . the modularity of peptoid synthesis establishes a versatile chemical platform to generate an array of three - dimensional architectures to target and modulate the activity of different biomolecular targets . generation of peptidomimetic conjugates capable of antagonizing ar via distinct mechanisms of action could circumvent drug resistance in ar pharmacology . peptoids offer a chemical platform that can be utilized to optimize biological activity and hold significant promise as next generation therapeutics for prostate cancer . the estrogen receptor ( er ) has a well - characterized mechanism of action . it is known that native ligand ( estradiol ) binding to the er ligand binding domain induces a conformational rearrangement that promotes dimerization , as determined by site - specific mutational analysis . additionally , x - ray crystal structures of er dimers in the presence of ligand and other er modulators have been reported , establishing a template for molecular design by elucidating the structural parameters of the er dimer complex . pioneering work from the katzenellenbogen lab has probed er function with various bivalent conjugates tethered by different linkers . using high - resolution structural information , the first steroidal constructs aimed toward targeting the er dimer initial studies focused on developing a correlation between linker length and binding affinity . the authors concluded that bivalent conjugates incorporating a 35 linker were most suitable for enhancing er binding affinity . steroidal bivalent conjugates modulate estrogen receptor ( er ) activity through bivalent binding interactions . crystal structure of the er ligand binding domain ( gray ribbon , pdb code 1ere ) is bound to native ligand ( estradiol , red spheres ) . nonsteroidal bivalent conjugates that induce agonistic and antagonistic er conformations ( figure 10 ) were designed and synthesized in order to distinguish intra- from intermolecular binding events . bivalent agonist conjugates displayed weak binding affinity , presumably due to burial of the hydrophilic linker within the protein interior . in contrast , it was determined that antagonist conjugates incorporating a 14.4 linker induced an intramolecular binding event ( i.e. , one targeting moiety optimized for competitive binding and the other for binding to additional hydrophobic pockets such as activation function 2 , figure 11 ) . additionally , a 29 linker was found to induce an intermolecular binding event . increases in linker length above 29 resulted in reducing binding affinities , presumably due to unfavorable entropic effects . importantly , most antagonistic nonsteroidal bivalent conjugates were more potent at inhibiting cell proliferation in breast cancer cells ( mcf7 ) than a monovalent pharmacophore control . er conformation is dependent upon ligand binding : ( a ) er bound in an agonist conformation ( gray ribbon ; diethylstilbestrol , colored spheres ; helix-12 in orange ; coactivator peptide in red ; pdb code 3erd ) ; ( b ) er bound in an antagonist conformation ( gray ribbon ; hydroxytamoxifen , colored spheres ; helix-12 in orange ; pdb code 3ert ) . diagram depicting intra- and intermolecular er binding events that are dependent on linker length . a critical objective from a molecular design approach is the ability to induce different er conformations that are dependent upon ligand binding . as discussed above , the conformation induced upon agonist or antagonist ligand binding to er ( figure 10 ) plays a critical role in the biological outcome . in an antagonist conformation , an intra- or intermolecular binding event can occur between two distinct targeting moieties . an open question is whether structure - based design can be utilized to generate heterobifunctional conjugates that target two distinct binding sites on ar ( i.e. , one targeting moiety optimized for competitive binding at the ligand binding domain and the other for binding to an additional hydrophobic pocket , such as af-2 or bf3 ) . over the past decade , targeting canonical or membrane - associated ar with heterobifunctional or multivalent constructs displaying anti - androgen drug ligands has emerged as a potential family of therapeutics . these compound classes hold great promise as effective therapeutic agents due to their ability to modulate ar activity through unique mechanisms of action . because of the large number of reports , we highlight only a few representative examples of promising strategies that have been used to target ar with nonsteroidal conjugates . recently , the oyelere lab reported a nonsteroidal heterobifunctional conjugate outfitted with histone deacetylase inhibitors ( figure 12a ) . histone deacetylase inhibitors show great promise in preclinical cancer models , but their inability to selectively target malignant tissue has restricted therapeutic development . by conjugating histone deacetylase inhibitors to nonsteroidal anti - androgen ligands , selective modulation of ar activity at concentrations lower than clinical anti - androgens was achieved . these results introduce a novel method to antagonize the ar and pave the way for next generation therapeutics . nonsteroidal conjugates targeting ar : ( a ) chemical structure of heterobifunctional conjugate displaying histone deacetylase inhibitor linked to a nonsteroidal antiandrogen ligand ; ( b ) chemical structure of heterobifunctional conjugate displaying doxorubicin linked to a nonsteroidal antiandrogen ligand ; ( c ) schematic depiction of a multivalent gold nanoparticle displaying nonsteroidal antiandrogen ligands that target membrane - associated ar . figure is obtained from refs ( 98 ) , ( 99 ) , and ( 101 ) . in similar studies , the koch lab reported a nonsteroidal heterobifunctional conjugate containing doxorubicin , a nonselective cytotoxic therapeutic dna intercalator used in the clinic ( figure 12b ) . to enhance selectivity , doxorubicin was conjugated to a nonsteroidal anti - androgen ligand through a salicylamide linker that can be hydrolyzed ( t1/2 = 57 min under physiological conditions ) to yield a doxorubicin formaldehyde schiff base . the anti - androgen conjugate successfully delivered the doxorubicin - formaldehyde schiff base to cells overexpressing ar , highlighting the ability of this approach to enhance selectivity by releasing the pharmacophore in prostate cancer cells . lastly , the el - sayed lab introduced the first nonsteroidal multivalent conjugates that selectively target membrane - associated ar ( figure 12c ) . bicalutamide was conjugated to gold nanoparticles , generating architectures that display approximately ( 2.25 0.02 ) 10 ligands / particle . the multivalent compounds enhanced potency by 1 order of magnitude , in comparison to the monovalent ligand , in prostate cancer cells . these results establish that conjugation of numerous copies of a known pharmacophore to a molecular scaffold can significantly increase antiproliferative effects and may overcome resistance that arises from monovalent treatment . there is a growing appreciation for the design of potent and selective therapeutic agents targeting the ar for prostate cancer patients . targeted drug therapy is beginning to play a pivitol role in new drug discovery efforts . classically , small molecules identified via chemical screening efforts have been considered to offer a relatively straightforward path for clinical implementation . in certain cases , extensive high - resolution structural information enables structure activity relationship profiles that can be utilized for optimization , facilitating translation into the clinic . unfortunately , their therapeutic responses can be short - lived because of acquired resistance . the studies highlighted in this review indicate how new chemical entities are being designed to engage ar with high potency . additional preclinical studies will be required to validate their potential for clinical translation . in many cases , it will be necessary to evaluate critical parameters such as selectivity , in vivo potency , and binding affinity . as discussed , chemical modifications at certain positions on the steroid core can result in diminished binding affinities , potentially limiting their utility in ar pharmacology . these molecular architectures have been demonstrated to elicit potent biological responses and more importantly target the ar in novel ways . in the future , we may begin to see examples of monodisperse homo- and heterogeneous bivalent or multivalent displays in which high - resolution structural data enable evaluation of structure activity relationships that have propelled many small molecule drug discovery efforts . more importantly , heterobifunctional displays will likely be designed to target two distinct binding sites on ar , enhancing potency and establishing new modes of ar antagonism . these constructs could potentially address the challenge of overcoming resistance in prostate cancer patients .

backgroundcatheter - related bloodstream infection ( crbsi ) is a life - threatening condition encountered in patients with long - term central venous catheter ( cvc ) indwelling . the objective was to investigate the clinical characteristics , treatment , and prognosis of crbsi in the intensive care unit ( icu ) in a chinese center , as well as the risk factors for early crbsi.material/methodsa total of 73 crbsi patients were retrospectively studied in relation to patients clinical and epidemiological data , microbiological culture , and treatment . patients were treated at the taizhou hospital of integrated traditional chinese and western medicine in zhejiang ( zhejiang wenlin , china ) between january 2010 and december 2012.resultsin this chinese center , the most common pathogens were gram - positive cocci , followed by gram - negative bacilli and fungi . a high prevalence of antibiotic - resistant pathogens was detected , and a higher percentage of non - candida albicans spp . was observed . multivariate analysis showed that an acute physiology and chronic health evaluation ii ( apache ii ) score > 20 and > 3 types of underlying diseases were independent factors associated with crbsi occurring within 14 days of cvc indwelling . untimely cvc removal and/or inappropriate use of antibiotics led to significantly longer time to defervescence and time to negative conversion of blood culture ( all p<0.05).conclusionsin this chinese center , gram - positive bacteria are predominantly detected in crbsi . apache ii score > 20 and the presence of > 3 types of diseases were associated with earlier crbsi onset . timely removal of cvc and appropriate use of antibiotics resulted in improved outcomes . the androgen receptor ( ar ) is a major therapeutic target in prostate cancer pharmacology . progression of prostate cancer has been linked to elevated expression of ar in malignant tissue , suggesting that ar plays a central role in prostate cancer cell biology . potent therapeutic agents can be precisely crafted to specifically target ar , potentially averting systemic toxicities associated with nonspecific chemotherapies . in this review , we describe various strategies to generate steroid conjugates that can selectively engage ar with high potency . analogies to recent developments in nonsteroidal conjugates targeting ar are also evaluated . particular focus is placed on potential applications in ar pharmacology . the review culminates with a description of future prospects for targeting ar .

for patients with acute coronary syndrome ( acs ) , the key to increased survival and the preservation of myocardial function is rapid diagnosis followed by appropriate early intervention , which will enable timely management strategies and intervention to be carried out . delays at any level of management , from the prehospital setting to the emergency department ( ed ) and , subsequently , to the cardiac catheterisation laboratory , must be minimised . the diagnosis of acute myocardial infarction requires the combination of a good and prompt clinical history , a 12-lead electrocardiogram ( ecg ) and also appropriate cardiac markers ( in cases of st - elevation ami [ stemi ] , the results of cardiac markers will not be required for decisions on management and intervention ) [ 15 ] . prehospital 12-lead ecgs are now being performed and transmitted to eds to save precious time in diagnosis . institutions set targets where the door - to - ecg time should not exceed 10 min , the door - to - needle time must be within 30 min and the door - to - intervention time should be within 90 min of arrival [ 611 ] . the acquisition of a 12-lead ecg is an important step to assess the cardiac status in routine and emergency situations . in a cardiac emergency , the time and accuracy of information are important elements that have a critical effect on patient outcomes . accurate ecg measurements are essential for diagnostic purposes , as well as serial comparison to evaluate changes in cardiac status and help in the prediction of future events . in clinical practice , the relevance of the ecg depends upon its reproducibility in an individual . with all of these points in mind the application of single - lead electrodes to the torso for standard 12-lead ecg acquisition takes time . a relatively new praecordial v1v6 , v - quick patch , has been developed and it provides a one - piece template for the placement of all six praecordial leads , which makes application easy . the person performing the 12-lead ecg need only to reach out for one single item to be applied to the patient s torso , instead of six separate pieces of items , as in the traditional case . the patch has characteristics that enable it to conform to variations in thoracic dimensions while retaining the ability of providing accurate electrode placement . training in the use of this new patch requires a minimal amount of time , as the template provides cues to the user on placement as it is being performed . the v - quick patch has the potential to be used anywhere and in any setting where a 12-lead ecg is carried out . this study will assess : the time required to obtain a 12-lead ecg using the v - quick patch system versus the standard single - electrode systemthe agreement between the 12-lead ecgs acquired by both techniques as assessed by two assessors the time required to obtain a 12-lead ecg using the v - quick patch system versus the standard single - electrode system the agreement between the 12-lead ecgs acquired by both techniques as assessed by two assessors the null hypothesis would be that there is no difference between the times taken and the level of agreement or quality of the ecgs acquired by the two different techniques . one hundred and fifty each of healthy male and female volunteers , at least 18 years of age , with various body builds and weights were enrolled . any volunteer who was incidentally found to have significant ecg changes were given an appointment with a cardiologist for the necessary evaluation . preparation consisted of cleaning the skin with alcohol wipes at the site for lead placement . electrodes were placed in the routine standard positions for the single - electrode 12-lead ecg . following the removal of the standard electrodes , the v - quick template was attached to the praecordium for the acquisition of the next 12-lead ecg . this was done using their unique identification number ( in ) and if this number ended with an even digit , they would have the v - quick technique ecg performed first . the volunteers may experience some minor dermatological effects from the applications and removal of the electrodes the time was measured with a stopwatch from the instance the lead application commenced for both techniques . for the v - quick technique , this would be from the moment the staff tears the pack open and for the standard method , it would be from the time the staff picks up the card of the single - electrode adhesive . the stopwatch was stopped once the last electrode had been applied satisfactorily and the ecg printout was complete . the two ecgs acquired by the different techniques , on the same volunteer , was performed by the same staff . the two ecgs were then compared in a blinded fashion by two senior emergency physicians . both the inter - assessor ( consistency between the two readers of the ecgs ) and intra - assessor ( same interpretation for both ecgs in the same volunteer ) agreement were analysed . the following cases were excluded : known electrolytes abnormalities or ingestion of medication which had the potential to cause electrolyte imbalance ( e.g. diuretics , potassium supplements)congestive heart failurehistory of renal and hepatic impairmentacute illness on the day that the ecgs were to be acquiredingestion of medication which could affect the heart rate and rhythmthe presence of dermatological disease which may affect the skin sites for electrode applicationimplanted pacemaker or automated implanted cardiac defibrillator ( aicd ) known electrolytes abnormalities or ingestion of medication which had the potential to cause electrolyte imbalance ( e.g. diuretics , potassium supplements ) congestive heart failure history of renal and hepatic impairment acute illness on the day that the ecgs were to be acquired ingestion of medication which could affect the heart rate and rhythm the presence of dermatological disease which may affect the skin sites for electrode application implanted pacemaker or automated implanted cardiac defibrillator ( aicd ) these patients were excluded because this was the first time we were using the v - quick device in singapore and there was no prior literature available on its use in asian patients and also because of how long it was going to take our nurses to obtain the 12-lead ecg . despite the null hypothesis of no difference for both techniques , we decided to first perform this trial on a normal , healthy cohort of volunteers . in the next phase , we plan to include all patients requiring ecg presenting to the ed . an agilent pagewriter 300pi and hewlett packard pagewriter xli was used to obtain all of the ecgs . these machines utilise a computerised analysis system and were the ones used in the ed . the v - quick patch system is packaged with one guide template with the four replaceable limb lead electrodes . the electrodes were pre - gelled for a simple peel - and - stick application . the guide template has the flexibility to accommodate different body sizes and geometries by allowing the movement of each ( praecordial ) electrode along the guide to attain the best suit for the recommended position ( figs . 1 and 2 ) . 2the standard single - lead electrocardiogram ( ecg ) system the v - quick patch placement system the standard single - lead electrocardiogram ( ecg ) system spss version 10.0 ( spss inc . p - values and confidence intervals were analysed using student s t - test . in the collection of data , some of the information gathered included : bra cup size for ladies : this was thought to be a good subjective assessment of breast size and volumeshirt size for men : this would give an approximate indication of the shoulder widthdistance between leads v2v4 and v4v6 : these measurements may indicate how broad the chest is or the expanse of the chest over the area where the ecg electrodes are placed bra cup size for ladies : this was thought to be a good subjective assessment of breast size and volume shirt size for men : this would give an approximate indication of the shoulder width distance between leads v2v4 and v4v6 : these measurements may indicate how broad the chest is or the expanse of the chest over the area where the ecg electrodes are placed the ecgs were analysed and compared in a blinded fashion by the two assessors . the following were examined : heart rate and rhythmspecial patterns , such as bundle branch block and ventricular hypertrophyr - wave amplitude and r - wave progressions - wave depthpresence of a q - wave and its morphology heart rate and rhythm special patterns , such as bundle branch block and ventricular hypertrophy r - wave amplitude and r - wave progression presence of a q - wave and its morphology the study was approved by the institutional review board and each volunteer was given a written informed consent form to sign for participation in the study . one hundred and fifty each of healthy male and female volunteers , at least 18 years of age , with various body builds and weights were enrolled . any volunteer who was incidentally found to have significant ecg changes were given an appointment with a cardiologist for the necessary evaluation . preparation consisted of cleaning the skin with alcohol wipes at the site for lead placement . electrodes were placed in the routine standard positions for the single - electrode 12-lead ecg . following the removal of the standard electrodes , the v - quick template was attached to the praecordium for the acquisition of the next 12-lead ecg . this was done using their unique identification number ( in ) and if this number ended with an even digit , they would have the v - quick technique ecg performed first . the volunteers may experience some minor dermatological effects from the applications and removal of the electrodes the time was measured with a stopwatch from the instance the lead application commenced for both techniques . for the v - quick technique , this would be from the moment the staff tears the pack open and for the standard method , it would be from the time the staff picks up the card of the single - electrode adhesive . the stopwatch was stopped once the last electrode had been applied satisfactorily and the ecg printout was complete . the two ecgs acquired by the different techniques , on the same volunteer , was performed by the same staff . the two ecgs were then compared in a blinded fashion by two senior emergency physicians . both the inter - assessor ( consistency between the two readers of the ecgs ) and intra - assessor ( same interpretation for both ecgs in the same volunteer ) agreement were analysed . the following cases were excluded : known electrolytes abnormalities or ingestion of medication which had the potential to cause electrolyte imbalance ( e.g. diuretics , potassium supplements)congestive heart failurehistory of renal and hepatic impairmentacute illness on the day that the ecgs were to be acquiredingestion of medication which could affect the heart rate and rhythmthe presence of dermatological disease which may affect the skin sites for electrode applicationimplanted pacemaker or automated implanted cardiac defibrillator ( aicd ) known electrolytes abnormalities or ingestion of medication which had the potential to cause electrolyte imbalance ( e.g. diuretics , potassium supplements ) congestive heart failure history of renal and hepatic impairment acute illness on the day that the ecgs were to be acquired ingestion of medication which could affect the heart rate and rhythm the presence of dermatological disease which may affect the skin sites for electrode application implanted pacemaker or automated implanted cardiac defibrillator ( aicd ) these patients were excluded because this was the first time we were using the v - quick device in singapore and there was no prior literature available on its use in asian patients and also because of how long it was going to take our nurses to obtain the 12-lead ecg . despite the null hypothesis of no difference for both techniques , we decided to first perform this trial on a normal , healthy cohort of volunteers . in the next phase , we plan to include all patients requiring ecg presenting to the ed . an agilent pagewriter 300pi and hewlett packard pagewriter xli was used to obtain all of the ecgs . these machines utilise a computerised analysis system and were the ones used in the ed . the v - quick patch system is packaged with one guide template with the four replaceable limb lead electrodes . the electrodes were pre - gelled for a simple peel - and - stick application . the guide template has the flexibility to accommodate different body sizes and geometries by allowing the movement of each ( praecordial ) electrode along the guide to attain the best suit for the recommended position ( figs . 1 and 2 ) . 2the standard single - lead electrocardiogram ( ecg ) system the v - quick patch placement system the standard single - lead electrocardiogram ( ecg ) system p - values and confidence intervals were analysed using student s t - test . in the collection of data , some of the information gathered included : bra cup size for ladies : this was thought to be a good subjective assessment of breast size and volumeshirt size for men : this would give an approximate indication of the shoulder widthdistance between leads v2v4 and v4v6 : these measurements may indicate how broad the chest is or the expanse of the chest over the area where the ecg electrodes are placed bra cup size for ladies : this was thought to be a good subjective assessment of breast size and volume shirt size for men : this would give an approximate indication of the shoulder width distance between leads v2v4 and v4v6 : these measurements may indicate how broad the chest is or the expanse of the chest over the area where the ecg electrodes are placed the ecgs were analysed and compared in a blinded fashion by the two assessors . the following were examined : heart rate and rhythmspecial patterns , such as bundle branch block and ventricular hypertrophyr - wave amplitude and r - wave progressions - wave depthpresence of a q - wave and its morphology heart rate and rhythm special patterns , such as bundle branch block and ventricular hypertrophy r - wave amplitude and r - wave progression presence of a q - wave and its morphology the study was approved by the institutional review board and each volunteer was given a written informed consent form to sign for participation in the study . the mean ages for the male and female volunteers were 4310.8 and 5212.7 years , respectively , with the female volunteers having a higher body mass index ( bmi ; 26.54.2 versus 24.35.8 , p=0.0003 ) ( table 1 ) . the race distribution is also shown in table 1 . table 1demographic and comparison data between the male and female volunteers male ( n=150)female ( n=150)age range19682372mean age4310.85212.7racechinese10895malay2015indian1827other413bmi range18.939.517.538.8mean24.35.826.54.2standard methodmean time ( s)58.610.866.012.2range44.968.554.082.1v - quick methodmean time ( s)40.56.750.25.6range35.145.941.353.9v2v4 distance ( cm)12.49.2v4v6 distance ( cm)10.69.8range of shirt size1524 range of bra cup size a : 20b : 47c : 69d : 14 demographic and comparison data between the male and female volunteers when comparing the mean time taken for both techniques , the v - quick method proved to be faster in both the male ( 40.56.7 versus 58.610.8 s , p<0.0001 ) and female volunteer ( 50.25.7 versus 66.012.2 s , p<0.0001 ) groups ( table 2 ) . even when the male and female volunteers were taken together ( i.e. all 300 volunteers were compared for the two techniques ) , the v - quick method was also shown to be significantly faster ( 45.43.8 versus 61.93.5 s , p<0.0001 ) ( table 2 ) . table 2comparison of the methods with p - values standard methodv - quick methodp - values ( 95% ci)mean time for all 300 volunteers ( s)61.93.545.43.8p<0.0001 ( 15.922 , 17.078)mean time for 150 female volunteers ( s)66.012.250.25.7p<0.0001 ( 13.636 , 17.964)mean time for 150 male volunteers ( s)58.610.840.56.7p<0.0001 ( 16.058 , 20.142 ) comparison of the methods with p - values when the different methods were compared between the genders ( table 3 ) , the v - quick method was shown to be significantly faster in male volunteers compared to female volunteers . the same results were also noted for the standard method ; it was faster to acquire the 12-lead ecg by the standard method in male volunteers . table 3comparison between genders of the two methods malefemalep - values ( 95% ci)mean time for standard method ( s)58.610.866.012.2p<0.0001 ( 10.018 , 4.782)mean time for v - quick method ( s)40.56.750.25.7p<0.0001 ( 11.113 , 8.287 ) comparison between genders of the two methods in the blinded analyses of the ecgs , there was 100% agreement between the two assessors when comparing ecgs of the same person ( in both male and female volunteers ) acquired by the two different techniques ( intra - assessor agreement ) and when each ecg was read by the two assessors separately ( inter - assessor agreement ) . the ecg continues to be a critical component of the evaluation of patients with emergency cardiac symptoms . this tool is now more than a hundred years old and has been a standard in clinical practice for more than half a century . the applications of new signal processing techniques and the expansion of the use of additional leads and innovative techniques have offered more information on cardiac electrical activity . even though it is one of the most common tests being performed today , there are hardly any published studies on the time taken to acquire it . often , ecgs are acquired by ecg technicians or nurses who , over the years , have become very versatile and familiar with the importance of accurate lead placement according to the thoracic landmarks . there has also been developments with the aim of making praecordial lead placement easier and faster . the v - quick technique showed a favourable result from this study in terms of timing , as well as agreement with the standard 12-lead ecg in 300 normal volunteers . the technique is simple and the skill relatively easy to acquire when one already knows the concept of the 12-lead ecg . the fact that the template is manufactured to fit the average standard size adult torso will make adjustment rarely necessary . however , having said that , the onus will still lie with the operator carrying out the ecg to strive for the most accurate placement . besides accuracy , timing is the other important factor , as we all try to meet the targets of door - to - ecg time at our institutions . it is important to highlight that there are multiple factors that affect this timing : how busy the department is at any particular point in time , the first contact time or triage time , manpower and staffing issues , as well as the availability of ecg machines throughout the ed , among other factors . it is , thus , crucial for each institution to look at the wider picture when trying to address timing issues to meet key performance indicators ( kpis ) . in female volunteers , the longer time required can be explained by the presence of breast tissue , which requires more attention to be paid to the standard landmarks and whether the leads should be placed over or under the breast tissue . comparatively , with one template to deal with , it is still faster to apply the v - quick system rather than to apply the leads singly over the praecordium , as shown in this study . in this study , the v - quick system gave a stable baseline on ecg recordings , unlike some techniques such as the ecg belt , where this was a common problem . praecordial electrode position is important , as the variability in placement may result in ecg changes . here , the nurses performing the ecgs on every individual marked the points of application with a skin marker to reduce the variability in placement between the two techniques . . there has been increasing practice to place the limb leads on the torso itself , as this has been thought to reduce the application time . however , it has been shown to have significant amplitude and waveform changes when compared to the ecg acquired with the limb leads placed in the usual recommended limb positions [ 19 , 20 ] . this trial was a first of its kind conducted in singapore using the v - quick patch system . looking at the results and also from past experience of the utilisation of the system , it appears to be very useful for consideration for use in the prehospital environment , where conditions are more unpredictable and stressful . as it is relatively easy to apply , using it whilst in a moving ambulance will also be less of a challenge when compared to the application of the small , single adhesives that the paramedics would have to apply at six different sites over the praecordium . many prehospital patients are also cold , clammy and sweaty and , very often , the adhesives from these small electrodes do not work well . this is where the one , single , larger template of the v - quick system would be a plus point . we will , in future , consider a trial using the v - quick system in all patients who present to the ed and also the prehospital group of patients requiring a 12-lead ecg . the v - quick patch was manufactured by a us - based company and it is likely the standardisation of the template was for the average american torso size . in this study , the template system was applied to asian volunteers . in the application , adjustments had to be made to the template , thus , creating more this may have been due to the slightly smaller chest expanse of the average asian compared to their caucasian counterpart . in the data collection , the distances between v2v4 and v4v6 were noted , as these were thought to be useful as a simple measure of chest or thoracic expanse . the shirt size may too be a reflection of this , but may be less constant or objective , as it would be very much affected by personal choice . the same goes for female volunteers , where the bra cup size may reflect breast tissue size and volume . if such similar studies were to be conducted in the caucasian population , it would be interesting to see the results and compare them to ours . in this trial , the nurses performing the ecgs were not blinded , as this would have been impossible to achieve . it would have been optimal to do so , except for the fact that , logistically , it was rather challenging and would have incurred the extra cost of videotaping . in this study of 300 male and female volunteers , the v - quick patch system was proved to be significantly faster than the single - electrode standard 12-lead system for the acquisition of electrocardiograms ( ecgs ) . the time taken was also faster in male compared to female volunteers for the reasons discussed . phrynoderma , meaning toad skin , is a type of follicular keratosis coined and described by nocholls in 1933 . various nutritional deficiencies such as vitamin ( vit ) a , vit b - complex , vit e and essential fatty acid ( efa ) deficiency , as well as protein - calorie malnutrition have been suggested as possible etiological factors . most of the studies on phrynoderma were conducted in the early and middle of the last century . therefore , clinical features of phrynoderma and its association with nutritional deficiency signs were studied to elucidate the relationship between phrynoderma and nutritional status of patients . a cross - sectional descriptive study of 125 consecutive phrynoderma patients attending the outpatient department ( opd ) of dermatology was conducted in a tertiary care hospital during a period of two years . in all patients , a detailed history with particular reference to age , gender , seasonal variations , socioeconomic status , and family history of disease and cutaneous examination findings such as distribution , sites of involvement , morphology of the lesions , and signs of nutritional deficiencies were noted . phrynoderma was diagnosed clinically if a patient presents with discrete , brown or skin colored , acuminate , keratotic papules with central keratin plug predominantly distributed over elbows , knees , extensor extremities , and/or buttocks . all patients with phrynoderma examined by the investigator , irrespective of age and gender , were included in the study . the proportion of patients with phrynoderma attending opd was 0.51% . among 125 patients , 79 ( 63.2% ) were male and 46 ( 36.8% ) , female . the age of the patients was in the range of 3 - 26 years , with a mean of 10 4.3 years [ table 1 ] . a majority of the patients ( 88% ) were from low socioeconomic group and the remaining ( 12% ) were from middle socioeconomic group . phrynoderma was common in students ( 94.4% ) , followed by lactating mothers ( 4.8% ) and laborers ( 0.8% ) . the disease was recurrent in 17 ( 13.6% ) patients . during a calendar year , 8.33% of the patients presented during summer , 46.7% in the rainy , and 45% in winter season . age distribution of phrynoderma cases the lesions were asymptomatic in 114 ( 91.2% ) patients and mild itching was present in 11 ( 8.8% ) patients . the disease was localized ( elbows , knees , extensor extremities , and/or buttocks ) in 106 ( 84.8% ) patients and generalized ( trunk and/or face ) in 19 ( 15.2% ) patients . elbows and buttocks alone were affected in 16 ( 12.8% ) and 2 ( 1.6% ) patients , respectively [ table 2 ] . the site of onset was elbows in 106 ( 84.8% ) patients , followed by knees ( 8% ) , buttocks ( 4% ) , and extensor extremities ( 4% ) . in all ( 100% ) patients , the lesions were discrete , keratotic , acuminate , and brown to skin colored papules with central keratinous plug [ figure 1 ] . the surrounding skin was dry and scaly in 44 ( 35.2% ) patients and pigmented in 72 ( 57.6% ) patients . various diseases or conditions sites of involvement in phrynoderma discrete brown to skin colored keratotic , follicular , acuminate papules with central keratinous plug localized to elbows associated diseases or conditions in phrynoderma histopathological changes in phrynoderma phrynoderma is a disease occurring in children and adolescents aged between 5 and 15 years . the disease is uncommon ( < 5% ) in children below 5 years of age . in the present study , thus , increased nutritional demand during childhood and lactation , and the lack of good nutrition due to low socioeconomic status may be responsible for occurrence of phrynoderma in these patients . the proportion of phrynoderma cases in the present study is less compared to other studies where it was found to be 1.3% , 3% , and 5% . these studies were conducted in the middle of last century . the decline in the proportion of phrynoderma patients over the past few decades therefore , nutrition seems to play an important role in the pathogenesis of phrynoderma . in many studies including the present one , the family history of phrynoderma was very low ( 0% , 3.57% , and 5% ) . the absence of the disease in siblings who are also taking the same diet suggests the role of other factors in the manifestation of phrynoderma . the distribution and site of onset of lesions indicate importance of pressure and friction in the development of lesions . the incidence of phrynoderma is higher in the cooler months of the year , as is documented in the present study . the flare up of the disease duirng winter may be due to follicular prominence , which generally occurs in otherwise normal children during cold weather . the typical case of phrynoderma presents with bilaterally symmetrical discrete , keratotic , follicular , brown or skin colored , acuminate papules with central keratinous plug localized to elbows , knees , buttocks , and extensor extremities . in generalized disease these distinct clinical features help in differentiating phrynoderma from other common follicular keratotic disorders such as keratosis pilaris , lichen spinulosis , pityriasis rubra pilaris , and follicular lichen planus . although the occurrence of phrynoderma has been related to nutritional status of the patient , in the present study , the signs and symptoms of nutritional deficiency the ocular manifestations of vit a deficiency in patients with phrynoderma have been reported to be 5% . it has also been demonstrated that the incidence of phrynoderma is less where the prevalence of signs of vit a deficiency is high and vice versa . extremely low levels of serum vit a , 0.1 mol / l ( normal , 1.4 - 4 mol / l ) has been reported secondary to vit a malabsorption , following small bowel bypass surgery for obesity , colectomy , and in pancreatic insufficiency . diet survey and serum vit a levels in apparently healthy phrynoderma patients and normal controls revealed no statistically significant difference between the two groups . the vit a deficiency as a cause of phrynoderma was largely based on the therapeutic response to cod liver oil , which was considered to be a source of vit a. however , later it has also been demonstrated as a source of efa . the patients presenting with signs of vit b - complex deficiency were less in number when compared to other studies ( 47.6% , 50% , 65% , 92% , and 25% ) . none of the patients in the present study presented with nutritional deficiency signs of efa , vit e , and vit c or protein calorie malnutrition . regarding efa intake in india , the computation based on revised figures of fat content in cereals and pulses has shown that the invisible fat present even in the poorest of indian diet provides minimum amount of linoleic acid needed , ie , 3% of total energy . the ratio of eicosatrienoic acid and arachidonic acid > 0.4 is considered as an accurate indicator of efa deficiency . the studies showing efa deficiency in phrynoderma patients used alkaline isomerization ( ai ) method for estimation of fatty acid levels . in ai , the fatty acids are estimated according to the number of double bonds and their chain length is not taken into consideration , thereby overestimating the dienoic and trienoic acids , and underestimating the tetraenoic and pentaenoic acids . this results in high trienoic to tetraenoic acid ratio . in gas exchange chromatography ( gc ) method , fatty acids are estimated according to both double bonds and chain length . thus , the ratio obtained by gc method reflects efa deficiency . in phrynoderma patients , the levels of linoleic acid and phospholipids ( sensitive indicators of efa deficiency ) , and ratio of linoleic to arachidonic acid ( indicator of efficient conversion of linoleic acid to arachidonic acid ) were also normal in phrynoderma patients . the cutaneous manifestations of riboflavin , pyridoxine , niacin , and efa deficiency states have a number of similarities . riboflavin is required for pyridoxine metabolism , which , in turn , is required for tryptophan - niacin metabolism , and niacin is necessary for fatty acid synthesis . the combination therapy of vit b - complex and efa or vit b - complex and vit e have shown better results when compared to single drug therapy . the serum levels of linoleic acid and vit e also increased significantly during the combination therapy . vitamins of b - complex group when used alone failed to show any therapeutic response . in addition , deficiency of a single vitamin of b - complex group is rare because poor nutritional diets or malabsorption are more often associated with multiple nutritional deficiencies . impaired balance or threshold levels of these nutrients with or without a deficiency state seem to alter the intersecting biochemical pathways and the local milieu of these nutrients resulting in phrynoderma . this can be compared with the formation of comedones in acne where a low level of linoleic acid in sebum has been attributed in the pathogenesis of follicular hyperkeratinization . follicular hyperkeratosis with keratin plugging along with epidermal hyperkertosis and acanthosis are the characteristic features of phrynoderma . the clinical and histopathological skin changes closely resembling those of phrynoderma have been noted in rats , which were fed with fat - deficient diet . hence , an abnormality in the efa metabolism in the pilosebaceous unit caused by vit b - complex appears to be the important step in the pathogenesis of phrynoderma . the main drawback of the present study is lack of biochemical evidence of nutritional status ( serum levels of nutrients ) of patients . these investigations were not done because of their non - availability in a resource poor set - up . normal serum levels of nutrients ( vit a , vit b - complex , efa ) have been reported in phrynoderma patients . however , with the available literature and our findings , it can be hypothesized that phrynoderma may be a multifactorial disease involving multiple nutrients , local factors like pressure and friction , and environmental factors manifesting in the setting of increased nutritional demand . further prospective case - control studies investigating the levels of vit b - complex , vit a , vit e , and efa in the blood and skin , and also in relation to the treatment are required to delineate the exact pathogenesis of phrynoderma .

introductionthe v - quick patch template system is compared with the standard 12-lead electrocardiogram ( ecg ) acquisition technique in this paper . the objectives of the study were : ( a ) to study and compare the time taken to produce the printed 12-lead ecg and ( b ) to look at the level of agreement when the ecgs were compared by two blinded , independent assessors.methodsone hundred and fifty each of male and female volunteers signed an informed consent form to participate in the clinical study . nurses were put through a 4-h training session to familiarise themselves with the v - quick patch system . the timings were measured with a stopwatch with the specific start and end points defined . the final ecg printouts were then compared by two blinded , independent assessors for several set criteria.resultsthe v - quick patch system was proved to be significantly faster than the standard 12-lead system in the acquisition of the ecg in both male and female volunteers . the time taken in male volunteers was also noted to be significantly faster than in female volunteers.conclusionthe two assessors shared a 100% agreement level when comparing the ecgs acquired by both techniques in the same individual ( intra - assessor agreement ) and when each ecg was read by the two assessors separately ( inter - assessor agreement ) . background : phrynoderma is a type of follicular hyperkeratosis . various nutritional deficiency disorders have been implicated in the etiology of phrynoderma.aim:to determine clinical features of phrynoderma and its association with nutritional deficiency signs.materials and methods : a cross - sectional descriptive study of 125 consecutive patients with phrynoderma attending the outpatient department ( opd ) of dermatology was conducted in a tertiary care hospital . in all patients , a detailed history was taken and cutaneous examination findings such as distribution , sites of involvement , morphology of the lesions , and signs of nutritional deficiencies were noted.results:the proportion of patients with phrynoderma attending the opd was 0.51% . there were 79 males and 46 females . age of the patients was in the range of 3 - 26 years with a mean of 10 4.3 years . the lesions were asymptomatic in 114 ( 91.2% ) patients . the distribution of lesions was bilateral and symmetrical in 89 ( 71.2% ) patients . the disease was localized ( elbows , knees , extensor extremities , and/or buttocks ) in 106 ( 84.8% ) patients . the site of onset was elbows in 106 ( 84.8% ) patients . the lesions were discrete , keratotic , follicular , pigmented or skin colored , acuminate papules in all patients . signs of vitamin a and vitamin b - complex deficiency were present in 3.2% and 9.6% patients , respectively . epidermal hyperkeratosis , follicular hyperkeratosis , and follicular plugging were present in the entire biopsy specimen.conclusion:phrynoderma is a disorder with distinctive clinical features and can be considered as a multifactorial disease involving multiple nutrients , local factors like pressure and friction , and environmental factors in the setting of increased nutritional demand .

dieulafoy lesion is an aberrant submucosal vessel that lies in close contact with the mucous membrane , which may lead to its exposure , causing massive gastrointestinal ( gi ) bleeding.1 it is the cause of approximately 6% of upper gi bleeding.2 it is most commonly found in the proximal stomach and especially within 6 cm of the gastroesophageal junction , predominantly on the lesser curvature . however , it can occur anywhere in the gi tract , such as the small bowel.3 due to difficult access , diagnosis of small bowel bleeding is often delayed ; therefore , a multidisciplinary approach is needed to obtain its proper diagnosis and treatment . we experienced two cases of ileal dieulafoy lesion bleeding that presented with massive hematochezia and instability of vitals . the two cases were diagnosed and treated successfully using single balloon enteroscopy ( sbe ) with hemoclips . we discuss the clinical features as well as useful diagnostic and therapeutic modalities of ileal dieulafoy lesion through a computer - assisted search of the english language literature . a 47-year - old man was admitted to our hospital for hematochezia of 8 hours . he denied previous medical history as well as taking any medication such as non - steroidal anti - inflammatory drugs . on initial physical examination , initial blood hemoglobin level was 10.2 g / dl , but all other values were within normal limits . we tried to perform colonoscopy but failed due to poor bowel preparation and fresh blood . on the second day of admission , he developed hypovolemic shock due to massive hematochezia , and blood hemoglobin level dropped to 6.2 g / dl . he underwent abdominal computed tomography ( ct ) , which showed contrast media that filled the bowel lumen at the terminal ileum . the contrast media had leaked out of the vessel supplied by a branch of the right ileocolic artery of the superior mesenteric artery , and the bowel lumen around the terminal ileum was filled with contrast media ( fig . 1 ) . however , we failed to control the bleeding , because the artery was too small for embolization treatment . he received transfusions of 10 units of packed red blood cells . while he was prepared for emergency surgery , his vital signs became stabilized , so he underwent colonoscopy . we found fresh blood throughout the entire colon and terminal ileum but could not find the bleeding focus . for small bowel evaluation , we performed sbe with a retrograde approach and found the dieulafoy lesion on the distal ileum , approximately 20 cm from the ileocecal valve ( fig . for evaluation of other small bowel lesions , we performed capsule endoscopy but could not find any abnormal lesion except the previously placed hemoclips ( fig . she had obstructive coronary artery disease and took aspirin and an antiplatelet agent . on admission , blood hemoglobin level was 8.7 g / dl . on the day of admission , she underwent egd and colonoscopy , but we could not find the bleeding focus . she then underwent abdominal ct , but there was no specific lesion in the gi tract suspicious for bleeding . we recommended enteroscopy for evaluation of the obscure bleeding focus , but she refused because her melena ceased . vital signs were stabilized , and she was discharged home on the ninth admission day . six days after discharge , she revisited our hospital due to recurrent fresh hematochezia . at that time , blood pressure dropped to 88/56 mm hg , and pulse was 107 beats / min . as she had underwent egd and colonoscopy , we performed sbe with a retrograde approach . we found a fresh blood clot adherent on the mucosa of proximal ileum and , after saline irrigation , we detected the active bleeding focus at a narrow point of normal - appearing mucosa ( fig . she was discharged home and had no evidence of recurrent gi bleeding at her 2 month follow - up visit . a 47-year - old man was admitted to our hospital for hematochezia of 8 hours . he denied previous medical history as well as taking any medication such as non - steroidal anti - inflammatory drugs . on initial physical examination , initial blood hemoglobin level was 10.2 g / dl , but all other values were within normal limits . we tried to perform colonoscopy but failed due to poor bowel preparation and fresh blood . on the second day of admission , he developed hypovolemic shock due to massive hematochezia , and blood hemoglobin level dropped to 6.2 g / dl . he underwent abdominal computed tomography ( ct ) , which showed contrast media that filled the bowel lumen at the terminal ileum . the contrast media had leaked out of the vessel supplied by a branch of the right ileocolic artery of the superior mesenteric artery , and the bowel lumen around the terminal ileum was filled with contrast media ( fig . 1 ) . however , we failed to control the bleeding , because the artery was too small for embolization treatment . he received transfusions of 10 units of packed red blood cells . while he was prepared for emergency surgery , his vital signs became stabilized , so he underwent colonoscopy . we found fresh blood throughout the entire colon and terminal ileum but could not find the bleeding focus . for small bowel evaluation , we performed sbe with a retrograde approach and found the dieulafoy lesion on the distal ileum , approximately 20 cm from the ileocecal valve ( fig . for evaluation of other small bowel lesions , we performed capsule endoscopy but could not find any abnormal lesion except the previously placed hemoclips ( fig . she had obstructive coronary artery disease and took aspirin and an antiplatelet agent . on admission , blood hemoglobin level was 8.7 g / dl . on the day of admission , she underwent egd and colonoscopy , but we could not find the bleeding focus . she then underwent abdominal ct , but there was no specific lesion in the gi tract suspicious for bleeding . we recommended enteroscopy for evaluation of the obscure bleeding focus , but she refused because her melena ceased . vital signs were stabilized , and she was discharged home on the ninth admission day . six days after discharge , she revisited our hospital due to recurrent fresh hematochezia . at that time , blood pressure dropped to 88/56 mm hg , and pulse was 107 beats / min . as she had underwent egd and colonoscopy , we performed sbe with a retrograde approach . we found a fresh blood clot adherent on the mucosa of proximal ileum and , after saline irrigation , we detected the active bleeding focus at a narrow point of normal - appearing mucosa ( fig . she was discharged home and had no evidence of recurrent gi bleeding at her 2 month follow - up visit . dieulafoy lesion can be associated with massive , life - threatening hemorrhage and accounts for between 1% and 2% of cases of major gi bleeding.4 dieulafoy lesion is used to describe the finding of a large submucosal artery , which is otherwise histologically normal , lying in close contact with the mucous membrane . the etiology of this abnormally tortuous submucosal artery is unknown.5 the incidence varies from 0.5% to 14% , depending upon selection criteria.2 as documented above , the diagnosis of dieulafoy lesion is based upon classical histologic features . however , histologic evaluation is usually unavailable in most case , because recently most vascular lesions have been treated by nonsurgical modalities.6 endoscopists rely entirely on endoscopic features to make their diagnosis from a critical review of many published articles . endoscopic criteria for the diagnosis of dieulafoy lesion are : 1 ) active arterial spurting or micropulsatile streaming from a minute ( less than 3 mm ) mucosal defect or through normal - surrounding mucosa ; 2 ) visualization of a protruding vessel with or without active bleeding within a minute mucosal defect or through normal - surrounding mucosa ; or 3 ) a fresh , densely adherent clot with a narrow point of attachment to a minute mucosal defect or to normal - appearing mucosa.7 although endoscopic features could not be proven to be a true dieulafoy lesion by the historical criteria mentioned above , the endoscopic finding remains convincing for dieulafoy lesion . in one study , the authors classified vascular lesions in the small bowel into the following six groups : 1 ) type 1a , punctulate erythema ( < 1 mm ) , regardless of oozing ; 2 ) type 1b , patchy erythema ( a few mm ) , regardless of oozing ; 3 ) type 2a , punctulate lesions ( < 1 mm ) with pulsatile bleeding ; 4 ) type 2b , pulsatile red protrusion without surrounding venous dilatation ; 5 ) type 3 , pulsatile red protrusion with surrounding venous dilatation ; and 6 ) type 4 , unclassified lesions . being different in size from one another , types 1a and 1b were considered angioectasia . type 2a and 2b were considered dieulafoy lesion , and type 3 represented an arteriovenous malformation.6 according to that classification , our first case is considered to be type 2b , and the second case is 2a . angioectasia is a venous / capillary lesion and thus is likely to be treated by endoscopic cauterization . however , dieulafoy lesion and an arteriovenous malformation may cause arterial bleeding , which require endoscopic treatment with a clip or with laparotomy for large lesions . the authors concluded that this classification would be useful for selecting the hemostatic procedure.6,8 small bowel bleeding continues to be difficult to visualize directly on routine endoscopy . therefore , diagnosis of bleeding in the small bowel is often delayed . to localize a source of small bowel bleeding , multidisciplinary approaches such as abdominal ct , angiography , radionuclide scan , and capsule endoscopy is needed . with the development of endoscopy , push enteroscopy is performed to diagnose and treat a bleeding source . push enteroscopy has two types , sbe and double - balloon enteroscopy ( dbe ) , and two approaches , anterograde ( per oral ) and retrograde ( per anal ) . in some studies , sbe is as effective as dbe for the appropriate diagnosis and management of obscure gi bleeding cases by identifying a bleeding source and providing a means of treatment in a high percentage of patients.9,10 however , in some studies , dbe had a higher total enteroscopy rate than sbe accompanied by a higher diagnostic yield.11,12 enteroscopy has a low risk of complications . however , acute pancreatitis after enteroscopy is the most frequent complication and has to be taken into consideration in the written informed consent.13 several cases of ileal dieulafoy lesion bleeding have been reported . most of them were treated surgically and diagnosed with histology.14 - 16 however , endoscopic treatment will replace surgery in a significant portion of small bowel dieulafoy lesion cases . since the widespread availability of endoscopic or angiographic treatment , surgery will play a minor role and often be left as the last therapeutic option for rebleeding lesions or after failed nonsurgical treatment.16 in summary , for the appropriate evaluation of small bowel bleeding , an aggressive multidisciplinary approach , such as radiologic intervention , enteroscopy , and surgery , should be performed . with advances in endoscopic techniques , enteroscopy could play a major role in the diagnosis and treatment of ileal dieulafoy lesion bleeding . obesity stigma and negative stereotypes of obese people are widespread and damaging to the health , dignity , human rights , and quality of life of obese individuals . standard media and biomedical depictions of obese individuals contribute to this stigmatization by positing that obesity incidence is nearly entirely dependent on individualistic actions . furthermore , obese individuals ' may occupy numerous intersecting social roles and identities based on their gender , class , race , and other social positions . biomedical and media depictions invariably refer to obesity as a crisis or epidemic . obesity 's multifactorial and multilevel etiology is reduced to an energy balance model of causation , which inadequately explains weight trajectories . despite the oft - reported inefficacy of weight loss dieting , public health interventions may be unsuccessful due to a narrow focus on weight loss , an overly simplistic notion of how obese individuals live ; experience their bodies ; the contexts they inhabit ; and the opportunities available to them in seeking wellness , happiness , and a full life . furthermore , while health is posited as the ultimate goal , these projects frequently focus on weight and deem fatness or higher weights as necessarily pathological . this is especially important given that the populations often targeted by such campaigns may differ culturally and socioeconomically from dominant groups . these diverse factors may affect their lifeways , priorities , and health conceptualizations in manners which may require in - depth exploration to produce truly beneficial and sensitive programming . qualitative social scientists , such as anthropologists or sociologists , trained in methods such as ethnography , may be uniquely suited to explore the lived experiences of obese individuals . they may aid in developing a public health strategy that is suited to the priorities and lifestyles of all individuals and is implemented in a manner consistent with a salutogenic , positive , and holistic understanding of health promotion . this paper discusses the potential for in - depth , qualitative social science research to concretely contribute to program delivery . even within the expanding fields of critical obesity research , as warin and gunson note , the compiling of actual obese people 's experiences and perspectives has been limited . through actual collaboration between disciplines , rather than public health researchers and programmers and qualitative social scientists operating in separate silos importantly , rather than reliance on stereotypes , such an approach would facilitate compassionate and evidence - based policy and programming . understanding how both patients and providers view health - related topics and how these actors must negotiate these views in a care - setting context is critical to planning effective and respectful public health care delivery . biomedical research nearly invariably posits obesity as a health crisis , despite evidence that obese individuals may be metabolically healthy ; overweight people live longer than normal weight persons , as do obese individuals in chronic disease populations and fit obese persons compared to unfit normal weight persons [ 2 , 6 ] . weight bias is moderate to high among healthcare professionals and trainees , including those specializing in obesity or nutrition - related practice [ 711 ] . a recent systematic review of physicians ' views on treating adult obesity found that physicians believed it was important to treat obesity . they were confident in their skills respecting obesity treatment , although obesity knowledge was actually limited . physicians believed they were largely unsuccessful in treating obesity but attributed this to patient noncompliance and lack of motivation , which coincides with their general view of overweight and obese individuals as lazy . similar results were attained by foster and colleagues in 2 nationally representative american surveys ( n = 5000 ) . physicians felt treatment for obesity was ineffective ; held negative views of obese patients ' appearance and compliance ; attributed obesity causation to lifestyle factors ; and sought greater compensation in delivering obesity treatment . similar to other studies , physicians in new york state expressed frustration in attempting to treat obesity . this frustration was based on the extent to which obesity - contributing factors were outside their control , low sense of self - efficacy in treating obesity , and a perceived lack of reimbursement . however , they also feel they are unsuccessful in treating obesity , largely as a result of noncompliance and lack of motivation of patients . reimbursement appeared to be less of an issue regarding physicians ' views of childhood obesity . studies have also presented more nuanced views of clinicians ' attitudes relating to obesity treatment . for example , physician 's bmi may mediate physicians ' likelihood of counseling obese patients . among primary care physicians , weight loss discussions were more likely to be initiated by physicians who believed clients had a higher bmi than themselves . normal weight physicians were also more likely to feel confident administering said advice , to feel physicians were responsible for serving as normal weight role models and to doubt patients would trust weight - related advice from overweight or obese clinicians . another study conducted in new york city involved a chart review and patient survey , and its results suggest little focus on obesity in practice . it was found that physicians were relatively unlikely to enter an official diagnosis of overweight or obesity on a patient 's chart , to advice weight loss or refer to a dietitian . patients generally wanted to lose weight and receive physician advice and referral to a dietitian . a qualitative study on german physicians ' and patients ' views on obesity management found that doctors were concerned with a potential overemphasis on obesity . both physicians and patients emphasized the need for multidisciplinary approaches to obesity management , the excess burden on primary care centres , and emphasized respectful trusting relationships between practitioners and patients . the need for more services and professional involvement , delivered by physicians or other providers , either separate from or within a primary care setting , was also referred to by both groups . a recent debate in the canadian family physician journal has highlighted that practitioners may be developing a more critical view on the orthodoxy of advocating weight loss for every obese patient . bosomworth presents a review on possible negative mortality , morbidity , and quality - of - life outcomes of weight loss . it is suggested that metabolically healthy obese individuals should strive to remain weight stable , not to gain or lose weight . an accompanying editorial encourages promoting self - acceptance and healthy lifestyles for obese patients , as weight loss is nearly impossible . havrankova presents the argument that weight loss as a public health goal is futile and contends that the focus should be on preventing obesity [ 21 , 22 ] . garrel posits that obesity prevention is largely outside the practitioner 's purview and argues instead for obesity treatment [ 23 , 24 ] . based on these guidelines , garrel supports urging weight loss only in obese individuals who have comorbid conditions ; physicians should work with obese individuals without comorbid conditions to prevent weight gain [ 23 , 24 ] . treatment of obese individuals with comorbid conditions would involve treating these comorbidities , setting realistic weight goals with patients , and warning them of unsafe weight loss methods . it allows for the possibility that some obese patients may be healthy and not benefit from treatment . physicians are more likely to refer to dietitians than to gastric bypass surgeons or to prescribe medication . dietitians agree on their primacy in obesity treatment . in a sample of 514 canadian dietitians , about 90% felt obesity contributed to ill - health and a large majority felt obese individuals should be encouraged to lose weight . however , they also emphasized the importance of health measures other than weight in obesity treatment , and the majority advised their clients against weighing themselves . indeed , many dietitians were positively disposed toward a weight - neutral , health - at - every - size ( haes ) approach ; however , there was variation in plans to adopt more structured eating plans and abandoning weight loss as a goal . some also argued that certain ( larger ) sizes exceeded healthy limits . in examining clinician perspectives on obesity , what appears to be essential , therefore , is the establishment of trusting and respectful relationships between clinicians and their obese patients in designing interventions for obese persons . these relationships must be sustained in light of obesity 's likely intractability and potential nonpathological nature . a greater understanding of clinicians ' and obese patients ' health perspectives , perceptions , and priorities over the life course is paramount for achieving these aims . better understanding of the experience of visiting healthcare providers as a larger patient may also provide valuable insight into sensitive care delivery and health ( not weight)-centric treatment approaches . a greater awareness of the stigma obese individuals encounter in day - to - day life , as well as in the healthcare system , is of particular importance to improve health and quality of life . examining these issues in depth is essential to rectify a social justice issue that has immense health and quality - of - life implications for a significant population . ethnographers and other applied social science researchers may be especially adept at exploring this issue and learning from obese persons about the oppression they may endure . this discrimination occurs in both interpersonal encounters and in institutional settings such as social situations , places of employment , and health care settings . this discrimination is particularly notable among heavier individuals ( bmi > 35 kg / m ) , 40% of whom reported experiencing discrimination in the american 1995 - 96 national midlife development survey . women and younger adults were also at considerably higher relative risk of experiencing weight stigma . these findings coincide well with reports of high levels internationally of stigmatizing attitudes toward obese persons . the public emphasizes the presumed causal role of the individual in developing obesity , and this was the single strongest predictor of possessing a stigmatizing attitude . the pervasiveness of obesity stigma in health care settings is of particular importance for obese individuals ' health and public health planning [ 7 , 8 ] . awareness of these sentiments may make it especially difficult for obese individuals to find adequate medical care . social scientists could explore these issues and seek to deploy an in - depth , exploratory perspective from obese individuals ' own ( emic ) perspective . the depiction of obesity in biomedical , media , and other popular accounts contributes to the manifestation and degree of obesity stigma present among the public . gard and wright identified that obesity research was communicated to the public in a way that erases the inherent uncertainty and imprecision of epidemiological studies and presents obesity as necessarily a health crisis , despite nondefinitive evidence . as this work is generated from within a biomedical institution , it is presumed to be unquestionably objective and lacking in any moralizing , political , or ideological thrust . such a frame allows for the dismissal of alternate views as fallacious , whether they originate from alternate epistemological scholarly arenas , lay perspectives , or embodied experience . it also disqualifies concerns of how this research may affect or be affected by antifat bias . these portrayals may inadvertently silence the source most important to understanding the public health programming needs of obese individuals , that is , obese individuals themselves , by presenting a homogenizing and blaming view of such individuals ' lifestyles and experiences . these views are likely to be reliant on assumptions , rather than data on obese persons ' lived experiences . bringing forth obese individuals ' perspectives and learning from their embodied knowledge through multidisciplinary research may enhance public health programming efforts . media commentators ' particular moral values help to establish which component of life they choose to implicate as causing obesity . often these values are related and presumed to be unique to modern life , such as diminished quality family time or the slothful nature of the current generation of children . similar to these accounts are evolutionary explanations for obesity stressed in media , which may present an inaccurate understanding of the biological or archaeological evidence of humanity 's evolutionary past . regardless of the validity of such claims , these and other arguments that invoke a golden era of nonobesity and health continue to deploy a moralistic frame that denigrates modern lifestyles as decadent , slothful , and glutinous . particularly , adult commentators often direct their ire toward children as embodying the presumed deterioration of societal values and lifestyles . child health prevention programs reliant on this framing may risk stigmatizing , disempowering , and harming children , rather than on trying to understand the children 's own perspectives . these portrayals posit that individuals are responsible for both the cause and cure for obesity . this depiction contributes to stigmatizing attitudes toward obese individuals , who come to be viewed as embodying a remediable social and health burden , borne , in part , by others . it further discounts that obese individuals may be healthy , may choose to emphasize more holistic , less weight - centric conceptualizations of health , or may have lives that preclude investment or engagement in self - care . interestingly , since 2003 , lawrence has detected a shift in american media coverage of obesity . personal responsibility framing dominates ; however , increasing focus is being placed on environmental factors that may contribute to obesity , particularly the fast food industry . unfortunately , this emphasis has not significantly affected antifat attitudes [ 28 , 29 ] . while environmental risks for obesity are acknowledged , individuals are assumed to willingly incur these risks and thus still be largely culpable for their body size and worthy of disapprobation . this discrimination has spread to previously nonstigmatizing nations . rather than castigating obese individuals for their size , qualitative social science researchers may be in a position to rewrite common stereotypical assumptions of obese individuals ' lives by working collaboratively to better understand the health and experiences of an often maligned group and addressing the health concerns most relevant to them . in addition to referring to obesity as an epidemic or crisis [ 2 , 31 ] , an even more extreme figurative device is employing military metaphors . this discourse effectively constructs obese individuals as targets in a war or even as domestic terrorists . terms like contagious are also used when describing obesity in epidemiological studies using methods such as social network analysis [ 33 , 34 ] . this frame seems to be particularly detrimental to the potentially health - enhancing and stress - reducing benefits of social support networks for individuals . as developing obesity is extremely feared [ 35 , 36 ] , it may be very isolating for obese individuals to have their friendship portrayed as a risk for developing obesity . it has been suggested that stigma may serve as a motivator for weight loss among obese individuals [ 1 , 38 ] . however , the evidence provides a far bleaker picture ; stigmatizing obese individuals is an ineffective tactic in reducing obesity rates . rather than stimulating healthful behaviors , stigma is more likely to produce poor eating habits and inactivity and thus may actually augment both obesity prevalence and disordered eating [ 1 , 3942 ] . additionally , the catastrophic rhetoric used to describe the obesity epidemic has been suggested as a potential contributor to rising rates of eating disorders . interventions designed to ameliorate childhood obesity have also been implicated in the development of eating disorders [ 4449 ] . given the adverse psychological outcomes produced from weight discrimination , stigma may also compromise physical and psychological health through stress - induced neuroendocrine dysregulation [ 1 , 50 ] . muennig and colleagues found that the difference between ideal weight and actual weight had a stronger effect on mentally and physically unhealthy days than bmi in american adults , suggesting that body dissatisfaction may have a potent impact on health , over and above objective fatness . the health effects of stigma - induced stress will likely be exacerbated by healthcare discrimination [ 7 , 8 ] , consequent inadequate care , and subsequent avoidance of healthcare providers . obesity may increase the risk of poverty , downward social mobility , and subsequent ill - health through prejudice and discrimination affecting education , employment , income , housing , and healthcare opportunities . thus , weight stigma may be far more health - damaging than previously thought and far more damaging than excess weight . for example , formerly obese adolescent girls continued to suffer from the lower self - esteem characteristic of chronically obese adolescents . additionally , there is qualitative evidence that weight loss efforts and concomitant lifestyle , dynamic , and emotional changes may result in dissolutions of friendships . this deterioration of social support may be an unacknowledged mechanism through which weight stigma affects health , which may previously have been erroneously attributed to the weight itself . in such a stigmatizing context , individuals may utilize a variety of stigma management techniques . these methods may include attempting to lose weight as both an act of contrition and to minimize the fatness for which obese individuals are oppressed [ 5458 ] . monaghan conducted ethnography on male members of a united kingdom slimming club and explored the effects of stigma on their lives . one method of managing stigma utilized by these individuals , who varied in their acceptance of obesity as a discredited state , involved the accounts they related concerning their weight issues [ 56 , 58 ] . some accounts entailed offering excuses that mitigated individual responsibility for obesity , such as appeals to a genetic condition or environmental issues . other individuals justified their size through appeals to their enjoyment of food or pride in their powerful size . individuals who rejected the discredited nature of obesity emphasized natural body diversity or deemphasized the importance of weight regarding health [ 56 , 58 ] . while one account may suit a particular context , a different account may be more useful in different situations [ 56 , 58 ] . to cope with stigmatizing environments being aware of the everyday effects of stigma on individuals ' lives , lifestyles , and health is an essential factor in planning salutogenic , engaging , beneficial , and inclusive public health strategies . for example , understanding obese persons ' decisions to avoid particularly stigmatizing physical fitness venues may allow public health planners to design an atmosphere conducive to supporting individuals of all sizes to engage in enjoyable , health - conducive movement . the literature thus suggests that obese individuals experience substantial stigmatization and exist in an environment saturated with nonproblematized information concerning the health risks of excess weight . this information is often presented in a manner that assumes control for health resides in the individual , the possibility of health and obesity are mutually exclusive , and that achieving wellness constitutes a moral imperative . to a greater or lesser extent , individuals appear to have adopted this discourse and allowed it to influence their perspectives and lifestyles . individuals ' health perceptions may be influenced by pervasive mainstream weight discourse but also be mediated by somatic understandings of wellbeing . as evidence suggests that obese individuals may be healthy , obese individuals may not absorb popular obesity - related messaging , based on their own somatic signaling or knowledge of their lifestyles . individuals may also have differing priorities regarding wellness that supersede weight concerns , such as an emphasis on experiencing pleasure or mitigating income - related food insecurity . regardless of the health risks associated with obesity , a greater understanding of how obese individuals feel concerning their health and quality of life , what obese individuals regard as their priorities concerning health , and what they feel would most benefit their quality of life and wellness is necessary . these views may also alter over time as different weight trajectories are experienced , and this is essential to consider given the chronicity of obesity . in crafting public health messaging , programs , and policies is considering the potentially multifaceted effects of stigma on obese individuals ' lives [ 59 , 60 ] . this includes incorporating that which is most at stake for actors in a local social world within the social dimensions of stigma [ 60 , p. 1525 ] . for example , this may involve stigma impeding life chances , financial and life opportunities , and the fulfillment of individual or familial role functions [ 59 , 60 ] . this view of stigma also considers the manner through which stigma is sociosomatic and how through psychobiological , moral - somatic , and moral - emotional pathways , stigma may have direct physiological consequences . these data on obese people 's perspectives and health will add to the rich work conducted in critical obesity and fat studies , theoretical understandings of the body , and qualitative research done on discursive bodily and health perspectives of individuals ' of all sizes , for example , [ 2 , 32 , 3648 , 5658 , 6268 ] . recent movements have emerged that are critical of a weight - centric public health model . one such undertaking is the health - at - every - size ( haes ) approach . haes advocates are critical of weight loss dieting 's very low rates of sustained weight loss and potentially negative effects on physical and mental health . such effects can include lowered self - esteem , heightened stress , weight cycling , and bone loss . furthermore , haes advocates are critical of the overestimation of excess weight 's effects on morbidity and mortality ; the discounting of the existence of healthy obese individuals ; and the ethics of promoting weight loss , given its low levels of success and possible harms . instead , the haes movement promotes the benefits of engaging in enjoyable physical activity and body acceptance . haes practitioners advice eating nutritionally according to an intuitive eating model , based on intuitive cues of hunger and satiety . clinical trials of haes lifestyle interventions have demonstrated improvements in psychosocial , clinical , physiological , and behavioral measures , independent of weight loss . critically , participants did not experience negative consequences , including weight gain , and these results compared favorably with diet - focused intervention groups [ 3 , 4 ] . the inclusion of social scientists into public health strategizing would facilitate the inclusion of true insiders ' ( i.e. , obese individuals ' ) perceptions on the advantages and disadvantages of these novel developments . these scholars , often from outside health fields , have raised critiques concerning dominant obesity discourse . studies critiquing the biological and epidemiological underpinnings of the prevailing view of obesity as a major health concern and the product of individual behavior have been published . a variety of issues have been raised by these scholars and include the use of terms such as epidemic or crisis in referring to obesity ; the moralization of a presumed health issue and resultant ethical and stigmatizing implications of interventions and messaging ; and the dominant obesity discourse 's effects on individuals ' bodily understandings , [ 2 , 32 , 3648 , 5658 , 6268 ] . findings from these and similar studies would benefit intervention planners in better understanding the daily lives of obese persons , obese individuals ' perspectives on health , and how to plan the most beneficial interventions . anthropology 's traditional focus on the subaltern has become more cognizant and reflective of power relations in research than in past , more colonially complicit , eras of anthropology . a focus today on stigmatized populations , such as obese persons , would draw upon emerging strengths in anthropology , including the utilization of anthropology in social critique and the development of critical ethnography . while in the contemporary social climate obese individuals are often stigmatized , obese individuals and their allies have also marshaled resistance to dominant obesity messaging . monaghan conducted ethnography on the lives of male members of a united kingdom slimming club [ 56 , 58 ] . this provided invaluable information on the experience and management of stigma in these individuals ' lives . further investigations into the experience of weight stigma for men , women , and children are necessary to grapple with a source of discrimination , which can be life and health - damaging but has attracted limited attention and censure . indeed such forms of stigma may operate largely unconsciously , and participant observation may be a valuable tool in detecting this stigma , its effects , and providing empirical support for effecting change . further ethnography on obese individuals , with a focus on health and with applied aims , would be immeasurably useful to health care providers , public health professionals , and the general public of all sizes . thus , collaboration between ethnographers and health researchers in planning health interventions would serve the interests of both disciplines and more importantly the needs of obese individuals . ethnographers could study individuals ' experiences in health care settings , fitness centers , or in employment and educational settings , in which weight bias has been reported . in documenting the manner in which obese individuals ' live ; the choices they make ; their priorities in wellness and quality of life ; their contributions to society and their potential experiences with others ' biases ; applied social scientists could put a human face on obesity and work toward producing a positive , life - affirming , and inclusive public health focused on reducing stigma and blame and helping all people achieve a high quality - of - life . applied social science can enhance obese individuals ' quality of life by providing a greater understanding and problematization of taken for granted assumptions regarding obese individuals ' health , lives , and lifestyles . this epitomizes what rabinow views as a particularly salient and novel area for anthropological exploration . the problematization of serious speech acts and practices , things , and classifications . anthropological experimentation in these areas could move aspects of a culture from being viewed as natural , to contingent , and finally , from a reflexive perspective [ 70 , p. 67 ] . the problematization of taken - for - granted social attitudes and processes is particularly relevant in studying weight stigma , which is largely considered unremarkable , or even acceptable , in current sociocultural climes . prevailing attitudes suggest that obese individuals are necessarily unhealthy , lazy , self - indulgent , and lacking in will power . internalization of such accounts may make individuals feel unhealthy , unworthy , immoral , or disempowered based on their body size or innocuous lifestyle choices . emic understandings of obese individuals ' lives , through ethnography or similar in - depth research methods , could lead to a less - stigmatizing , more healthful view of how obese individuals navigate their everyday lives this more in - depth understanding may contribute invaluably to public health approaches in the future . a greater understanding would be available on what constitutes health to obese individuals , what they prioritize in terms of wellbeing , and their experiences in seeking to live well lives . this perspective would help counter potentially traumatic past experiences of dieting or healthcare discrimination ; produce a more patient - centered approach more congruent to obese individuals ' lives and wishes ; and help establish trusting relationships and bonds between obese individuals , health care providers , and public health officials . applied social scientists , particularly those with access to health practitioner and policy - related audiences , may be especially valuable in this research endeavor with respect to disseminating findings and facilitating progress . these individuals may circulate a critique to key stakeholders in health - related fields , who are in a position to effect change . specific opportunities for qualitative researchers could involve participant observation of obese persons ' interactions with the healthcare system to identify what these persons find the most challenging and most promising aspects of their care in these settings . critical ethnography could also be conducted within existing prevention programs to identify successful , sensitive , or problematic components of existing programs . multisited ethnography could allow obese persons to identify sites of stigmatization and places of support for undertaking self - care . needless to say , challenges would arise in attempting a critical ethnography of the lived experience of obese individuals . investment in orthodox obesity understandings are entrenched among biomedical , public health , and lay audiences . seeking to conduct and disseminate such research this may be particularly evident in trying to demonstrate its value to those largely unaware of obesity stigma 's pervasiveness , its effects , or who have internalized negative stereotypes of obese persons . researchers would also have to be reflexive concerning their own biases and truly and accurately reflect the findings of research participants . in order to best stimulate change , these researchers would also have to strive to create positive , open - minded , and collaborative relationships with a variety of biomedical , public health , and lay audiences in order to exchange findings in a manner most conducive to cooperation and reform . otherwise , researchers risk merely circulating critique among those already critical of dominant obesity discourse or incapable of initiating reform to public health interventions and messaging . facilitating the collaboration of partnerships between patient - providers and policy makers would be of great value . this would allow policy makers to learn from patients ' experiences and desires to rewrite public health messaging , programs , and clinical guidelines to better address obese persons needs and priorities in terms of healthcare system allocation ; address systemic and interpersonal discrimination ; and establish weight industry regulations more in keeping with respect for consumers and truth in advertising . these possibilities , along with numerous others , would allow researchers and public - health planners to learn what health priorities are pertinent to a needlessly patholigized and stigmatized population . it may open the doors to previously nonconsidered structural reforms and the provision of holistic , local health needs . these individuals are inundated daily with messages concerning the risk that they embody , solely by virtue of their size . these risks are thought to extend beyond their own health , to the health of others , the sustainability of the health care system , and even society 's future . furthermore , society deems them as culpable for their presumed poor health and for straining the health care system . within this environment , exposure to such messaging renders obese individuals as a population worthy of special consideration in planning public health programs to avoid reinforcing demeaning stereotypes . what are lacking , despite this oppressive focus on obesity , are the perspectives of obese individuals themselves concerning their health , goals , wishes , and quality of life . furthermore , limited attempts have been made to bridge communicative gaps between physicians and obese patients and to gain a more thorough view of physicians ' perceptions on obesity . given applied social scientists ' dedication to understanding emic perspectives and expertise in in - depth exploratory methods such as ethnography , they may be particularly suited to aid in this pursuit . the outcome may be beneficial , inclusive , and nonstigmatizing public health programs from which all individuals would greatly benefit .

ileal dieulafoy lesion is an unusual vascular abnormality that can cause gastrointestinal bleeding . it can be associated with massive , life - threatening hemorrhage and requires urgent angiographic intervention or surgery . ileal dieulafoy lesion is hard to recognize due to inaccessibility and normal - appearing mucosa . with advances in endoscopy , aggressive diagnostic and therapeutic approaches including enteroscopy have recently been performed for small bowel bleeding . we report two cases of massive ileal dieulafoy lesion bleeding diagnosed and treated successfully by single balloon enteroscopy with a review of the literature . obesity is viewed as a major public health concern , and obesity stigma is pervasive . such marginalization renders obese persons a special population . weight bias arises in part due to popular sources ' attribution of obesity causation to individual lifestyle factors . this may not accurately reflect the experiences of obese individuals or their perspectives on health and quality of life . a powerful role may exist for applied social scientists , such as anthropologists or sociologists , in exploring the lived and embodied experiences of this largely discredited population . this novel research may aid in public health intervention planning . through these studies , applied social scientists could help develop a nonstigmatizing , salutogenic approach to public health that accurately reflects the health priorities of all individuals . such an approach would call upon applied social science 's strengths in investigating the mundane , problematizing the taken for granted and developing emic ( insiders ' ) understandings of marginalized populations .

celiac disease ( cd ) is a common autoimmune disorder estimated to affect approximately 1% of the us population . many studies have evaluated methods for identifying high - risk populations of patients in need of proactive screening for case identification . as with many chronic diseases , quality improvement approaches can be applied to populations of patients with cd to standardize care and track outcomes . effectively identifying and tracking outcomes of these cohorts of patients however , the development of cd patient registries often requires significant resources including practitioners , informatics teams , and administrative support . our center has developed and maintained a patient registry including all newly diagnosed patients since early 2012 . prior studies have evaluated the reliability of international classification of diseases-9 ( icd-9 ) searches to identify patients with cd with only moderate success yielding only 17% of sensitivity . contrast experiments have been done using manually defined keywords to classify high - risk cd cases generating much more accurate results at 73% of sensitivity . few efforts combining different aspects of available information to further improve classification accuracy including the use of data from visits , laboratory tests and pathology reports have been described . pathology reports have been previously used in building natural language processing ( nlp ) systems for automated chart review of patients . here , we define nlp broadly as methods to process textual data . one study found pathology reports to be insufficient for detecting breast cancer when using nlp approach compared with manual approaches while others found the use of nlp for analyzing pathology reports to be effective on detection of colon cancer and manifestation of prostatectomy details . currently , a lack of research exists on investigating the usefulness of pathology reports on automated cd detection . based on a subset of patients prescreened using icd-9 code 579.0 , we evaluated the effectiveness of using natural language features from pathology reports to improve the identification of cd patients . this paper was among the first to adopt a machine learning approach to further increase the accuracy of cd patient identification based on a combination of pathological and clinical metrics . in contrast to prior research , we conducted contrast experiments using both natural language features and clinical features under different machine learning configurations for celiac identification . usa ) clinical system on patients who visited our hospital from 2012 to 2015 and had icd-9 code of 579.0 assigned indicating concern for cd . our subsequent analysis was , therefore , based on patients with concerns rather than general patient population . a laboratory test result was marked with a nominal value of either high ( 20 eu / ml ) or normal ( < 20 eu / ml ) . an upper endoscopy ( egd ) may also be performed then resulted in a text - based pathology report , which could be used for nlp analysis . the registry that we developed are all confirmed diagnosis of cd based on confirmation with both abnormal laboratory / serology testing and biopsy confirmation after detailed chart review . for each of our patients in the registry , cd most confirmed cd patients ( confirmed by our experts for having cd ) only had one pathology report while false - positive cd patients ( confirmed by our experts for not having cd ) had either one or zero pathology report . twenty - nine patients who had two or more pathology reports were excluded from our experiment as they presented a challenge to our analysis if inconsistent information about cd diagnoses existed at different periods of time . in patients with multiple ttg laboratories , the most elevated laboratory result obtained within the past 6 months of the egd was chosen to represent the laboratory status of that cd patient . variables used for celiac classification are listed in table 1 . for the report category , the total number of pathology reports was used with one indicating patients having egd done and 0 otherwise ( v1 ) . both user - define phrases ( high - risk phrases , or v2 ) and automatically extracted phrases ( n - gram phrases generated from our machine learning program , or v3 ) were chosen to evaluate the effectiveness of utilizing pathology reports . for the laboratory category , both the laboratory results ( v4 ) and total number of labs done were used ( v5 ) . for the icd-9 category , the total number of cd icd-9 codes assigned ( v6 ) was used for classification . variables for celiac disease classification to evaluate the effectiveness of using different combinations of variables and feature selection methods , we further developed two additional set of variables based on the aforementioned six sets of variables above ( v1v6 ) . combining all first six categories of variables created the variable set 7 ( v7 ) . automatically selecting features out of v6 using a feature selection algorithm resulted in the variable set 8 ( v8 ) [ table 1 ] . for expert - knowledge - driven feature selection , we identified a list of nine key phrases described by clinicians as commonly used in pathology reports as indicators of high - risk cd . these nine key phrases , which were all converted to lower cases , included brunner 's gland hyperplasia , flattening villi , intraepithelial lymphocytes , marsh gland stage , marsh lesion , marsh s3 lesion , shortened villi , villous blunting , and villous atrophy . each key phrase was first converted to a binary variable with the value of one indicating the phrase existed in the pathology report and 0 otherwise . automatically extracted n - gram features were phrases generated using weka , an open source java - based machine - learning program , developed by the university of waikato , new zealand . the maximum number of words included in n - gram feature was chosen to be three resulting in a large set of text features consist of uni- , bi- and tri - grams . we only collected phrases up to trigrams because the length of the longest user - provided high - risk phrase was also three . we kept the first 4000 text features with all stop words ( i.e. , a list of common words such as the , skipped . during the process of converting documents into n - gram features , term frequency - inverse document frequency ( tf - idf ) was calculated on each n - gram feature . tf - idf is a data transformation method commonly used in text classification to show the importance of a word to a document in a collection of documents . it has been found useful in several recent classification and information retrieval studies using medical documents . finally , all n - grams were converted to lowercase to avoid duplications during tf - idf transformation . therefore , we only chose the three most representative classifiers from four major categories to experiment with in this paper . the four classifier categories included bayes , function - based , lazy model and tree classifiers . in each category previous studies on classifying medical documents showed that the performance variation among different models could be relatively small within the category compared with across the categories . this was another main reason we only chose to experiment with three classifiers rather than all available classifiers from each category . selected classification models for experiments there are different ways to configure a classifier . in most cases of our experiments , the default configuration was chosen as long as the setting was compatible with the type of data we classify . for example , for libsvm the standard algorithm of regularized c - support vector classification was selected as well as the default radial kernel type [ table 2 ] . for each one of the twelve classification models [ table 2 ] , we also experimented with the use of the eight different variable configurations ( v1v8 ) [ table 1 ] to find the best variable configuration to produce the best possible classifier . each variable configuration was used as the inputs to each one of the twelve classification models . resulting in twelve experiments for each variable configuration . the classification model with the best performance overview of the architectural design of experiments feature selection ( or variable selection ) is critical for optimizing a classification model . appropriately selected features could minimize the chance of overfitting as well as reduce feature redundancy and training time in the face of high - dimensional variables . a typical feature selection process included two separate steps available in the weka tool we used . the first step is to define a feature evaluation method using criteria based on which features are selected . the second step is to define a search method where features will be searched and selected into the final feature set . there is not a single rule of thumb for choosing the best possible feature selection method . after performing several experiments , we found the default feature selection method in weka worked the best for us . the feature selection model we used implemented a correlation - based feature evaluation model called cfssubseteval . the cfssubseteval feature evaluation model ranks the features based on their correlation with the class while minimizing the redundancy of variables . the underlying feature search model was the best first feature search model which searches the attribute space by allowing backtracking . detailed discussion on cfssubseteval and best first is beyond the scope of this paper and we recommend researchers to refer to the original work from the original authors . each classifier was validated using a 10-fold cross - validation method on our entire dataset . confirmed cases and controls the best model with the highest area under the curve ( auc ) for each variable configuration was reported . auc was a commonly used metric for machine learning - based evolution as a chance corrected measure and independent of class distributions . chi - square test was conducted to test the statistical association between each of the variables and celiac status . in addition to auc , we also reported precision , recall , f1 , and kappa . precision , recall , and f1 were classic machine learning measures for evaluating overall accuracy with 1 being the best and 0 being the worst for predicting positive cases . the kappa score measured the level of agreement between the user and the classifier with 1 being complete agreement and 0 being complete disagreement . for expert - knowledge - driven feature selection , we identified a list of nine key phrases described by clinicians as commonly used in pathology reports as indicators of high - risk cd . these nine key phrases , which were all converted to lower cases , included brunner 's gland hyperplasia , flattening villi , intraepithelial lymphocytes , marsh gland stage , marsh lesion , marsh s3 lesion , shortened villi , villous blunting , and villous atrophy . each key phrase was first converted to a binary variable with the value of one indicating the phrase existed in the pathology report and 0 otherwise . automatically extracted n - gram features were phrases generated using weka , an open source java - based machine - learning program , developed by the university of waikato , new zealand . the maximum number of words included in n - gram feature was chosen to be three resulting in a large set of text features consist of uni- , bi- and tri - grams . we only collected phrases up to trigrams because the length of the longest user - provided high - risk phrase was also three . we kept the first 4000 text features with all stop words ( i.e. , a list of common words such as the , of , is , etc . ) skipped . during the process of converting documents into n - gram features , term frequency - inverse document frequency ( tf - idf ) tf - idf is a data transformation method commonly used in text classification to show the importance of a word to a document in a collection of documents . it has been found useful in several recent classification and information retrieval studies using medical documents . finally , all n - grams were converted to lowercase to avoid duplications during tf - idf transformation . therefore , we only chose the three most representative classifiers from four major categories to experiment with in this paper . the four classifier categories included bayes , function - based , lazy model and tree classifiers . in each category previous studies on classifying medical documents showed that the performance variation among different models could be relatively small within the category compared with across the categories . this was another main reason we only chose to experiment with three classifiers rather than all available classifiers from each category . selected classification models for experiments there are different ways to configure a classifier . in most cases of our experiments , the default configuration was chosen as long as the setting was compatible with the type of data we classify . for example , for libsvm the standard algorithm of regularized c - support vector classification was selected as well as the default radial kernel type [ table 2 ] . for each one of the twelve classification models [ table 2 ] , we also experimented with the use of the eight different variable configurations ( v1v8 ) [ table 1 ] to find the best variable configuration to produce the best possible classifier . each variable configuration was used as the inputs to each one of the twelve classification models . resulting in twelve experiments for each variable configuration . the classification model with the best performance was chosen as the best model for the underlying variable configuration used . overview of the architectural design of experiments feature selection ( or variable selection ) is critical for optimizing a classification model . appropriately selected features could minimize the chance of overfitting as well as reduce feature redundancy and training time in the face of high - dimensional variables . a typical feature selection process included two separate steps available in the weka tool we used . the first step is to define a feature evaluation method using criteria based on which features are selected . the second step is to define a search method where features will be searched and selected into the final feature set . there is not a single rule of thumb for choosing the best possible feature selection method . after performing several experiments , we found the default feature selection method in weka worked the best for us . the feature selection model we used implemented a correlation - based feature evaluation model called cfssubseteval . the cfssubseteval feature evaluation model ranks the features based on their correlation with the class while minimizing the redundancy of variables . the underlying feature search model was the best first feature search model which searches the attribute space by allowing backtracking . detailed discussion on cfssubseteval and best first is beyond the scope of this paper and we recommend researchers to refer to the original work from the original authors . therefore , we only chose the three most representative classifiers from four major categories to experiment with in this paper . the four classifier categories included bayes , function - based , lazy model and tree classifiers . in each category previous studies on classifying medical documents showed that the performance variation among different models could be relatively small within the category compared with across the categories . this was another main reason we only chose to experiment with three classifiers rather than all available classifiers from each category . selected classification models for experiments there are different ways to configure a classifier . in most cases of our experiments , the default configuration was chosen as long as the setting was compatible with the type of data we classify . for example , for libsvm the standard algorithm of regularized c - support vector classification was selected as well as the default radial kernel type [ table 2 ] . for each one of the twelve classification models [ table 2 ] , we also experimented with the use of the eight different variable configurations ( v1v8 ) [ table 1 ] to find the best variable configuration to produce the best possible classifier . each variable configuration was used as the inputs to each one of the twelve classification models . resulting in twelve experiments for each variable configuration . the classification model with the best performance was chosen as the best model for the underlying variable configuration used . appropriately selected features could minimize the chance of overfitting as well as reduce feature redundancy and training time in the face of high - dimensional variables . a typical feature selection process included two separate steps available in the weka tool we used . the first step is to define a feature evaluation method using criteria based on which features are selected . the second step is to define a search method where features will be searched and selected into the final feature set . there is not a single rule of thumb for choosing the best possible feature selection method . after performing several experiments , we found the default feature selection method in weka worked the best for us . the feature selection model we used implemented a correlation - based feature evaluation model called cfssubseteval . the cfssubseteval feature evaluation model ranks the features based on their correlation with the class while minimizing the redundancy of variables . the underlying feature search model was the best first feature search model which searches the attribute space by allowing backtracking . detailed discussion on cfssubseteval and best first is beyond the scope of this paper and we recommend researchers to refer to the original work from the original authors . each classifier was validated using a 10-fold cross - validation method on our entire dataset . confirmed cases and controls the best model with the highest area under the curve ( auc ) for each variable configuration was reported . auc was a commonly used metric for machine learning - based evolution as a chance corrected measure and independent of class distributions . chi - square test was conducted to test the statistical association between each of the variables and celiac status . in addition to auc , we also reported precision , recall , f1 , and kappa . precision , recall , and f1 were classic machine learning measures for evaluating overall accuracy with 1 being the best and 0 being the worst for predicting positive cases . the kappa score measured the level of agreement between the user and the classifier with 1 being complete agreement and 0 being complete disagreement . our data extraction resulted in identification of 1498 unique patients with icd-9 codes suggesting possible cd . we found 363 confirmed cases ( positive celiac cases [ table 3 ] ) matching the previously establish celiac registry and 1135 false positive cases ( negative celiac cases [ table 3 ] ) . demographic data are shown in table 3 . chi - square test found statistically significant associations between age , gender , race , and celiac status . classification variables such as those about visit , laboratory and pathology report counts and laboratory results were all found to be significant for differentiating cd patients . as this study mainly focused on using clinical variables for celiac classification we did not include any demographic variables in building classifiers [ table 3 ] . patient characteristics and their association with celiac disease table 4 listed the results summarizing the best classification model out of each variable configuration . models were ranked based on auc score from the highest to the lowest . among all twelve classification models , we experimented with the libsvm was found to be the top performer in five experiments ( experiments 3 , 4 , 5 , 6 , 8) , followed by logistic model in two experiments ( experiments 1 , 7 ) and nave bayes in one experiment ( experiment 2 ) . the best performing model was the logistic model using all available features filtered by an automatic feature selection process . it achieved auc of 0.94 , kappa of 0.78 , and f1 of 0.92 [ table 4 ] . statistics of the best classification models for each variable configuration among experiments using single category of variables ( experiment 2 and 48 ) , nave bayes model based on automatically selected n - gram features generated the best results : auc of 0.92 , kappa of 0.73 , and f1 of 0.90 , which was also the overall second best performing model . here , we also conducted feature selection on executing the nave bayes model as we aimed to find the best possible performance of using text features . despite the slightly lower performance , the nave bayes model was much faster than other models in terms of model building and model evaluation time . in our case , it only took nave bayes a few seconds to build and evaluate the model in 10-fold cross - validation while it took a few minutes for libsvm and logistic model to finish in the case of using all 4000 features . using only high - risk phrases , number of laboratories and number of icd-9 ( experiment 6 , 7 , 8) resulted in the lowest model accuracy . using laboratory resulted in higher accuracy than the previous three . it was also intriguing that using the number of pathology reports alone only slightly underperformed using report text features alone . however , additional features resulted in higher accuracy given the results based on a combination of different features . table 5 listed some of the automatically selected n - gram phrases in contrast to user - provided high - risk phrases . it was found that automatically created n - gram features not only covered the majority of user - provided high - risk phrases but also had the potential of being more accurate and specific in finding relevant information . for example , automatically generated n - grams such as celiac and abdominal pain were directly relevant features to cd but were not initially on our clinicians list . comparison of user - defined high - risk phrases and automated key phrases some of the provided high - risk features were not included in our automatically selected feature list such as the phrase of marsh lesion ( converted to lower case for processing ) . we found this may be due to the extreme low frequency of the usage of such phrases in our pathology reports ( only 1 out of all 363 confirmed cases had this exact phrase ) . in table 5 , the italicized phrases highlighted some of the overlaps between the two lists : user - provided and automatically extracted . the automatically extracted list only represented a subset of most relevant n - gram features [ table 5 ] . our machine learning approach could quickly and effectively choose the most relevant variables from various clinical sources for automated cd classification . the level of accuracy we achieved was higher compared with previous studies on the same topic . n - gram features performed well in our cd detection experiment which agreed with and further improved the results from previous studies using text features . in contrast to previous studies , our automated feature generation and selection approaches were more efficient in finding relevant classification features in a short period of time and more effective than user - provided high - risk phrases . this result showed the automated list was more effective in providing a comprehensive list of relevant keywords than manual methods . the number of laboratories done was as effective as the actual lab results reported in our case for cd classification ( auc of 0.71 vs. 0.83 ) . this could be because an increased number of labs done or the fact that the laboratory was ordered may suggest a higher likelihood of abnormal laboratory result . our experiments also agreed with previous studies on the fact that icd-9 code alone produced very poor results ( kappa of 0.28 , f1 of 0.75 and auc of 0.63 ) . therefore , we suggested researchers only use icd-9 code as a prescreening tool for subsetting patients as in this study . in addition , our results added further evidence to previous studies that nave bayes model generated the most accurate results even if only pathology report features were used ( kappa of 0.73 , f1 of 0.90 , and auc of 0.92 ) . this further indicates that pathology report , as an important pierce of synoptic reporting of celiac status , contains invaluable information for celiac classification . in addition , by combining both clinical and text features altogether , classification accuracy and kappa score could be further improved ( kappa of 0.78 , f1 of 0.92 , and auc of 0.94 ) . this suggests the complementary effect of using both structured data and unstructured data for celiac classification . finally , feature selection was found to be a crucial step in celiac detection for further improving the efficiency and accuracy of classification . feature selection dramatically removed redundant and noninformative terms ( from about 4000 to 60 ) . although new to the task of cd classification , feature selection methods have been found widely used in other disease classification studies including alzheimer 's disease and asthma . in our case , some variables ( i.e. , number of pathology reports and lab results ) could individually predict celiac at a reasonably high accuracy . however , this result may be problem dependent . other studies implementing the same metrics may or may not result in similar performance . even that , we still observed performance gain by adding nlp features with both accuracy and agreement increased by a reasonable amount . the automated tool we developed could speed up the process of refining the subset population initially identified based on icd-9 code 579.0 . this refining process can automatically confirm the patient as either positive or negative cd case based on the knowledge of the patient obtained from the laboratories and pathology notes . the classification accuracy was over 90% of correctness ( f1 ) in our case while it greatly eliminated the need of often time - consuming process of manual review . first , since the cd classification task was performed on a subset of patients prescreened by icd-9 code rather than on the general patient population , the performance of final classification results may depend on the accuracy of initial icd-9 assignment . for example , if actual cd patients were not assigned an icd-9 code 579.0 initially , they will not have the chance of being further identified by our machine learning system . it is , therefore , important to make sure the initial icd-9 code assignment will include as many high - risk cases as possible . second , although feature selection method was found to be critical in yielding high performance , we have not conducted systematic experiments with all possible feature selection methods provided in the weka machine learning system . given our experience , it was likely that some methods may further improve accuracy while others may even lower the accuracy . finally , for most machine learning algorithms we experimented , only the default configuration was used for classification . it was likely different configurations of parameter setting may lead to very different results of accuracy . therefore , researchers are encouraged to experiment with different classifier settings to achieve the best possible results for their tasks . since the cd classification task was performed on a subset of patients prescreened by icd-9 code rather than on the general patient population , the performance of final classification results may depend on the accuracy of initial icd-9 assignment . for example , if actual cd patients were not assigned an icd-9 code 579.0 initially , they will not have the chance of being further identified by our machine learning system . it is , therefore , important to make sure the initial icd-9 code assignment will include as many high - risk cases as possible . second , although feature selection method was found to be critical in yielding high performance , we have not conducted systematic experiments with all possible feature selection methods provided in the weka machine learning system . given our experience , it was likely that some methods may further improve accuracy while others may even lower the accuracy . finally , for most machine learning algorithms we experimented , only the default configuration was used for classification . it was likely different configurations of parameter setting may lead to very different results of accuracy . therefore , researchers are encouraged to experiment with different classifier settings to achieve the best possible results for their tasks . in this paper , we compared results from 96 machine - learning experiments for cd identification based on 12 classification model variations and eight feature set variations . the logistic model that used a combination of clinical and pathology report features generated the best results : kappa of 0.78 , f1 of 0.92 , and auc of 0.94 . our results were in agreement with previous studies on the insufficiency of using icd-9 codes alone and the merits of using pathology report features for cd identification . this study provides new evidence on adopting feature selection techniques to further improve classification efficiency and accuracy based on a subpopulation of prescreened patients using icd-9 code . this study demonstrated a viable computational approach to automatically reviewing and confirming prescreened cd patients based on icd-9 code at the state of the art accuracy much improved from previous research on the same topic . the possible presence of infection in cervical smear tests is usually reported by the cytologists based on cytological criteria . therefore , non - specific cervicitis or inflammatory changes in a smear report are common . these findings are usually unclear for clinical approaches and there is not an appropriate guideline for management of such patients . it is not known if these women should be recalled for cultures and treatment or inflammatory change should be considered as minor . changes in balance of normal vaginal flora can cause an overgrowth of pathogens that lead to discharge . although it is a common complain among sexually active women , there are still gaps in our knowledge about it . bacterial vaginosis ( bv ) , vaginal candidiasis ( vc ) and trichomoniasis are the three infections most commonly associated with vaginal discharge ( 1 ) . a number of studies have indicated the association of bv with postoperative complications after gynecologic surgery as well as pregnancy complications ( 25 ) . trichomonas vaginalis ( tv ) is also another common cause of vaginal discharge , but some studies have not found its association with pregnancy complications ( 3 ) or some other indicated that tv during pregnancy was a predisposing factor for preterm delivery and delivering low - birth weight infants ( 6 ) . the pap smear test for detection of bv has showed a sensitivity of 88.2% , a specificity of 98.6% , and a positive predictive value ( ppv ) of 96.8% ( 7 ) . the sensitivity , specificity , and ppv of pap test for tv diagnosis has been reported 98% , 96% , and 88% , respectively ( 8) . also , some recent studies have demonstrated the co - infection of vaginal pathogenic organisms in pap tests ( 2 , 9 , 10 ) . this study aimed to investigate the prevalence of pathogenic vaginal microorganisms and the presence of inflammation in pap smear among an iranian women sample and to evaluate the possible co - infection of these organisms . this cross - sectional study was carried out on pap smear samples of women referred to gynecological clinic of taleghani hospital in tehran , iran , between october 2008 and march 2009 . exclusion criteria were pregnancy , smoking , use of oral contraception and/or corticosteroids , regular use of vaginal douche , and women with chronic systemic diseases or systemic immunosuppression . prior to data collection , the objectives of the study were explained in community meetings . all women received their laboratory results in written form , and results were explained during a consultation . the study protocol was also approved by the institutional review boards of department of gynecology and obstetrics . data were obtained in privacy using an information form applied by one investigator , followed by gynecological examination and specimen collection all done by one gynecologist . cervical smear samples , based on the conventional method , were prepared with a spatula and fixed on glass slides and were stained with the papanicolaou technique . tv was defined as the presence of trophozoites in pap smears ; vc was diagnosed if fungal hyphae or budding yeasts were present in pap smears . the presence of inflammation in the smears was divided into mild , moderate , and severe categories . mild inflammation was defined as less than 30 inflammatory cells / high - power field ; moderate inflammation was defined as 30 to 100 inflammatory cells / high - power field ; and severe inflammation was defined as more than 100 inflammatory cells / high - power field . the frequency of pathogenic microorganism included bv , tv , and vc were determined in specimens . statistical significance was analyzed using statistical package for social sciences ( spss software version 17 ) . the comparison between data was performed with the pearson 's chi square or fisher 's exact test . mild inflammation was noted in 136 ( 26.1% ) , moderate in 155 ( 29.4% ) and severe in 117 ( 22.2% ) of them ( table 1 ) . the frequency of pathogenic vaginal microorganisms and inflammation among pap smear samples among all samples , bv ( the presence of clue cells ) was the most common pathogenic microorganism which was seen in 91 samples ( 17.2% ) , followed by vc ( n = 56 ; 10.6% ) . tv ( n = 2 ; 0.4% ) was shown to have a low prevalence among our patients ( table 1 ) . the overall prevalence of pathogenic microorganisms was greater among reproductive age participants , meanwhile the prevalence of bv as well as vc was significantly different ( p = 0.042 and p = 0.006 , respectively ) between non- and post- menopausal women . inflammation was detected in 76% ( n = 402 ) of pap smears , in which 80.1% ( n = 322 ) were reported among reproductive age women . however , the severity of inflammation was not significantly different between non- and post- menopausal women . overall , the presence of inflammation was significantly associated with vc ( p = 0.002 ) , but its severity was not correlated to the infection . although , there was no significant relation between the presence of inflammation and bv , the severity of inflammation in specimen was significantly increased in association with bv ( p < 0.001 ) . in our study , the prevalence of bv among pap smear specimens of women living in tehran was 17% which is similar to azargoon 's study ( 16% ) on a population in semnan ( another city in center of iran ) ( 3 ) , but it had a higher rate in hamadan ( a western city of iran ) and has been reported 28.5% ( 11 ) . in comparison with other asian countries , in india , but it was 38% in an african study in botswana on 703 pregnant women ( 13 ) . some predisposing factors for bv include having sexual relationship with more than one partner , early onset of sexual activity , orogenital sexual contacts , coitus during menstruation , use of iud , and smoking ( 2 , 14 ) . differences in sexual behavior and risk factors in different societies can justify the differences in bv prevalence of mentioned studies . reproductive age women indicated that the prevalence of bv in muslim women was lower than non - muslims . although the prevalence of tv in pap samples of our study was much less than several studies , similar reports demonstrated by depuydt et al in flanders detected by real - time pcr ( 0.37% ) ( 15 ) . however , this rate was 18% in the study in hamedan ( 11 ) , 8.5% among indian women ( 16 ) , and 19% in the african study ( 13 ) . a probable reason for these variations could be differences in pap sampling quality and cytologists skill . several studies show an increased vaginal ph ( 5 ) in both bv and tv infection ( 3 , 5 , 17 ) . demirezen et al in turkey on 600 pap tests detected tv in 6% of women and bv in 44% of tv cases . they also found a significant relationship between tv and bv infection , which might be due to this hypothesis that tv by phagocytosis the vaginal lactobacilli increases the vaginal ph and generates an anaerobic environment , providing an appropriate condition for the growth of anaerobic microorganisms ( 18 , 19 ) . also , it was confirmed by heller 's study on 400 pap samples which concluded that bv diagnosis should be considered at the presence of tv in pap smears ( 2 ) . in our study , we found no significant association between bv and tv in conventional pap smear sampling , which might be because of low prevalence of tv in our samples . the effect of habits and behaviors on vaginal flora among different population can be evaluated in further assessments . in our study , only the presence of candida in pap smear samples was associated with the report of inflammation . therefore , reporting inflammation in pap smear test might be considered for candidiasis evaluation and treatment . several studies investigated the association between infection and existence of inflammation in cervical smears ( 2023 ) . some of them found that inflammation on pap smear had a relatively low predictive value for the presence of infection ( 21 ) . they stated that the inflammation was not exactly a consequence of infection and other causes might be responsible for this appearance ( 20 ) . in contrast , others indicated a significant association between inflammatory smear and reproductive tract infections ( 22 , 24 ) . as burke et al ( 22 ) in their study on 256 women found an evidence of inflammation in 9.7% of smears and genital tract infection in 29.2% of their participants overall . they found infection in 48% of women having inflammatory changes on smear test vs. 27.3% of women whose smear tests showed no evidence of inflammation . they discovered that the prevalence of infection with chlamydia trachomatis , candida , bacteroides and gardnerella vaginalis was higher in the inflammatory smear group . they concluded that women with an inflammatory smear were more likely to mask the infection than women whose smear shows no evidence of inflammation ( 22 ) . prevalence of bv infection ( 17% ) in this study was similar to most other studies , which was not true for tv ( < 1% ) . the use of other diagnostic methods may be probably more valuable than conventional pap smear for evaluation of tv among iranian women and it needs further investigations . as the report of inflammation in our samples was very high , use the newer pap smear techniques such as thinprep ( liquid base preparation ) in the further studies is recommended . based on our results , inflammation reported in the pap smears demands the evaluation for vc and its proper treatment .

background : celiac disease ( cd ) is a common autoimmune disorder . efficient identification of patients may improve chronic management of the disease . prior studies have shown searching international classification of diseases-9 ( icd-9 ) codes alone is inaccurate for identifying patients with cd . in this study , we developed automated classification algorithms leveraging pathology reports and other clinical data in electronic health records ( ehrs ) to refine the subset population preselected using icd-9 code ( 579.0).materials and methods : ehrs were searched for established icd-9 code ( 579.0 ) suggesting cd , based on which an initial identification of cases was obtained . in addition , laboratory results for tissue transglutaminse were extracted . using natural language processing we analyzed pathology reports from upper endoscopy . twelve machine learning classifiers using different combinations of variables related to icd-9 cd status , laboratory result status , and pathology reports were experimented to find the best possible cd classifier . ten - fold cross - validation was used to assess the results.results:a total of 1498 patient records were used including 363 confirmed cases and 1135 false positive cases that served as controls . logistic model based on both clinical and pathology report features produced the best results : kappa of 0.78 , f1 of 0.92 , and area under the curve ( auc ) of 0.94 , whereas in contrast using icd-9 only generated poor results : kappa of 0.28 , f1 of 0.75 , and auc of 0.63.conclusion:our automated classification system presented an efficient and reliable way to improve the performance of cd patient identification . objectivenon - specific cervicitis or inflammatory changes in a smear report are common which are usually unclear for clinical approaches . to investigate the frequency of inflammation and pathogenic vaginal microorganisms in cervical smears among an iranian population sample.materials and methodsthis cross - sectional study was carried out on pap smear samples of women referred to gynecological clinic of taleghani hospital in tehran , iran , between october 2008 and march 2009 . this study was conducted on 528 conventional papanicolaou cervical smears . the frequency and severity of inflammation and prevalence of bacterial vaginosis ( bv ) , trichomonas vaginalis ( tv ) , and vaginal candidiasis ( vc ) was determined in the samples . also co - infection of the microorganisms in pap samples was evaluated . percentage , meanstandard deviation of the outcome parameters were calculated . the comparison between data was performed with the pearson 's chi square or fisher 's exact test.resultsthe prevalence of bv , vc , and tv in pap samples was 17% , 11% , and 0.4% respectively . overall , the prevalence of these microorganisms in women of reproductive age was higher than menopausal women . there was a significant association between vc and the presence of inflammation in our samples.conclusionbased on our results , inflammation in the pap smears can suggest an infection of vc and the patients should be considered for proper vc treatment .

brucellosis is a highly contagious zoonosis caused by bacteria of the genus brucella and is listed in class b animal epidemics by the world organization for animal health ( oie ) . the genus has six classic species : b. abortus , b. melitensis , b. suis , b. canis , b. ovis and b. neotomae . recently , the marine species b. ceti , b. pinnipedialis , b. microti and b. inopinata were included in the genus ( foster et al . the incidence of human brucellosis is estimated by the oie at 500,000 new cases per year worldwide . however , official statistics is widely acknowledged to be underestimated and , in a considerable number of cases , the origin of the infection is not identified . the majority of human cases worldwide is attributed to b. melitensis ( pappas et al . , 2005 ) . in general , b. melitensis and b. suis are more virulent in humans than b. abortus or b. canis ( who , 2006 ) . other species can cause infection in humans , but only rarely ( diaz aparicio , 2013 ) . the disease is more prevalent in western parts of asia , india , middle eastern , southern european and latin american countries . the transmission of brucella infection and its prevalence in a region depend on several factors , such as dietary habits , methods of processing milk and milk products , social customs , husbandry practices , climatic conditions , socioeconomic status and environmental hygiene ( mantur and amarnath , 2008 ) . as the infectious dose is very low , infections are an occupational risk for farmers , veterinarians , abattoir workers ( schneider et al . , 2013 ) , laboratory personnel and others who work with animals and consume their products ( pepin et al . , 1997 ) . human brucellosis is transmitted by inhalation , animal contact and the consumption of dairy products and undercooked meat products . the consumption of traditional dishes , such as raw liver , can cause human infection ( malik , 1997 ) . however , it has been estimated that even 10 - 100 microorganisms are sufficient to cause the disease in humans ( pappas et al . , 2006 ) . thus , contaminated meat and meat products could represent a source of infection , especially if they come from animals slaughtered during the acute phase of the disease ( fao / who , 1986 ) and if they are consumed raw or undercooked . the handling and preparation of infected meat and offal may give rise to the contamination of other foodstuffs and kitchen utensils ( who / cds / epr/2006.7 ; who , 2006 ) . tests carried out in the us on the carcasses of bovine and swine that were slaughtered because infected revealed that 1.2% of the bovine carcasses and 3.5% of the swine carcasses were contaminated by brucella spp . ( sadler , 1960 ) . in a similar study conducted in india on 100 carcasses of goats , two of the 700 neck muscle samples analysed tested positive for brucella melitensis ( randhawa and karla , 1970 ) . other studies have shown that the offal of slaughtered ruminants constitutes a risk for transmission of the infection ( fatma and mahdey , 2010 ; sekulovski , 2008 ) . in orderto evaluate the prevalence of contamination of meats and to assess the risk of infection for consumers and professionally exposed workers , an investigation on 307 carcasses of bovines , sheep and goats that were slaughtered because they had resulted positive on serological testing for brucella spp . was performed . the study was carried out within the framework of the national programmes for the eradication and monitoring of animal diseases and the prevention of zoonoses in italy , approved by decisions 2011/807/ue and 2012/761/ue of the european commission ( 2011,2012 ) . the study lasted 12 months and was carried out in 10 abattoirs in southern italy , where brucellosis infection is still widespread on cattle , sheep and goat farms . after slaughter , swab samples were taken from 307 carcasses : 40 cattle , 60 sheep and 207 goats . these animals came from 24 different farms and were slaughtered because they had proved positive on serological tests for brucella spp . in previous studies ( randhawa and karla , 1970 ; fatma and mahdey , 2010 ) brucella spp . contamination of the carcasses of slaughtered animals had been evaluated by means of microbiological tests carried out on samples of muscle or organ tissue . in the present study , swab samples were taken in accordance with the procedure indicated by the decision of the european commission ( 2001 ) : duplicate samples were taken by means of swabs both dry and moistened in a sterile aqueous solution which were wiped over the external and internal surfaces of the carcasses immediately after slaughter and before storage in refrigerators . a total of 608 swab samples were taken ; no transport medium was deemed necessary , as the samples were promptly consigned to the laboratory , where they underwent polymerase chain reaction ( pcr ) analysis for brucella spp . in accordance with the following procedure . for dna extraction , a commercially available kit ( qiaamp dna mini kit ; qiagen , valencia , ca amplification was carried out by means of a nested pcr technique , based on a published protocol ( romero et al . , 1995 ; the second step utilised a pair of oligonucleotides that are internal to those used in the first step and produce a smaller fragment ; they prove functional only if the result of the first pcr is specific . ( 5-tcgagcgcccgcaagggt - gagcgg-3 ) and r2 ( 5-aaccatagtgtctc - cactaacc-3 ) allowing an amplification product of 905 bp . in the second step of pcr , the primer pairs r0 ( 5-tagctagttg - gtggggtaaaggc-3 ) and r1 ( 5-caggcttgcgcccattgtcc-3 ) were used allowing an amplification product of 144 bp . to prepare the reaction mixture for the first pcr , 12.5 l of a commercially available pcr master mix ( pcr master mix ; promega corp . , madison , wi , usa ) was used at a 1x concentration for each sample . the reaction mixture contains 50 units / ml of taq polymerase , 400 mm of datp , dgtp , dctp , dttp and 3 mm of mgcl2 . to the reaction mixture , 0.5 l of each primer f4 and r2 at a concentration of 0.2 m and 6.5 l of h2o were added , with a final reaction volume of 25 l . as a positive control in pcr , the negative control was made up only of master mix and water . to prepare the reaction mixture for the second pcr , the thermal profile of the first pcr involved an initial denaturation at 95c for 4 followed by 35 cycles of denaturation ( at 94c for 1 ) , annealing ( at 54c for 1 ) and extension ( at 72c for 1 ) and a final extension at 72c for 7. the thermal profile of the second pcr was the same as the first one except for the cycle number that consisted of 30 cycles in the second pcr . in accordance with italian law and with the aim of eradicating brucellosis in cattle , sheep and goats , the carcasses of infected animals may be freely marketed if the outcome of post - slaughter examination is favourable , while all the internal organs and the udders must be destroyed . parts of these organs were therefore removed and subjected to culture tests for the bacteriological identification and typing of brucella spp . by means of the technique described in the manual of the office international des epizooties , ch . this involves : streaking on brucella agar medium added with a selective supplement and horse serum ; incubation at 37c in an atmosphere enriched with 5 - 10% co2 ; and observation for up to 10 days . subsequent species typing of the isolated strains was carried out at the national reference centre for brucellosis in teramo ( italy ) by means of the pcr - restriction fragment length polymorphism technique , in accordance with the procedure prescribed in vol . the biomolecular tests revealed the presence of brucella spp . in 25/307 carcasses ( 8% ) : 1 bovine carcass ( 2.5% ) of a 9-year - old cow ; 9 carcasses of sheep ( 15% ) aged between 18 months and 5 years , all from the same farm ; and 15 carcasses of goats ( 7.2% ) aged between 12 months and 6 years and coming from 5 different farms ( figure 1 ) . the 25 carcasses found to be contaminated with brucella spp . belonged to 25 animals slaughtered in 6 different slaughterhouses . the bacteriological tests carried out on the organs of all 307 animals examined allowed isolating brucella spp . in 136/307 cases , i.e. 44% the typing analysis allows detecting type 3 brucella abortus in the cattle organs , and type 3 brucella melitensis in the sheep and goats organs . the organs , specifically the lymph nodes , from all the carcasses that had tested positive on pcr on swabs , also proved positive on bacteriological testing for type 3 brucella abortus ( the bovine carcass ) and for type 3 brucella melitensis ( the sheep and goat carcasses ) . the results obtained show that the carcasses ( i.e. , the meat ) of animals slaughtered due to brucella spp . this situation is the same as that seen in the case of other pathogens , like salmonella spp . , for which specific laboratory tests are prescribed and measures have to be taken . in the present study , the percentage of meats that proved to be contaminated was far higher than in the studies quoted above ( sandler , 1960 ; randhawa and karla , 1970 ) . contamination by brucella spp . was detected in a higher percentage of sheep carcasses than cattle or goat carcasses , while the bacteriological tests conducted on the organs detected the highest percentage of contamination among goats . the only brucella serotypes isolated from the organs were type 3 brucella abortus in cattle and type 3 brucella melitensis in sheep and goats . these are the only serotypes that have been isolated in recent years in the geographical area considered ( casalinuovo et al . , 2011 ; de massis et al . , 2014 ) thus , the environments and facilities of slaughterhouses may also be severely contaminated , thereby constituting a risk of contagion for staff involved in butchery and meat handling , and a risk of contamination of the carcasses of other animals that are not infected by brucellosis . indeed , the positivity of the carcasses involved 6/10 slaughterhouses . our samples were taken at the end of the butchery process , when the carcasses were ready for storage in refrigerators or delivery to retail outlets . this means that each contaminated carcass could in turn contaminate equipment and utensils and other meats or foodstuffs . consequently , even in domestic kitchens , contamination could further involve other equipment , utensils and food products . indeed , as brucella spp . survives well at the temperatures reached by refrigerators or freezers , other refrigerated or frozen foods may become contaminated . pcr testing of swab samples taken from the carcasses of animals that had tested positive for brucella proved to be useful . this practice could be applied systematically , since this noninvasive sampling technique does not damage commercial meat cuts , is economically advantageous , and requires far less time ( 24 - 48 h ) than detection by means of culture media . carcasses that resulted positive for pcr might be sequestered and potentially submitted to different confirmation tests . annex i , section iv , chapter ix , f , of regulation ( ce ) n. 854/2004 ( european commission , 2004 ) deals with what should be done with meat from infected animals , and lays down preventive measures to be taken with regard to the specific risks of brucellosis . indeed , the regulation requires that animals proving positive or uncertain for brucellosis have to be slaughtered separately , and that precautions have to be taken to avoid the risk of contaminating other carcasses , the butchery process and the abattoir staff . the udders , genital organs and blood of such animals are classified as unfit for human consumption , while their meat can be freely marketed . only if postmortem examination reveals lesions attributable to acute infection by brucella spp . otherwise , the meat of these animals , just like the meat of healthy animals , is allowed to enter the food chain . these measures are justified by the need to avoid the destruction of large amounts of meat , which is a valuable food source . nevertheless , the results of the present study show that the carcasses , and consequently the meat , of animals positive for brucellosis may be contaminated by brucella spp . this means that consumers may be exposed to the risk of infection , so they should be sufficiently informed with regard to the origin of the meat at the time of purchase . behet 's disease ( bd ) is a chronic inflammatory disease that is characterized by recurrent oral and genital ulceration and uveitis1 ) , the other clinical manifestations include skin lesions , arthritis , thrombosis , neurologic symptoms and intestinal ulceration . polychondritis is known as a rare complication of bd and no case of bd combined with polychondritis had been reported on until firestein et al . reported on five patients with both bd and relapsing polychondritis ( rp ) in 19852 ) . we recently experienced a case of a 40-year - old male patient with both bd and polychondritis , and we report herein on this unusual case along with a review of the literature . a 40-year - old man was referred to our hospital for further evaluation of his fever , oral ulcers and skin rashes that had lasted for 10 days . he had previously suffered from intermittent oral ulceration and uveitis that had been treated with oral prednisolone and intraocular injections of triamcinolone at another hospital 1 year prior to this admission . on the physical examination , his blood pressure was 130/80 mmhg , the pulse rate was 80 per minute and the body temperature was 38.4. he had conjunctival injections in both eyes and several aphthous ulcers on the tongue ( figure 1a ) and buccal mucosa . a small round ulcer with tenderness was found on the penile root ( figure 1b ) . several round erythematous skin lesions were present on the chest and thigh ( figure 2a ) . his left knee joint was swollen ( figure 2b ) and tender , and needle aspiration revealed turbid inflammatory synovial fluid . the laboratory findings were as follows : hemoglobin 14.8 g / dl , white blood cell count 10,400/l , platelet count 291,000/l , erythrocyte sedimentation rate 104 mm / hr and c - reactive protein 205 mg / l ( normal range , 0~3.4 mg / l ) . the renal and liver function tests were normal and the tests for rheumatoid factor and antinuclear antibody were negative . the urinalysis was normal and no microorganisms were grown on both the blood and urine cultures . his chest radiography showed no abnormal findings and mild splenomegaly was observed on abdominal sonography . skin biopsy was performed and the histologic examination showed perivascular mononuclear cell infiltrations , extravasation of the red blood cells and fibrinoid necrosis of the blood vessel walls ; all of these findings were compatible with cutaneous vasculitis combined with bd ( figure 3 ) . on the 5th day of admission , both ear auricles became swollen , red and tender ( figure 4 ) . he remembered having a similar auricular inflammation 2 years ago that had improved after taking non - steroidal anti - inflammatory drugs . after he was administered with colchicine 1.2 mg / day and prednisolone 30 mg / day , his clinical symptoms improved and he was discharged on the tenth day of admission . polychondritis is an inflammation of the various catilagenous structures such as the auricle , nose and trachea , and it has also been known to involve the eye and the joints . when polychondritis is relapsing , it is called relapsing polychondritis ( rp)8 ) . in rp , various cutaneous manifestations are also seen , including palpable purura , erythema nodosum , panniculitis , livedo reticularis and urticaria . there have been case reports on polychondritis combined with other rheumatologic diseases such as systemic lupus erythematosus ( sle ) , rheumatoid arthritis ( ra ) and systemic vasculitis9 - 11 ) . however , there have been no reports of polychondritis combined with bd until firestein et al . first reported on five patients who had the features characteristic of both bd and rp , and those authors termed this as mouth and genital ulcers with inflamed cartilage ( magic ) syndrome2 ) . polychondritis is still known as a rare complication of bd and only five more cases of bd combined with polychondritis have been reported on since firestein 's report3 - 7 ) . in our patient , bd was diagnosed based upon the recurrent oral and genital ulcers , the skin vasculitis , the arthritis and the past history of uveitis . the chondritis developed at the bilateral auricles , eye and the joints during the acute exacerbation period of bd , and this resolved with the improvement of the symptoms of bd . this suggests that the development of polychondritis may have been associated with bd in our case . as for treatment for the combined cases of bd and rp , colchicine , steroid and dapsone skin involvement in bd includes various lesions such as erythema nodosum , pustules , papules , pseudofolliculitis , acneiform folliculits , sweet 's syndrome , or pyoderma gangrenosum12 ) . the histopathology of the papulopustular lesions usually shows vasculopathy that is characterized by the perivascular infiltration of mononuclear cells , and this infiltrate is mainly composed of lymphocytes . the erythema nodosum - like lesion is known to be characterized by extravascular neutrophilic infiltration and panniculitis , and this can be followed by lymphocyte infiltration during the evolution of the skin lesions14 ) . in our case , the main histopathylogic finding of the skin lesions was perivascular mononuclear cell infiltration and fibrinoid necrosis of the vessel walls , which are the characteristic findings of vasculopathy . although cases of bd combined with polychondritis have rarely been reported , bd and polychondritis do not seem to be separate entities . compared the frequency of the various clinical features in bd and rp ( table 1 ) , and they showed that some of the manifestations that are considered unique for one disease could be found in the other disease2 ) . these similarities between the clinical manifestations of bd and rp imply that there seems to be something in common for the pathogenesis of bd and polychondritis . the pathogenesis of both bd and rp are associated with autoimmunity . in patients with rp , the antibodies to collagen type ii , ix and xi are elevated in the sera15 , 16 ) and the frequency of human leukocyte antigen ( hla)-dr4 is increased17 ) . additionally , rp has been associated with other autoimmune diseases such as ra and sle9 , 10 ) . in patients with bd , autoantibodies such as antiendothelial cell antibody and antibody to -tropomyosin have been found18 - 20 ) and it has been postulated that cross - reactivity and molecular mimicry between the peptides from the streptococcal proteins or the viral heat shock proteins ( hsps ) , which are homologues of human hsps , and the mucosal antigens result in the selection of autoreactive t cells21 ) . have proposed that autoimmunity to components of the cartilage other than type ii collagen , such as proteoglycans or elastic tissue , could be the common mechanism of bd and rp2 ) . in particular , elastic tissue is ubiquitous in the human mucosa that is mainly involved in bd , and in the cartilage too , which is the major target of rp . however , because of the rarity of the combined cases , there have not yet been any investigations about the common pathogenesis of the two diseases , and so additional research is clearly needed . although there have been only a few reports about polychondritis combined with bd , we think this combination of diseases might have been overlooked so far and closer scrutiny would reveal more cases .

a study was conducted in order to evaluate the contamination by brucella spp . of meat from animals slaughtered because they had resulted positive for brucellosis at some time during their life . after slaughter and before delivery to market outlets , swab samples were taken from 307 carcasses of infected animals : 40 cattle , 60 sheep and 207 goats . the swabs were subsequently analysed by means of polymerase chain reaction ( pcr ) tests . in addition , bacteriological tests were carried out on the lymph nodes and internal organs of the same animals . brucella spp . was detected by means of pcr in 25/307 carcasses ( 8% ) : 1 bovine ( 2.5% ) , 9 sheep ( 15% ) and 15 goats ( 7.2% ) and was isolated by means of a cultural method in 136/307 carcasses ( 44% ) . moreover , additional analysis , performed on lymph nodes from the same carcasses that had proved positive by pcr , allowed highlighting type 3 brucella abortus in the bovine carcass and type 3 brucella melitensis in the sheep and goat carcasses . the study shows that cattle , sheep and goats meat of animals slaughtered because they had tested positive for brucellosis may be contaminated by brucella spp . as this could constitute a real risk of transmission to both butchery personnel and consumers , the meat of animals infected by brucella spp . should be analysed before being marketed . in this respect , pcr technique performed on swabs proved to be more useful , practical and faster than the traditional bacteriological method . polychondritis is an inflammatory disorder that affects various catilagenous structures , and the clinical features include auricular , nasal and respiratory tract chondritis . it also involves the eyes , audiovestibular apparatus , joints and vascular structures . polychondritis can be associated with several rheumatologic diseases such as systemic lupus erythematosus , rheumatoid arthritis and systemic vasculitis . however , polychondritis is a rare complication of behet 's disease ( bd ) and only ten cases with combined bd and polychondritis have been reported on around the world . in this report , we describe a 40-year - old korean man with bd who suffered from polychondritis that manifested as bilateral auricular chondritis , conjunctivitis and arthritis .

before discussing the methodology , a detailed understanding of finite element method and its significance is essential . the basic idea in finite element method is to find the solution for a complicated problem by replacing it by a simpler one . in finite element method , the actual continuum or body of the matter these elements are considered to be interconnected at specified points , which are called nodes or nodal points . a variety of element types and shapes are available to provide users with the required flexibility to meet the compatibility and completeness requirements . it is useful in solving complex structural problems by dividing the complex structures into many simpler and smaller segments . the technical improvement in computers and finite element software , has improved accuracy and speed of this analysis . in the general field of medicine microsoft windows 98 se ( operating system)computer aided designing ( cad ) or modeling software proe-2001msc microsoft windows 98 se ( operating system ) computer aided designing ( cad ) or modeling software proe-2001 msc the creation of geometric model , mesh generation , and boundary conditions were applied . in the case of postprocessing simulating , creation of geometric model before developing a finite element model of any system , a geometric model should be generated using cad software . the three - dimensional model to create is a maxillary four incisors and its surrounding structures such as alveolar bone and its periodontal ligament . the morphological dimensions of the teeth used were as given by wheeler 's dental anatomy ( 6 edition ) . from the standard dimensions , the bounding points of the cross - section ( in space x , y , and z coordinates ) were determined and plotted in the cad graphics screen . with the bounding points called the key points , smooth lines were drawn connecting all the key points simulating the profile of the natural tooth . with the sketch model , volume for the section of the body is created ( solid modeling ) [ figures 1 and 2 ] . in a similar manner , the periodontal ligament surrounding the tooth and alveolar bone as it was difficult to obtain the hourglass shape of the periodontal ligament in our study , it was assumed to be 0.25 mm thickness around the radicular portion , although the thickness varies from the alveolar crest to the apex . the alveolar bone was modeled as a rectangular block of structure with labial thickness of 3 mm apically and 5 mm distally to the existing tooth [ figure 4 ] . sectioned natural tooth sectioned natural tooth periodontal ligament surrounding alveolar bone and tooth are modeled alveolar bone modeled in rectangular block finite element model generation ( mesh generation ) this forms the backbone of the finite element analysis . in this stage , the geometric model is converted into the finite element model . the cad model was meshed or divided into several three - dimensional tetrahedral shaped finite elements [ table 1 ] . tetrahedral elements are used in order to precisely mesh the curved and irregular regions present in the tooth [ figure 5 ] . the mechanical properties of each material such as bone , periodontal ligament and the tooth are taken from the previously published values [ table 2 ] . number of elements and nodes in the model mechanical properties for tooth , periodontal ligament and alveolar bone superior surface of bone model the boundary conditions with the areas where the model was restrained from any further movements were assigned as follows , at the superior surface of the bone model ( area representing the palatal surface ) , the area was constrained at their bases to avoid overall rigid body motion 3 [ figure 5 ] . the three - dimensional model was oriented in such a way that the mesiodistal plane was represented by x axis , the vertical plane by the y axis and the labiolingual plane by the z axis . to evaluate the center of resistance the force was applied at a distance of 5.2 mm from the incisal edge of the modeled tooth in the labiolingual direction ( along the z axis ) . the magnitude of force given was 15 g of intrusive force and 120 g of retractive force for each tooth . determination of center of resistance center of resistance of a tooth is a point through which a pure force if acts will produce linear movement without rotation . since , it is a geometric property the finite element analysis itself can easily locate it . microsoft windows 98 se ( operating system)computer aided designing ( cad ) or modeling software proe-2001msc microsoft windows 98 se ( operating system ) computer aided designing ( cad ) or modeling software proe-2001 msc the creation of geometric model , mesh generation , and boundary conditions were applied . in the case of postprocessing simulating , the tooth movement and determination of center of resistance were done . creation of geometric model before developing a finite element model of any system , a geometric model should be generated using cad software . the three - dimensional model to create is a maxillary four incisors and its surrounding structures such as alveolar bone and its periodontal ligament . the morphological dimensions of the teeth used were as given by wheeler 's dental anatomy ( 6 edition ) . from the standard dimensions , the bounding points of the cross - section ( in space x , y , and z coordinates ) were determined and plotted in the cad graphics screen . with the bounding points called the key points , smooth lines were drawn connecting all the key points simulating the profile of the natural tooth . with the sketch model , volume for the section of the body is created ( solid modeling ) [ figures 1 and 2 ] . in a similar manner , the periodontal ligament surrounding the tooth and alveolar bone as it was difficult to obtain the hourglass shape of the periodontal ligament in our study , it was assumed to be 0.25 mm thickness around the radicular portion , although the thickness varies from the alveolar crest to the apex . the alveolar bone was modeled as a rectangular block of structure with labial thickness of 3 mm apically and 5 mm distally to the existing tooth [ figure 4 ] . sectioned natural tooth sectioned natural tooth periodontal ligament surrounding alveolar bone and tooth are modeled alveolar bone modeled in rectangular block finite element model generation ( mesh generation ) this forms the backbone of the finite element analysis . in this stage , the geometric model is converted into the finite element model . the cad model was meshed or divided into several three - dimensional tetrahedral shaped finite elements [ table 1 ] . tetrahedral elements are used in order to precisely mesh the curved and irregular regions present in the tooth [ figure 5 ] . the mechanical properties of each material such as bone , periodontal ligament and the tooth are taken from the previously published values [ table 2 ] . number of elements and nodes in the model mechanical properties for tooth , periodontal ligament and alveolar bone superior surface of bone model the boundary conditions with the areas where the model was restrained from any further movements were assigned as follows , at the superior surface of the bone model ( area representing the palatal surface ) , the area was constrained at their bases to avoid overall rigid body motion 3 [ figure 5 ] . the three - dimensional model was oriented in such a way that the mesiodistal plane was represented by x axis , the vertical plane by the y axis and the labiolingual plane by the z axis . to evaluate the center of resistance the force was applied at a distance of 5.2 mm from the incisal edge of the modeled tooth in the labiolingual direction ( along the z axis ) . the magnitude of force given was 15 g of intrusive force and 120 g of retractive force for each tooth . determination of center of resistance center of resistance of a tooth is a point through which a pure force if acts will produce linear movement without rotation . since , it is a geometric property the finite element analysis itself can easily locate it . creation of geometric model before developing a finite element model of any system , a geometric model should be generated using cad software . the three - dimensional model to create is a maxillary four incisors and its surrounding structures such as alveolar bone and its periodontal ligament . the morphological dimensions of the teeth used were as given by wheeler 's dental anatomy ( 6 edition ) . from the standard dimensions , the bounding points of the cross - section ( in space x , y , and z coordinates ) were determined and plotted in the cad graphics screen . with the bounding points called the key points , smooth lines were drawn connecting all the key points simulating the profile of the natural tooth . with the sketch model , volume for the section of the body is created ( solid modeling ) [ figures 1 and 2 ] . in a similar manner , the periodontal ligament surrounding the tooth and alveolar bone were also modeled [ figure 3 ] . as it was difficult to obtain the hourglass shape of the periodontal ligament in our study , it was assumed to be 0.25 mm thickness around the radicular portion , although the thickness varies from the alveolar crest to the apex . the alveolar bone was modeled as a rectangular block of structure with labial thickness of 3 mm apically and 5 mm distally to the existing tooth [ figure 4 ] . sectioned natural tooth sectioned natural tooth periodontal ligament surrounding alveolar bone and tooth are modeled alveolar bone modeled in rectangular block finite element model generation ( mesh generation ) this forms the backbone of the finite element analysis . in this stage , the geometric model is converted into the finite element model . the cad model was meshed or divided into several three - dimensional tetrahedral shaped finite elements [ table 1 ] . tetrahedral elements are used in order to precisely mesh the curved and irregular regions present in the tooth [ figure 5 ] . the mechanical properties of each material such as bone , periodontal ligament and the tooth are taken from the previously published values [ table 2 ] . number of elements and nodes in the model mechanical properties for tooth , periodontal ligament and alveolar bone superior surface of bone model the boundary conditions with the areas where the model was restrained from any further movements were assigned as follows , at the superior surface of the bone model ( area representing the palatal surface ) , the area was constrained at their bases to avoid overall rigid body motion 3 [ figure 5 ] . the three - dimensional model was oriented in such a way that the mesiodistal plane was represented by x axis , the vertical plane by the y axis and the labiolingual plane by the z axis . to evaluate the center of resistance the force was applied at a distance of 5.2 mm from the incisal edge of the modeled tooth in the labiolingual direction ( along the z axis ) . the magnitude of force given was 15 g of intrusive force and 120 g of retractive force for each tooth . determination of center of resistance center of resistance of a tooth is a point through which a pure force if acts will produce linear movement without rotation . since , it is a geometric property the finite element analysis itself can easily locate it . the location of center of resistance has been studied over the years with numerous model systems , these studies include , analytical models , direct measurement in vivo , photo elastic technique , strain gauge technique , laser reflection technique , and holographic method , etc . although these model systems have provided some insight into the displacement characteristics of the tooth to the applied forces , these studies have obvious limitations such as direct measurement in vivo have a distinct disadvantage of being invasive . furthermore , it is difficult to apply controlled force variables on human subjects because of anatomic constraints . the major limitation in studies that used physical models was that synthetic substances were used to simulate the periodontal ligament . many of those materials did not have physical properties exactly duplicating that of the periodontal ligament . in the analytical techniques , the root morphology has been approximated to parabolid , conical or wedge shape leading to different results . to overcome the essence of this method is that a predetermined force is idealized into an assemblage of separated finite regions or elements . these are then considered to be interconnected at specific points called nodes on their common boundaries.[5 . the results obtained in this study is in accordance with the study of matsui et al . the study protocol was approved by the animal care and use committee of juntendo university . male c57bl/6 mice ( 7 weeks old ) were purchased from oriental yeast ( tokyo , japan ) and housed in specific pathogen deficient ( apoe ) mice ( 6 weeks old ) were purchased from the jackson laboratory ( bar harbor , me ) and housed in specific pathogen mice were maintained under 12-h light / dark cycle , fed a standard rodent diet ( from clea japan at nihon bioresearch ) , and provided with water ad libitum , except where noted . mice were treated with either high - dose ( 24 nmol kg body wt day ) or low - dose ( 300 pmol kg body wt day ) exendin-4 ( sigma - aldrich , tokyo , japan ) , or with saline through a mini - osmotic pump ( alzest , model 1004 ; durect , cupertino , ca ) that delivered the solution continuously for up to 28 days . at the age of 8 weeks , the osmotic pump was implanted under the skin of the back of each mouse after local anesthesia . islets were isolated by a standard collagenase digestion method as described previously ( 16 ) . mouse aortic vascular endothelial cells were isolated and cultured as described previously ( 17 ) . von willebrand factor antibody ( dako , carpinteria , ca ) and negative immunostaining with anti-smooth muscle actin ( sigma - aldrich ) . mouse aortic vascular smooth muscle cells were isolated and cultured as described previously ( 18 ) . the cultured cells were verified as smooth muscle cells by immunostaining using anti-smooth muscle actin . peritoneal macrophages were harvested from the mice with cold pbs at 3 days after intraperitoneal injection of 3% thioglycolate media . the pooled macrophages from each mouse were cultured in rpmi 1640 , supplemented with 0.2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c to allow cell adhesion . human peripheral blood mononuclear cells were isolated from whole blood collected from overnight - fasted healthy volunteers , with mono - poly resolving medium ( ds pharma biomedical , osaka , japan ) after heparinization . monocytes were isolated from peripheral blood mononuclear cells by positive selection using macs cd14 microbeads ( miltenyi biotec , bergisch gladbach , germany ) . we confirmed that 95% of the isolated cells were cd14 monocytes by flow cytometry analysis . cells were cultured in rpmi 1640 , supplemented with 2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol at 37c under a humidified atmosphere with 5% co2 . thp-1 cells were cultured in cultured in rpmi 1640 , supplemented with 10% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c . human umbilical vein endothelial cells ( huvecs ) were cultured in 500-ml bottle of endothelial cell basal medium-2 and the following growth supplements : 0.2 ml hydrocortisone , 2 ml human fibroblast growth factor basic , 0.5 ml vascular endothelial growth factor , 0.5 ml r3-igf-1 , 0.5 ml ascorbic acid , 0.5 ml heparin , 10 ml fbs , 0.5 ml human epidermal growth factor , and 0.5 ml ga-1000 ( cambrex bioscience walkersville , charles city , ia ) under 95% relative humidity and 5% co2 at 37c . all samples were sonicated on ice and centrifuged at 15,000 g at 4c for 20 min . the supernatants were collected and western blot analysis was performed using anti glp-1r antibody ( ab3907 ; abcam , cambridge , u.k . ) or rabbit anti glyceraldehyde-3-phosphate dehydrogenase antibody ( cell signaling technology , beverly , ma ) as described previously ( 16 ) . the intraperitoneal glucose tolerance test ( ipgtt ) was performed at the age of 12 weeks ( 4 weeks after implantation of the osmotic pump ) . briefly , 1.0 g / kg body wt glucose was injected intraperitoneally after overnight fasting . blood glucose level was measured with a glucometer ( one - touch ultra ; life scan , burnaby , canada ) . plasma insulin levels were measured using an elisa kit ( morinaga , kanagawa , japan ) . the insulin tolerance test was performed at the age of 12 weeks with 0.75 units / kg body wt insulin ( humalin ; blood samples were collected from the retro - orbital venous plexus in awake mice to measure blood glucose and plasma insulin concentrations . after mice were killed by intraperitoneal injection of sodium pentobarbital ( 1 mg / kg ; abbott laboratories ) , tissue preparation was performed by systemic perfusion with saline and 10% buffered formalin . fixation was performed by immersion of the isolated thoracic aorta with 10% buffered formalin at 4c . for en face immunohistochemistry of the endothelial surface , the thoracic aorta was cut open longitudinally along the ventral side with scissors and placed on a glass slide . then immunohistochemistry was performed using anti - mouse mac-2 monoclonal antibody ( dako , cederlane , burlington , on , canada ) . next , each specimen was placed on a slide glass with the intimal side up , and covered with a coverslip . specimens were viewed under a microscope ( e800 ; nikon , tokyo , japan ) connected to an xyz controller and a digital camera ( sony , tokyo , japan ) . to count the number of endothelium - adherent monocytes , we set a rectangular area with sides that were twice the length of the long and short diameters of the vessel opening of the intracostal arteries , respectively , and that were centered on the opening . the total number of mac-2immunopositive cells within the entire rectangular areas were counted in each aorta . the cell density in each area was then calculated as the cell count ( determined by an examiner blinded to the treatment regimen ) divided by the total area ( 1921 ) . for fluorescent staining , the samples were embedded in optimal cutting temperature compound , then sectioned , air dried , and washed in pbs . after immersion in blocking solution of 10% goat serum in pbs for 30 min at room temperature , the sections were incubated overnight at 4c in a humidified chamber for labeling with rabbit polyclonal anti - glp1 receptor antibody ( 1:50 , ls - a1205 ; mbl international , woburn , ma ) , and rabbit anti mac-2 monoclonal antibody ( 1:200 ; dako ) . the specimens were placed in the appropriate goat secondary antibody conjugated with alexa fluor dyes ( invitrogen , carlsbad , ca ) and diluted 1:300 in pbs for 30 min at room temperature . the specimen was placed on a glass slide , dapi - containing mounting medium ( vector laboratories , burlingame , ca ) was added , and then the tissue was covered with a cover glass . samples were viewed by confocal laser scanning microscopy ( fluoview fv1000 ; olympus , tokyo , japan ) . the heart and the aorta were flushed with normal saline followed by 10% buffered formalin as described previously ( 20 ) . for quantitative analysis of arteriosclerotic lesions in the aortic sinus , the heart was cut in two halves and the top half was embedded in optimal cutting temperature compound , then cross - sectioned at 4-m thickness at 50-m interval with a cryostat . twelve consecutive sections were taken sequentially from just above the aortic valve throughout the aortic sinus and allowed to dry at room temperature for 30 min . the mean lesion area of those 12 sections was calculated and expressed in square millimeters . the isolated macrophages were washed once and then incubated with or without 0.03 , 0.3 , and 3 nmol / l exendin-4 or 10 mol / l forskolin ( an adenylate cyclase activator ; sigma - aldrich ) for 1 h , followed by incubation with or without lipopolysaccharide ( lps , 1 g / ml ; sigma - aldrich ) for 1 h. to inhibit the exenatide signal , macrophages were incubated with 5 mol / l mdl-12330a ( sigma - aldrich ) , a specific adenylate cyclase inhibitor , and 10 mol / l pki14 - 22 ( sigma - aldrich ) , a protein kinase a ( pka ) inhibitor , for 30 min before adding exendin-4 . control macrophages were incubated with the vehicle ( dmso , final concentration < 0.1% ) . after treatment , nuclear protein extracts were isolated from peritoneal macrophages and the content of nuclear factor-b ( nf-b ) p65 was determined using a specific elisa kit using the method recommended by the manufacturer ( imgenex , san diego , ca ) ( 22 ) . total rna was extracted from peritoneal macrophages using the rna easymicro kit ( qiagen , tokyo , japan ) and the instructions provided by the manufacturer . first - strand cdna was synthesized using 1 g of total rna with oligo - dt primers and superscript reverse transcriptase ( invitrogen ) as described previously ( 23 ) . the resulting cdnas were amplified using the sybr green pcr kit ( applied biosystems , foster city , ca ) . quantitative pcr was performed with an abi prism 7700 sequence detection system ( perkin elmer life sciences , boston , ma ) . the relative abundances of mrnas were calculated by the comparative cycle of threshold method with tata box after collection and stabilization for 6 h , monocytes were cultured in the presence or absence of different concentrations of exendin-4 for 24 h. the culture supernatants were removed and cells washed with pbs to remove nonadherent cells . the fc receptors were blocked by clear back ( human fc receptor blocking reagent ; mbl , nagoya , japan ) for 5 min at room temperature . then the cells were stained with phycoerythrin labeled antibodies to anti - human cd11b or the corresponding isotype control ( nonspecific mouse igg - phycoerythrin ) ( bd biosciences , cologne , germany ) and incubated at 4c for 15 min . bonferroni multiple comparison test was used for comparisons among multiple treatment groups and the control group . the study protocol was approved by the animal care and use committee of juntendo university . male c57bl/6 mice ( 7 weeks old ) were purchased from oriental yeast ( tokyo , japan ) and housed in specific pathogen deficient ( apoe ) mice ( 6 weeks old ) were purchased from the jackson laboratory ( bar harbor , me ) and housed in specific pathogen mice were maintained under 12-h light / dark cycle , fed a standard rodent diet ( from clea japan at nihon bioresearch ) , and provided with water ad libitum , except where noted . mice were treated with either high - dose ( 24 nmol kg body wt day ) or low - dose ( 300 pmol kg body wt day ) exendin-4 ( sigma - aldrich , tokyo , japan ) , or with saline through a mini - osmotic pump ( alzest , model 1004 ; durect , cupertino , ca ) that delivered the solution continuously for up to 28 days . at the age of 8 weeks , the osmotic pump was implanted under the skin of the back of each mouse after local anesthesia . islets were isolated by a standard collagenase digestion method as described previously ( 16 ) . mouse aortic vascular endothelial cells were isolated and cultured as described previously ( 17 ) . von willebrand factor antibody ( dako , carpinteria , ca ) and negative immunostaining with anti-smooth muscle actin ( sigma - aldrich ) . mouse aortic vascular smooth muscle cells were isolated and cultured as described previously ( 18 ) . the cultured cells were verified as smooth muscle cells by immunostaining using anti-smooth muscle actin . peritoneal macrophages were harvested from the mice with cold pbs at 3 days after intraperitoneal injection of 3% thioglycolate media . the pooled macrophages from each mouse were cultured in rpmi 1640 , supplemented with 0.2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c to allow cell adhesion . human peripheral blood mononuclear cells were isolated from whole blood collected from overnight - fasted healthy volunteers , with mono - poly resolving medium ( ds pharma biomedical , osaka , japan ) after heparinization . monocytes were isolated from peripheral blood mononuclear cells by positive selection using macs cd14 microbeads ( miltenyi biotec , bergisch gladbach , germany ) . we confirmed that 95% of the isolated cells were cd14 monocytes by flow cytometry analysis . cells were cultured in rpmi 1640 , supplemented with 2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol at 37c under a humidified atmosphere with 5% co2 . thp-1 cells were cultured in cultured in rpmi 1640 , supplemented with 10% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c . human umbilical vein endothelial cells ( huvecs ) were cultured in 500-ml bottle of endothelial cell basal medium-2 and the following growth supplements : 0.2 ml hydrocortisone , 2 ml human fibroblast growth factor basic , 0.5 ml vascular endothelial growth factor , 0.5 ml r3-igf-1 , 0.5 ml ascorbic acid , 0.5 ml heparin , 10 ml fbs , 0.5 ml human epidermal growth factor , and 0.5 ml ga-1000 ( cambrex bioscience walkersville , charles city , ia ) under 95% relative humidity and 5% co2 at 37c . all samples were sonicated on ice and centrifuged at 15,000 g at 4c for 20 min . the supernatants were collected and western blot analysis was performed using anti glp-1r antibody ( ab3907 ; abcam , cambridge , u.k . ) or rabbit anti glyceraldehyde-3-phosphate dehydrogenase antibody ( cell signaling technology , beverly , ma ) as described previously ( 16 ) . the intraperitoneal glucose tolerance test ( ipgtt ) was performed at the age of 12 weeks ( 4 weeks after implantation of the osmotic pump ) . briefly , 1.0 g / kg body wt glucose was injected intraperitoneally after overnight fasting . blood glucose level was measured with a glucometer ( one - touch ultra ; life scan , burnaby , canada ) . plasma insulin levels were measured using an elisa kit ( morinaga , kanagawa , japan ) . the insulin tolerance test was performed at the age of 12 weeks with 0.75 units / kg body wt insulin ( humalin ; eli lilly , indianapolis , in ) after 6 h of fasting . blood samples were collected from the retro - orbital venous plexus in awake mice to measure blood glucose and plasma insulin concentrations . after mice were killed by intraperitoneal injection of sodium pentobarbital ( 1 mg / kg ; abbott laboratories ) , tissue preparation was performed by systemic perfusion with saline and 10% buffered formalin . fixation was performed by immersion of the isolated thoracic aorta with 10% buffered formalin at 4c . for en face immunohistochemistry of the endothelial surface , the thoracic aorta was cut open longitudinally along the ventral side with scissors and placed on a glass slide . then immunohistochemistry was performed using anti - mouse mac-2 monoclonal antibody ( dako , cederlane , burlington , on , canada ) . next , each specimen was placed on a slide glass with the intimal side up , and covered with a coverslip . specimens were viewed under a microscope ( e800 ; nikon , tokyo , japan ) connected to an xyz controller and a digital camera ( sony , tokyo , japan ) . to count the number of endothelium - adherent monocytes , we set a rectangular area with sides that were twice the length of the long and short diameters of the vessel opening of the intracostal arteries , respectively , and that were centered on the opening . the total number of mac-2immunopositive cells within the entire rectangular areas were counted in each aorta . the cell density in each area was then calculated as the cell count ( determined by an examiner blinded to the treatment regimen ) divided by the total area ( 1921 ) . for fluorescent staining , the samples were embedded in optimal cutting temperature compound , then sectioned , air dried , and washed in pbs . after immersion in blocking solution of 10% goat serum in pbs for 30 min at room temperature , the sections were incubated overnight at 4c in a humidified chamber for labeling with rabbit polyclonal anti - glp1 receptor antibody ( 1:50 , ls - a1205 ; mbl international , woburn , ma ) , and rabbit anti mac-2 monoclonal antibody ( 1:200 ; dako ) . the specimens were placed in the appropriate goat secondary antibody conjugated with alexa fluor dyes ( invitrogen , carlsbad , ca ) and diluted 1:300 in pbs for 30 min at room temperature . the specimen was placed on a glass slide , dapi - containing mounting medium ( vector laboratories , burlingame , ca ) was added , and then the tissue was covered with a cover glass . samples were viewed by confocal laser scanning microscopy ( fluoview fv1000 ; olympus , tokyo , japan ) . the heart and the aorta were flushed with normal saline followed by 10% buffered formalin as described previously ( 20 ) . for quantitative analysis of arteriosclerotic lesions in the aortic sinus , the heart was cut in two halves and the top half was embedded in optimal cutting temperature compound , then cross - sectioned at 4-m thickness at 50-m interval with a cryostat . twelve consecutive sections were taken sequentially from just above the aortic valve throughout the aortic sinus and allowed to dry at room temperature for 30 min . the mean lesion area of those 12 sections was calculated and expressed in square millimeters . the isolated macrophages were washed once and then incubated with or without 0.03 , 0.3 , and 3 nmol / l exendin-4 or 10 mol / l forskolin ( an adenylate cyclase activator ; sigma - aldrich ) for 1 h , followed by incubation with or without lipopolysaccharide ( lps , 1 g / ml ; sigma - aldrich ) for 1 h. to inhibit the exenatide signal , macrophages were incubated with 5 mol / l mdl-12330a ( sigma - aldrich ) , a specific adenylate cyclase inhibitor , and 10 mol / l pki14 - 22 ( sigma - aldrich ) , a protein kinase a ( pka ) inhibitor , for 30 min before adding exendin-4 . control macrophages were incubated with the vehicle ( dmso , final concentration < 0.1% ) . nuclear protein extracts were isolated from peritoneal macrophages and the content of nuclear factor-b ( nf-b ) p65 was determined using a specific elisa kit using the method recommended by the manufacturer ( imgenex , san diego , ca ) ( 22 ) . total rna was extracted from peritoneal macrophages using the rna easymicro kit ( qiagen , tokyo , japan ) and the instructions provided by the manufacturer . first - strand cdna was synthesized using 1 g of total rna with oligo - dt primers and superscript reverse transcriptase ( invitrogen ) as described previously ( 23 ) . the resulting cdnas were amplified using the sybr green pcr kit ( applied biosystems , foster city , ca ) . quantitative pcr was performed with an abi prism 7700 sequence detection system ( perkin elmer life sciences , boston , ma ) . the relative abundances of mrnas were calculated by the comparative cycle of threshold method with tata box after collection and stabilization for 6 h , monocytes were cultured in the presence or absence of different concentrations of exendin-4 for 24 h. the culture supernatants were removed and cells washed with pbs to remove nonadherent cells . the fc receptors were blocked by clear back ( human fc receptor blocking reagent ; mbl , nagoya , japan ) for 5 min at room temperature . then the cells were stained with phycoerythrin labeled antibodies to anti - human cd11b or the corresponding isotype control ( nonspecific mouse igg - phycoerythrin ) ( bd biosciences , cologne , germany ) and incubated at 4c for 15 min . bonferroni multiple comparison test was used for comparisons among multiple treatment groups and the control group . as a first step to elucidate the antiatherosclerotic effects of exendin-4 , we first investigated the expression of glp-1 receptor in cells associated with atherosclerogenesis . similar to lung and pancreatic -cells ( 24 ) , mice peritoneal macrophages and vascular smooth muscle cells abundantly expressed glp-1 receptor protein , and the expression level was higher than in freshly isolated endothelial cells . similar to the expression level in macrophages , glp-1 receptor was abundantly expressed in thp-1 cells , which are derived from human monocytes , and freshly isolated human monocytes . in contrast to the freshly isolated endothelial cells , abundant expression of glp-1 receptor was detected in huvecs ( fig . in addition , immunohistochemical staining showed glp-1 receptor expression in cells that expressed mac-2 , a marker of macrophages located in the atherosclerotic lesions of the aortic valve of apoe mice ( fig . these results may suggest that glp-1 can directly act on monocytes or macrophages and affect the progression of atherosclerosis . a : expression of glp-1 receptor in murine lung and liver , isolated murine islets , isolated murine macrophages ( ms ) , cultured murine endothelial cells ( ecs ) , cultured murine smooth muscle cells ( smcs ) , human monocyte derived line , thp-1 cells , and huvecs . c : immunohistochemical staining of glp-1 receptor ( green ) and mac-2 , a marker of macrophages ( red ) in atherosclerotic lesions of apoe mice . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the effect of glp-1 receptor activation on atherosclerosis , c57bl/6 mice received continuous infusion of 300 pmol kg day ( low dose ) or 24 nmol kg day ( high dose ) exendin-4 for 28 days . during the treatment period , neither dose affected body weight ( fig . 2a ) . after the 24-day treatment , both doses of exendin-4 improved glucose tolerance without affecting insulin secretion ( fig . the results of the insulin tolerance test were similar in the two groups ( fig . treatment with high - dose exendin-4 , but not the low dose , slightly increased total cholesterol and hdl cholesterol compared with control group ( table 1 ) . the density of monocytes that adhered to the endothelial cells of the thoracic aorta was markedly suppressed in both the low- and high - dose treatment groups , compared with control ( fig . exendin-4 reduced monocytic adhesion to the endothelium in c57bl/6 mice . a : changes in body weight during treatment with exendin-4 in c57bl/6 mice ( n = 6 each ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 each ) . e : the density of adherent mac-2positive cells on endothelial cells at branching areas in each group of mice after 28-day treatment ( n = 6 ) with representative en face views of immunohistologic staining with mac-2 antibody . data are mean sem . * p < 0.05 versus high - dose group , + p < 0.05 versus low - dose group . ( a high - quality digital representation of this figure is available in the online issue . ) results of laboratory tests in c57bl/6 mice and apoe mice after 28-day treatment with exendin-4 data are mean se . blood samples were collected from c57bl/6j mice ( n = 6 ) and apoe mice ( n = 13 except for hba1c , n = 6 for hba1c ) in the fasting state after 28-day treatment with exendin-4 . * p < 0.05 vs. control group . p < 0.05 for low ex4 vs. high ex4 . low ex4 , low - dose exendin-4 ; high ex4 , high - dose exendin-4 ; tg , triglycerides ; cm , chylomicron ; sd - ldl , small , dense ldl ; hba1c , a1c ; na , not applicable . to explore the role of glp-1 receptor activation on the progression of atherosclerosis treatment with high - dose exendin-4 modestly reduced body weight gain and glucose tolerance and decreased serum total cholesterol level without affecting ldl cholesterol level ( fig . on the other hand , treatment with low - dose exendin-4 only modestly reduced glucose level at 30 min after glucose injection without affecting other parameters investigated ( fig . the density of monocytes that adhered to endothelial cells of the thoracic aorta was significantly lower in the low- and high - dose groups than the control group ( fig . quantification of mrna expression in the thoracic aorta showed that exendin-4 treatment significantly downregulated intercellular adhesion molecule-1 ( icam-1 ) and tended to downregulate vcam-1 ( fig . the oil red o positive area at the aortic valve level was significantly reduced in the high - dose group compared with the control group ( fig . the area of the atherosclerotic lesions in the low - dose group also tended to be smaller than the control group , however the difference was not significant . the metabolic effect of exendin-4 in apoe mice . a : changes in body weight during exendin-4 treatment in apoe mice ( n = 13 ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 ) . data are mean sem . * p < 0.01 versus high - dose group , + p < 0.01 versus low - dose group . exendin-4 reduced monocyte adhesion to the endothelium and atherosclerotic lesions in apoe mice . a : en face immunohistochemical staining with mac-2 antibody of the aorta of each group . the density of adherent mac-2positive cells on the endothelium at branching areas in each group of mice after 28-day treatment ( n = 7 ) and representative en face views of immunohistologic staining with mac-2 antibody . b : aortas harvested from each group of mice after 28-day treatment were used for isolation of total rna . the mrna expression levels of icam-1 and vcam-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in the test mice relative to that in the control group ( set at 1.0 , n = 57 ) . c : representative histologic sections of the aortic sinuses stained with oil red o after 28-day treatment . the mean area of oil red o positive lesions was determined ( n = 20 ) . ( a high - quality digital representation of this figure is available in the online issue . ) the data obtained from apoe mice and c57bl/6 mice suggested that exendin-4 could have beneficial effects against atherosclerosis without affecting the metabolic parameters and that it could potentially prevent the progression of atherosclerosis by its direct action on the cells involved in atherosclerogenesis . the abundant expression of glp-1 receptor in monocytes / macrophages and the inhibitory effects of exendin-4 on monocyte adhesion on endothelial cells encouraged us to investigate the effects of exendin-4 on the inflammatory response . indeed , incubation with 1 g / ml of lps for 1 h induced 10-fold increases in the expression levels of tnf- and monocyte chemoattractant protein-1 ( mcp-1 ) , a representative cytokine and a chemokine in isolated macrophages , respectively ( data not shown ) . thus , we investigated the effects of various concentrations of exendin-4 ( 0.033 nmol / l ) in counteracting this response . exendin-4 at all concentrations significantly suppressed lps - induced increases in the expression levels of tnf- and mcp-1 in macrophages ( fig . glp-1 receptor is well - known gs - protein coupled receptor , thus the activation of glp-1 receptor results in increased camp concentration due to activation of adenylate cyclase ( 25 ) . to explore the mechanism of exendin-4induced suppression of tnf- and mcp-1 expression in macrophages , we preincubated peritoneal macrophages with mdl-12330a , a specific adenylyl cyclase inhibitor , or forskolin , an adenylyl cyclase activator . the addition of mdl-12330a completely suppressed the inhibitory effect of exendin-4 on the expression levels of tnf- and mcp-1 ( fig . on the other hand , forskolin significantly suppressed lps - induced tnf- and mcp-1 expression in macrophages , and the levels of suppression by forskolin were similar to those of exendin-4 ( fig . these results suggest that the inhibitory effects of exendin-4 on the expression of tnf- and mcp-1 are largely dependent on the activation of adenylate cyclase . next , we investigated the downstream pathway of camp using pki14 - 22 , a specific pka inhibitor . similar to mdl-12330a , the inhibitory effect of exendin-4 was significantly reversed by pki14 - 22 ( fig . 5d ) , suggesting the involvement of pka in the anti - inflammatory effect of exendin-4 . exendin-4 reduced the inflammatory response through camp signaling pathway in macrophages , and reduced the expression of cd11b in human monocytes . a : peritoneal macrophages isolated from 8-week - old c57bl/6 mice were incubated with various concentrations of exendin-4 ( 0.033 nmol / l ) for 1 h followed by treatment with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna . the mrna expression levels of tnf- and mcp-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in peritoneal macrophages without the addition of exendin-4 set at 1.0 ( n = 45 ) . b : peritoneal macrophages were preincubated with 5 mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 46 ) . / l exendin-4 or 10 mol / l forskolin for 1 h followed by lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . mol / l pki14 - 22 for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of nuclear protein extracts . the nuclear level of nf-b p65 was determined by enzyme - linked immunosorbent assay ( elisa ) ( n = 34 ) . f : human monocytes isolated from healthy volunteers were incubated without or with various concentrations of exendin-4 ( 0.033 nmol / l ) for 24 h. then , the surface expression of cd11b was assessed by flow cytometry . although nf-b is a major regulator of the expression of tnf- and mcp-1 , vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide are known to inhibit nf-b dependent gene activation by activation of pka in cultured monocytic cell line thp-1 ( 26 ) . thus , we investigated the effect of exendin-4 on lps - induced nuclear translocation of nf-b p65 in macrophages . without any stimulation , nuclear nf-b p65 was not detected in peritoneal macrophages , however , stimulation with lps robustly induced nuclear translocation of nf-b p65 ( data not shown ) . such translocation was markedly suppressed by exendin-4 , and this inhibitory effect was completely abolished by mdl-12330a ( fig . these results indicate that exendin-4 inhibits nuclear translocation of nf-b p65 by activating camp , in parallel with the expression of tnf- and mcp-1 . finally , we investigated the effect of exendin-4 on human monocytes . thus , we investigated the effects of exendin-4 on their counterreceptors , cd11b , in isolated human peripheral monocytes . exposure to 0.3 and 3 nmol / l , but not 0.03 nmol / l , exendin-4 for 24 h significantly reduced the surface expression of cd11b , as assessed by flow cytometry ( fig . these results suggest that activation of the glp-1 receptor has the antiatherogenic effects on human circulating monocytes . as a first step to elucidate the antiatherosclerotic effects of exendin-4 , we first investigated the expression of glp-1 receptor in cells associated with atherosclerogenesis . similar to lung and pancreatic -cells ( 24 ) , mice peritoneal macrophages and vascular smooth muscle cells abundantly expressed glp-1 receptor protein , and the expression level was higher than in freshly isolated endothelial cells . similar to the expression level in macrophages , glp-1 receptor was abundantly expressed in thp-1 cells , which are derived from human monocytes , and freshly isolated human monocytes . in contrast to the freshly isolated endothelial cells , abundant expression of glp-1 receptor was detected in huvecs ( fig . in addition , immunohistochemical staining showed glp-1 receptor expression in cells that expressed mac-2 , a marker of macrophages located in the atherosclerotic lesions of the aortic valve of apoe mice ( fig . these results may suggest that glp-1 can directly act on monocytes or macrophages and affect the progression of atherosclerosis . a : expression of glp-1 receptor in murine lung and liver , isolated murine islets , isolated murine macrophages ( ms ) , cultured murine endothelial cells ( ecs ) , cultured murine smooth muscle cells ( smcs ) , human monocyte derived line , thp-1 cells , and huvecs . c : immunohistochemical staining of glp-1 receptor ( green ) and mac-2 , a marker of macrophages ( red ) in atherosclerotic lesions of apoe mice . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the effect of glp-1 receptor activation on atherosclerosis , c57bl/6 mice received continuous infusion of 300 pmol kg day ( low dose ) or 24 nmol kg day ( high dose ) exendin-4 for 28 days . during the treatment period , neither dose affected body weight ( fig . 2a ) . after the 24-day treatment , both doses of exendin-4 improved glucose tolerance without affecting insulin secretion ( fig . the results of the insulin tolerance test were similar in the two groups ( fig . treatment with high - dose exendin-4 , but not the low dose , slightly increased total cholesterol and hdl cholesterol compared with control group ( table 1 ) . the density of monocytes that adhered to the endothelial cells of the thoracic aorta was markedly suppressed in both the low- and high - dose treatment groups , compared with control ( fig . exendin-4 reduced monocytic adhesion to the endothelium in c57bl/6 mice . a : changes in body weight during treatment with exendin-4 in c57bl/6 mice ( n = 6 each ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 each ) . e : the density of adherent mac-2positive cells on endothelial cells at branching areas in each group of mice after 28-day treatment ( n = 6 ) with representative en face views of immunohistologic staining with mac-2 antibody . data are mean sem . * p < 0.05 versus high - dose group , + p < 0.05 versus low - dose group . ( a high - quality digital representation of this figure is available in the online issue . ) results of laboratory tests in c57bl/6 mice and apoe mice after 28-day treatment with exendin-4 data are mean se . blood samples were collected from c57bl/6j mice ( n = 6 ) and apoe mice ( n = 13 except for hba1c , n = 6 for hba1c ) in the fasting state after 28-day treatment with exendin-4 . * p < 0.05 vs. control group . p < 0.05 for low ex4 vs. high ex4 . low ex4 , low - dose exendin-4 ; high ex4 , high - dose exendin-4 ; tg , triglycerides ; cm , chylomicron ; sd - ldl , small , dense ldl ; hba1c , a1c ; na , not applicable . to explore the role of glp-1 receptor activation on the progression of atherosclerosis , we treated apoe mice with low- or high - dose exendin-4 . treatment with high - dose exendin-4 modestly reduced body weight gain and glucose tolerance and decreased serum total cholesterol level without affecting ldl cholesterol level ( fig . 3 , table 1 ) . on the other hand , treatment with low - dose exendin-4 only modestly reduced glucose level at 30 min after glucose injection without affecting other parameters investigated ( fig . the density of monocytes that adhered to endothelial cells of the thoracic aorta was significantly lower in the low- and high - dose groups than the control group ( fig . quantification of mrna expression in the thoracic aorta showed that exendin-4 treatment significantly downregulated intercellular adhesion molecule-1 ( icam-1 ) and tended to downregulate vcam-1 ( fig . the oil red o positive area at the aortic valve level was significantly reduced in the high - dose group compared with the control group ( fig . the area of the atherosclerotic lesions in the low - dose group also tended to be smaller than the control group , however the difference was not significant . the metabolic effect of exendin-4 in apoe mice . a : changes in body weight during exendin-4 treatment in apoe mice ( n = 13 ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 ) . * p < 0.01 versus high - dose group , + p < 0.01 versus low - dose group . exendin-4 reduced monocyte adhesion to the endothelium and atherosclerotic lesions in apoe mice . a : en face immunohistochemical staining with mac-2 antibody of the aorta of each group . the density of adherent mac-2positive cells on the endothelium at branching areas in each group of mice after 28-day treatment ( n = 7 ) and representative en face views of immunohistologic staining with mac-2 antibody . b : aortas harvested from each group of mice after 28-day treatment were used for isolation of total rna . the mrna expression levels of icam-1 and vcam-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in the test mice relative to that in the control group ( set at 1.0 , n = 57 ) . c : representative histologic sections of the aortic sinuses stained with oil red o after 28-day treatment . the mean area of oil red o positive lesions was determined ( n = 20 ) . ( a high - quality digital representation of this figure is available in the online issue . ) the data obtained from apoe mice and c57bl/6 mice suggested that exendin-4 could have beneficial effects against atherosclerosis without affecting the metabolic parameters and that it could potentially prevent the progression of atherosclerosis by its direct action on the cells involved in atherosclerogenesis . the abundant expression of glp-1 receptor in monocytes / macrophages and the inhibitory effects of exendin-4 on monocyte adhesion on endothelial cells encouraged us to investigate the effects of exendin-4 on the inflammatory response . indeed , incubation with 1 g / ml of lps for 1 h induced 10-fold increases in the expression levels of tnf- and monocyte chemoattractant protein-1 ( mcp-1 ) , a representative cytokine and a chemokine in isolated macrophages , respectively ( data not shown ) . thus , we investigated the effects of various concentrations of exendin-4 ( 0.033 nmol / l ) in counteracting this response . exendin-4 at all concentrations significantly suppressed lps - induced increases in the expression levels of tnf- and mcp-1 in macrophages ( fig . glp-1 receptor is well - known gs - protein coupled receptor , thus the activation of glp-1 receptor results in increased camp concentration due to activation of adenylate cyclase ( 25 ) . to explore the mechanism of exendin-4induced suppression of tnf- and mcp-1 expression in macrophages , we preincubated peritoneal macrophages with mdl-12330a , a specific adenylyl cyclase inhibitor , or forskolin , an adenylyl cyclase activator . the addition of mdl-12330a completely suppressed the inhibitory effect of exendin-4 on the expression levels of tnf- and mcp-1 ( fig . on the other hand , forskolin significantly suppressed lps - induced tnf- and mcp-1 expression in macrophages , and the levels of suppression by forskolin were similar to those of exendin-4 ( fig . these results suggest that the inhibitory effects of exendin-4 on the expression of tnf- and mcp-1 are largely dependent on the activation of adenylate cyclase . next , we investigated the downstream pathway of camp using pki14 - 22 , a specific pka inhibitor . similar to mdl-12330a , the inhibitory effect of exendin-4 was significantly reversed by pki14 - 22 ( fig . 5d ) , suggesting the involvement of pka in the anti - inflammatory effect of exendin-4 . exendin-4 reduced the inflammatory response through camp signaling pathway in macrophages , and reduced the expression of cd11b in human monocytes . a : peritoneal macrophages isolated from 8-week - old c57bl/6 mice were incubated with various concentrations of exendin-4 ( 0.033 nmol / l ) for 1 h followed by treatment with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna . the mrna expression levels of tnf- and mcp-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in peritoneal macrophages without the addition of exendin-4 set at 1.0 ( n = 45 ) . b : peritoneal macrophages were preincubated with 5 mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 46 ) . c : peritoneal macrophages were incubated with 0.3 nmol / l exendin-4 or 10 mol / l forskolin for 1 h followed by lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . mol / l pki14 - 22 for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . e : peritoneal macrophages were preincubated with 5 mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of nuclear protein extracts . the nuclear level of nf-b p65 was determined by enzyme - linked immunosorbent assay ( elisa ) ( n = 34 ) . f : human monocytes isolated from healthy volunteers were incubated without or with various concentrations of exendin-4 ( 0.033 nmol / l ) for 24 h. then , the surface expression of cd11b was assessed by flow cytometry . although nf-b is a major regulator of the expression of tnf- and mcp-1 , vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide are known to inhibit nf-b dependent gene activation by activation of pka in cultured monocytic cell line thp-1 ( 26 ) . thus , we investigated the effect of exendin-4 on lps - induced nuclear translocation of nf-b p65 in macrophages . without any stimulation , nuclear nf-b p65 was not detected in peritoneal macrophages , however , stimulation with lps robustly induced nuclear translocation of nf-b p65 ( data not shown ) . such translocation was markedly suppressed by exendin-4 , and this inhibitory effect was completely abolished by mdl-12330a ( fig . these results indicate that exendin-4 inhibits nuclear translocation of nf-b p65 by activating camp , in parallel with the expression of tnf- and mcp-1 . finally , we investigated the effect of exendin-4 on human monocytes . thus , we investigated the effects of exendin-4 on their counterreceptors , cd11b , in isolated human peripheral monocytes . exposure to 0.3 and 3 nmol / l , but not 0.03 nmol / l , exendin-4 for 24 h significantly reduced the surface expression of cd11b , as assessed by flow cytometry ( fig . these results suggest that activation of the glp-1 receptor has the antiatherogenic effects on human circulating monocytes . in the present study , we provide evidence that exendin-4 , a glp-1 receptor agonist , prevents the progression of atherosclerosis in apoe mice without major effects on metabolic parameters . our data suggest that exendin-4 markedly reduced the accumulation of monocytes / macrophages in the vascular wall at least in part by suppressing the inflammatory response in macrophages through the activation of the camp / pka pathway . the results showed that the exendin-4 decreased monocyte adhesion to endothelial cells in two nondiabetic mice , c57bl6 and apoe mice . in both mouse strains , exendin-4 reduced glucose level during ipgtt , however , because the two strains are nondiabetic , the effect of exendin-4 on glucose level should play only a minor effect on its antiatherosclerogenetic properties . in this study , we confirmed the expression of glp-1 receptor in endothelial cells , smooth muscle cells , macrophages , and monocytes . because these cells play critical roles in the progression of atherosclerosis , glp-1 receptor activation in these cells may be involved in atherosclerogenesis . indeed , several groups reported the beneficial effects of glp-1 receptor activation on endothelial cells ( 1013 ) . on the other hand , we confirmed in the present study the direct anti - inflammatory effect of glp-1 on monocytes / macrophages . indeed , treatment with exendin-4 at concentrations observed during treatment of humans ( 27,28 ) reduced the expression of inflammatory mediators tnf- and mcp-1 in activated macrophages . tnf- and mcp-1 are among the important cytokines and chemokines whose atherogenic effect has been established . both tnf- and furthermore , forced expression of tnf- and mcp-1 in leukocytes promotes advanced atherosclerotic lesions ( 29,31 ) . thus , in addition to the effect of glp-1 on endothelial cells , its effect on monocytes / macrophages may also have a major impact on the attenuation of atherosclerosis . it was reported previously that the main effects of glp-1 are mediated through the activation of adenylate cyclase and the production of camp ( 25 ) . using adenylate cyclase inhibitor and activator , we also demonstrated in this study that stimulation of camp by exendin-4 is critical for the attenuated production of proinflammatory mediators from macrophages . this result is consistent with previous studies in which increased intracellular levels of camp inhibited tnf- production and its transcription in macrophage ( 3234 ) . these data suggest that exendin-4 regulates inflammatory response of macrophages via the camp / pka pathway , which inhibits proinflammatory cytokine production as reported recently ( 35,36 ) . our results showed that pka activation and inhibition of nf-b p65 translocation mediate overexpression of inflammatory cytokines by increased camp level elicited by glp-1 receptor activation . the adhesion of circulating monocytes to the intimal endothelial cells is thought to be one of the earliest events in the complex pathologic process of atherosclerogenesis ( 14,15 ) . this can be mediated by the interaction of specific adhesion molecules on vascular endothelial cells with their integrin counterreceptors on monocytes . activation of monocytes by cytokines , chemokines , hypercholesterolemia , and hyperglycemia leads to increased expression of this integrin , and increased monocyte expression of cd11b correlates with adhesion of these cells to the endothelium in patients with hypercholesterolemia ( 37 ) . our results showed a potential suppressive effect of exendin-4 on the surface expression of cd11b on human monocytes . on the other hand , we demonstrated that exendin-4 decreased the expression of icam-1 , which interacts with cd11b on monocytes in apoe mice . these effects may contribute at least in part to the reduced monocyte adhesion to the endothelium in vivo . however , additional experiments are required to clarify the mechanism of exendin-4induced inhibition of cd11b expression . in conclusion , our data suggest that glp-1 receptor activation significantly reduced the accumulation of monocytes / macrophages in the vascular wall and eventually inhibited atherosclerogenesis by regulating inflammation in macrophages via the camp / pka pathway and the integrin - related gene expression on monocytes . these unique effects of glp-1 receptor activation may help design new therapies for cardiovascular disease in patients with type 2 diabetes .

aim : the aim of this study is to evaluate the center of resistance of maxillary incisors during simultaneous intrusion and retraction.subjects and methods : in this study , the following steps were employed namely , ( 1 ) preprocessing - the creation of geometric model , mesh generation and boundary conditions . ( 2 ) postprocessing - the tooth movement and determination of center of resistance.results:the center of the mid - sagittal plane approximately 6 mm apical and 4 mm posterior to a line perpendicular to the occlusal plane from the labial alveolar crest of the central incisor resistance for the maxillary four incisors was located within the.conclusion:finite element is a sound mechanical method of analysis as it was sufficient enough to provide insight into interactions between orthodontic forces , and dental tissues and reliability of this study . further by using this study clinically , the center of resistance can be precisely located in single rooted tooth during orthodontic treatment . objectiveexogenous administration of glucagon - like peptide-1 ( glp-1 ) or glp-1 receptor agonists such as an exendin-4 has direct beneficial effects on the cardiovascular system . however , their effects on atherosclerogenesis have not been elucidated . the aim of this study was to investigate the effects of glp-1 on accumulation of monocytes / macrophages on the vascular wall , one of the earliest steps in atherosclerogenesis.research design and methodsafter continuous infusion of low ( 300 pmol kg1 day1 ) or high ( 24 nmol kg1 day1 ) dose of exendin-4 in c57bl/6 or apolipoprotein e deficient mice ( apoe/ ) , we evaluated monocyte adhesion to the endothelia of thoracic aorta and arteriosclerotic lesions around the aortic valve . the effects of exendin-4 were investigated in mouse macrophages and human monocytes.resultstreatment with exendin-4 significantly inhibited monocytic adhesion in the aortas of c57bl/6 mice without affecting metabolic parameters . in apoe/ mice , the same treatment reduced monocyte adhesion to the endothelium and suppressed atherosclerogenesis . in vitro treatment of mouse macrophages with exendin-4 suppressed lipopolysaccharide - induced mrna expression of tumor necrosis factor- and monocyte chemoattractant protein-1 , and suppressed nuclear translocation of p65 , a component of nuclear factor-b . this effect was reversed by either mdl-12330a , a camp inhibitor or pki14 - 22 , a protein kinase a specific inhibitor . in human monocytes , exendin-4 reduced the expression of cd11b.conclusionsour data suggested that glp-1 receptor agonists reduced monocyte / macrophage accumulation in the arterial wall by inhibiting the inflammatory response in macrophages , and that this effect may contribute to the attenuation of atherosclerotic lesion by exendin-4 .

disability prevalence increases with advanced age , and disability in the elderly population is a major public health problem . this is especially crucial for chinese : there are 88 million persons aged 65 and above in china alone , and the number is projected to increase to 341 million in 2050 ; importantly , disability prevalence in chinese elderly population has been increasing [ 35 ] in contrast to a declining trend in the developed countries [ 6 , 7 ] . there is a big gap between the scale of the problem and the quantity and quality of available information . the first population - based report from china was published 16 years ago , and countable number of publications [ 35 , 812 ] exist in the literature . in this study , with large population - based data from shanghai ( the biggest city in china ) and singapore ( a city country in southeast asia , with majority of the residents being descendents of immigrants from south china ) , we aimed to examine the independent contributions of physical and cognitive health to disability in chinese older adults and explore the potential influences of environment . participants of the present study were identified from two big population - based studies : the shanghai survey of alzheimer 's disease and dementia ( ssadd ) and the singapore longitudinal ageing study ( slas ) . the ssadd was conducted in 1987 based on a probability sample of 5055 community - dwelling chinese older adults from jing'an district of shanghai city . the slas was conducted among a total population sample of 2808 older adults from south east region of singapore . baseline assessments of the slas were completed from september 2003 to december 2005 . details of the ssadd [ 8 , 13 ] and , we selected 4639 participants from the ssadd sample and 2397 participants from the slas cohort . all participants had complete data on demographic information , physical health , and cognitive function and obtained a mini - mental state examination ( mmse ) total score of at least 21 points . face - to - face interviews were conducted by psychiatrists / psychiatric nurses ( in shanghai ) or trained research nurses ( in singapore ) , and data were collected for a wide range of variables . for the present analysis , we extracted the following variables from the databases : age , sex , functional status , chronic diseases , self - rated health status , and mmse total score . functional status was assessed by the participant 's level of dependency in performing 8 activities of daily living ( adl ) : eating , grooming , dressing , transferring , walking , toileting , bathing , and climbing stairs . the participants were asked do you have or not have any of the following illnesses or conditions at the present time ? " in the slas , a list of 14 medical conditions was covered in the interview . the participants were asked do you have a history of this medical problem ? medical conditions that were not included in the list were recorded under any other problems . we selected ten chronic diseases on which data were available from both samples : hypertension , diabetes , heart diseases ( in singapore : defined as any of heart attack , heart failure , or atrial fibrillation ) , stroke ( in shanghai : effects of stroke ) , kidney disorder ( in singapore : kidney failure ) , chronic obstructive lung disease ( in shanghai : emphysema / bronchitis ) , asthma , arthritis ( in shanghai : arthritis or rheumatism ) , mental illness , and cancer ( in singapore , identified from any other problems ) . in statistical analysis , the number of chronic diseases was used as an objective measure of physical health . in singapore , self - rated health status was assessed using a single question : in general , would you say your health is : excellent , very good , good , fair , or poor ? in shanghai , the same question was asked but there were four choices : excellent , good , fair , poor . excellent and very good together and created a new variable self - rated health status : 1 = excellent or very good , 2 = good , 3 = fair , and 4 = poor . this was used as a continuous variable in multivariate analyses ( score ranged from 1 to 4 , with higher value representing poorer health status ) . the test has been validated in shanghai and singapore , respectively . summed scores of mmse range from 0 to 30 , higher values denoting better cognitive functioning . chi - square test ( for dichotomous variables ) or independent sample t test ( for continuous variables ) was used to compare the difference regarding various characteristics between the two study samples . multiple logistic regressions were used to examine the relationship between health factors and disability , and the influence from environment . in analysis based on the combined sample , age , sex , number of chronic diseases , self - rated health status , mmse total score , and environment ( singapore versus shanghai ) were included in the regression model . the same set of variables ( except environment ) was used in the stratified analysis . the prevalence of disability was much higher in shanghai sample ( 5.0% ) than that in the singapore sample ( 1.8% ) . however , the shanghai participants were also older , had more chronic diseases , had poorer self - rated health status , and obtained lower mmse total score as compared to their singapore counterparts ( table 1 ) . a scrutiny of the eight adl items revealed that the difference was only significant for bathing ( 3.5% versus 0.4% ) and climbing stairs ( 3.1% versus 1.2% ) . in multiple logistic model based on the combined sample , number of chronic diseases , self - rated health status , mmse total score , and environment the odds ratio was 1.35 ( 95% ci 1.221.50 ) for number of chronic diseases , 2.85 ( 95% ci 2.363.43 ) for self - rated health status ( continuous ) , 0.89 ( 95% ci 0.850.94 ) for mmse total score , and 0.68 ( 95% ci 0.480.96 ) for environment ( singapore versus shanghai ) , respectively . results from stratified analysis showed that the strength of associations between health variables and disability differed between the two samples ( table 3 ) . for example , in the shanghai sample , one point increase on the 4-point self - rated health status scale was associated with 3.13 times ( 95% ci 2.553.85 ) higher odds of having disability while the odds ratio was only 1.73 ( 95% ci 1.072.81 ) in the singapore sample . for mmse total score , the corresponding odds ratio was 0.91 ( 95% ci 0.860.96 ) in the shanghai sample and 0.83 ( 95% ci 0.730.94 ) in the singapore sample . based on data from two population - based studies , we found that the number of chronic diseases , self - rated health status , cognitive function , and unmeasured environment factors represented by study sample were significantly associated with functional disability among chinese older adults . the contributions of physical health [ 8 , 18 ] and cognitive function [ 19 , 20 ] to functional disability have been documented and were well replicated in our analysis . with large population - based data , we firstly quantified the strength of differential contributions of each of self - reported physical health , self - rated health status , and cognitive function to disability in chinese elderly population . the findings are relevant and important for policymakers , medical practitioners , and academics . participants in the singapore sample were 32% less likely to have disability compared with participants in the shanghai sample . participants from both studies were chinese older adults living in big city , and a number of important covariates were adjusted in multiple regression models . it is less likely that the observed difference was caused by unmeasured factors at individual level . however , a careful examination of the two most difficult adl tasks ( bathing and climbing stairs ) provided us with plausible explanations . in 1987 , most families in shanghai had no bathroom and shower , and bathing was considered as a complicated task . in contrast , the majority of slas participants lived in public houses that have standard bathroom and shower and easy - to - climb stairs . our study provided fresh evidence on the role of environmental factors in disablement process [ 2123 ] . as pointed out by verbrugge and jette : disability is not a personal characteristic , but is instead a gap between personal capability and environmental demand disability can be diminished swiftly and markedly if the physical or mental demands of a given task are reduced . our findings suggest that environment not only affects the prevalence of disability , but also modifies the strength of associations between disability and health variables . strengths of our study include concurrent measuring of various health factors and simultaneous inclusion of those factors in multivariate model . however , no causal relationship could be drawn given cross - sectional nature of the study design . in conclusion , physical health and cognitive function were significantly associated with disability among chinese older adults living in asian metropolises . all the authors contributed substantially to the design , analysis and interpretation of the data and participated in drafting or revising the paper . conventional nontargeted chemotherapeutics , such as antimetabolites , microtubule inhibitors , and dna intercalating / alkylating agents , are effective at killing cancer cells , but due to their indiscriminate penetration into nearly all cells , they can also damage healthy cells , causing such toxicities as myelosuppression , alopecia , mucositis , peripheral neuropathy , and cardiotoxicity . to minimize such collateral damage to healthy tissues , physicians must often either reduce the dosage or decrease the frequency of drug administration , leading to incomplete elimination of diseased tissue . on the basis of these considerations , recent approaches to cancer therapy have focused on developing methods that specifically target chemotherapeutic agents to cancer cells , allowing for improved tumor suppression with fewer adverse events . the most common approach to drug targeting has relied on the specificity of a monoclonal antibody for its tumor - specific antigen . through the conjugation of a highly cytotoxic drug to a tumor - specific antibody , tumor - selective drug delivery examples of such tumor - targeted antibody drug conjugates ( adcs ) include trastuzumab emtansine and brentuximab vedotin . although several adcs have shown significant success in preclinical and clinical settings , some questions as to their ability to penetrate solid tumors have been raised . a related strategy to achieve tumor - selective drug delivery involves the use of low molecular weight targeting ligands that can similarly deliver attached drugs specifically to cancer cells . drug conjugates ( figure 1a ) also target receptors that are overexpressed on malignant cells , and their much smaller sizes may permit more thorough tumor penetration . ( a ) general representation of ligand conjugated to cytotoxic payload via a peptide linker . the circle represents the cholecystokinin 2 receptor ( cck2r ) binding ligand , whereas the linker is represented by an oval . the cytotoxic drug , or payload , the solid black line represents a covalent bond between the ligand and the linker , and the dotted line symbolizes a cleavable self - immolative bond . ( b ) chemical structures of the cck2r ligand crl conjugated to the cytotoxic antimicrotubule agents desacetyl vinblastine hydrazide and tubulysin b hydrazide via a hydrophilic peptide linker . in this paper , z-360 , a low molecular weight ligand of the cholecystokinin 2 receptor ( cck2r ) , is modified to deliver two of the more potent antimicrotubule agents currently available ( desacetyl vinblastine monohydrazide and tubulysin b hydrazide ) . cck2r is a transmembrane receptor primarily found in epithelial cells of the gi tract and brain , where it can bind gastrin and other amidated peptides of the gastrin - cholecystokinin family . cck2r is overexpressed on many cancers of the lung , pancreas , liver , and gi tract ( esophagus , colon , and gastrointestinal stromal tumors ) . because cck2r in the brain is inaccessible to drugs that can not pass the blood brain barrier , this restricted expression pattern renders cck2r a possible candidate for ligand - targeted drug delivery . we report here the design , synthesis , and biological evaluation of two highly potent cck2r - targeted chemotherapeutic agents and demonstrate their efficacy and tolerability in treating cck2r - expressing human tumor xenografts in nu / nu mice . h - cys ( trt)-2-cl - trt resin was obtained from novabiochem ( san diego , ca ) . diisopropylethylamine ( dipea ) , piperidine , dimethylformamide ( dmf ) , isopropyl alcohol ( i - proh ) , and all other reagents were purchased from sigma - aldrich . tubulysin b and desacetyl vinblastine hydrazide and their activated derivatives were a kind gift from endocyte inc . dulbecco s modified eagle medium ( dmem ) , roswell park memorial institute medium ( rpmi ) , fetal bovine serum ( fbs ) , and g418 ( geneticin ) were all purchased from gibco ( grand island , ny ) . the hek 293 cell line transfected with wild type cck2r was a generous gift from dr . cells were cultured as monolayers in dmem supplemented with 10% fetal bovine serum , g418 disulfate ( 400 g / ml ) , 1% of 2 mm glutamine , and 1% of penicillin - streptomycin at 37 c in a 5% co2:95% humidified - air atmosphere . kb cells were cultured from frozen stocks in rpmi and supplemented with the same concentrations of fetal bovine serum , penicillin - streptomycin , and glutamine as the hek 293 cells . the cck2r ligand ( z-360 ) was synthesized as previously described and abbreviated crl to be consistent with previous publications . the peptide spacer was prepared using fmoc - protected solid phase peptide synthesis as outlined in scheme 1 ( supporting information ( si ) , figure 1 ) and named l1 . as shown in scheme 1 ( si , figure 1 ) , crl was coupled to the peptide spacer on the solid phase and cleaved from the resin using a standard cleavage cocktail solution . crude crl - l1 was purified by preparative rp - hplc [ a = 2 mm ammonium acetate buffer ( ph 5.0 ) , b = ch3cn , solvent gradient : 5% b to 80% b in 25 min ] to yield the requisite product . lrms - lc / ms ( m / z ) : [ m + h ] calcd for c72h110n12o27s , 1607.79 ; found , 1608 . a solution of saturated sodium bicarbonate ( 2 ml ) and hplc grade water was bubbled with argon continuously for 10 min . crl - l1 ( 36 mg , 0.0226 mmol ) was dissolved in argon - purged hplc grade water ( 2.0 ml ) , and the ph of the reaction mixture was increased to 7 using argon - purged sodium bicarbonate . a solution of disulfide activated - davbh ( 11.10 mg , mmol ) in thf ( 2.0 ml ) was then added to the reaction mixture ( si , figure 2 ) . the progress of the reaction was monitored using analytical lrms - lcms , and after stirring for 20 min , the reaction was found to reach completion . crude crl - l1-davbh was purified by preparative rp - hplc [ a = 20 mm ammonium acetate ( ph 7.2 ) , b = ch3cn , solvent gradient : 5% b to 80% b in 30 min ] , yielding the desired product . lrms ( lc / ms ) ( m / z ) : [ m + h ] calcd for c118h168n18o36s2 , 2478 ; found , 2478 . crl - l1-tubbh was synthesized from activated tubulysin b hydrazide following the same procedure used for the synthesis of crl - l1-davbh ( si , figure 2 ) . after removing the thf under reduced pressure , crl - l1-tubbh was purified by preparative rp - hplc [ a = 2 mm ammonium acetate buffer ( ph 7.0 ) , b = ch3cn , solvent gradient : 5% b to 80% b in 25 min ] to yield the requisite product . lrms - lc / ms ( m / z ) : [ m + h ] calcd for c117h177n19o38s3 , 2553.96 ; found , 2554 . l1-davbh and l1-tubbh were synthesized from activated davbh and tubbh , respectively , following the procedure outlined for the synthesis of crl - l1-davbh ( si , figure 1 and 3 ) . each compound was then purified by reverse phase hplc [ a = 2 mm ammonium acetate buffer ( ph 7.0 ) , b = ch3cn , solvent gradient : 5% b to 80% b in 25 min ] to yield the requisite product . lrms - lc / ms ( m / z ) : [ m + h ] calcd for l1-davbh , c89h134n14o32s2 , 1976.22 ; found , 1976 ; and ( m / z ) : [ m + h ] calcd for l1-tubbh c88h143n15o34s3 , 2051.35 ; found , 2051 . all conjugates were found to be stable in saline for at least four weeks as monitored by lc - ms . cck2r - transfected hek 293 cells ( 100,000 cells / well ) were seeded on amine - coated 24-well plates and allowed to form monolayers . the spent medium in each well was replaced with fresh medium containing increasing concentrations of the test agents . after incubating for 2 h at 37 c , cells were rinsed 3 with fresh medium and then incubated an additional 66 h at 37 c in fresh medium . spent medium in each well was again replaced with fresh medium ( 0.5 ml ) containing h - thymidine ( 1 ci / ml ) , and the cells were incubated for an additional 4 h. after washing the cells 3 with medium , they were dissolved in 0.5 ml of 0.25 m naoh . thymidine incorporation was then determined by counting cell - associated radioactivity using a scintillation counter ( packard , packard instrument company ) . the ic50 value was derived from a plot of the percent of h - thymidine incorporation versus log concentration using graph pad prism 4 and tablecurve 2d software . hek 293 cells expressing cck2r ( 5.0 10 in 50% hc matrigel ) were injected into the shoulders of 56 week old female nu / nu mice . an age - matched group of animals was similarly implanted with 1 10 kb cells in 100 l of cell culture medium . because kb cells do not overexpress cck2r , the kb xenograft model served as a negative control . tumors were measured in two perpendicular directions 3 per week with vernier calipers , and their volumes were calculated as 0.5 x l x w , where l is the longest axis ( in millimeters ) , and w is the axis perpendicular to l ( in millimeters ) . solutions were administered either intraperitonealy ( crl - l1-davbh , l1-davbh ) or intravenously ( crl - l1-tubbh , l1-tubbh , crl - l1 ) . each mouse received 2 mol / kg of the test or control agent in 100 l of saline per injection . injections were given 3 per week for 3 weeks , and the mice were weighed at each dosing as a measure of gross toxicity . all animal work was performed under the guidance of the purdue laboratory animal program and was reviewed by the purdue animal care and use committee . tumors were excised , fixed in formalin , embedded in paraffin , sectioned , and stained with hematoxylin and eosin by the purdue histology and phenotyping laboratory . proper design of a ligand - targeted chemotherapeutic agent requires ( i ) selection of a high affinity ligand with good selectivity for a cancer - enriched receptor , ( ii ) identification of a therapeutic agent with sufficient potency to kill cancer cells when captured by a cancer - specific receptor , and ( iii ) construction of a linker that will enable delivery and release of the attached drug preferentially within the targeted cells . because cholecystokinin receptor ligand ( crl ) has been shown to exhibit high affinity ( 0.47 nm ) and strong selectivity for cck2r ( > 600-fold specificity over cck1r ) , it was selected for exploration as a targeting ligand for drug delivery to cck2r - expressing cancer cells ( figure 1b ) . to avoid nonspecific adsorption to cck2r negative cells , we incorporated a water - soluble peptide spacer , referred to as l1 , between the ligand and its therapeutic payload ( figure 1b ) . previous results from our lab have shown only a slight loss of affinity with no effect on specificity when crl is conjugated to its payload via hydrophilic linkers . in the present study , two highly potent microtubule inhibitors , desacetyl vinblastine hydrazide ( davbh ) and tubulysin b hydrazide ( tubbh ) , were attached to the crl - l1 peptide spacer through a self - immolative disulfide linker . this linker allows for selective release of the cytotoxic agent upon entry into the reducing environment of cancer cells . detailed schemes for the synthesis of crl conjugates of davbh and tubbh are described in si figures 2 and 3 and complete chemical structures for all conjugates can be found in si figures 47 . to determine the cytotoxicity and targeting specificity of the crl - l1-davbh conjugate , we incubated crl - l1-davbh , free davbh , and nontargeted l1-davbh with cck2r - transfected hek 293 cells for 2 h followed by incubation of the cells in drug - free medium for 66 h. cell viability was then measured via incorporation of h - thymidine . as shown in figure 2 , the potency of free davbh and crl - l1-davbh was 9 and 29 nm , respectively , whereas the potency of the nontargeted conjugate l1-davbh was markedly reduced by a factor of > 1000 ( ic50 value > 50 m ) . importantly , crl by itself was found to show no cytotoxicity toward hek 293-cck2r cells ( si , figure 8) , demonstrating that the aforementioned cytotoxicity was due to the targeted therapeutic agent rather than obstruction of cck2r by crl . in vitro cytotoxicity of davbh derivatives . the cytotoxicity of free davbh ( circles ) , the nontargeted l1-davbh ( triangles ) , and the targeted crl - l1-davbh ( squares ) conjugates in hek 293 cells transfected with cck2r when pulsed for 2 h at 37 c , washed with culture medium three times , and then incubated for an additional 66 h. cells were incubated with h - thymidine for 4 h and washed three times . after the final wash , the cells were dissolved in 0.5 ml of 0.25 m naoh . viability of the cells was assessed by analyzing the incorporation of h - thymidine . , we injected 2 mol / kg crl - l1-davbh intraperitoneally into mice bearing cck2r - transfected hek 293 xenografts at a frequency of 3x per week for 3 weeks ( n = 5 ) . this dose was selected based on previous studies showing significant therapeutic benefit when similar folate- and dupa - targeted chemotherapeutics were administered at the same concentration . as shown in figure 3a , crl - l1-davbh was found to markedly delay tumor growth but not lead to complete tumor eradication . to determine whether the residual lesion was comprised predominately of cancer or stromal cells , h&e staining was performed on excised tumors ( day 33 ) . unlike the saline control , which was comprised of almost entirely of cancer cells ( figure 4a ) , crl - l1-davbh - treated tumors exhibited a greatly decreased ratio of cancer to stromal cells ( figure 4b ) . as expected , the nontargeted l1-davbh exerted little impact on tumor growth , demonstrating the critical role of ligand targeting on crl - l1-davbh efficacy ( figure 3a ) . importantly , body weights in the crl - l1-davbh - treated group remained essentially constant over the course of the study , suggesting that the targeted therapy was not grossly toxic to the animals ( figure 3b ) . in vivo therapeutic efficacy and gross toxicity of davbh conjugates . ( a ) volume of tumors and ( b ) percent weight change in nu / nu mice subcutaneously injected with cck2r - transfected hek 293 cells ( 5.0 10 in 50% hc matrigel ) on day 0 . mice were randomized to different treatment groups ( n = 5 ) and began treatment on day 15 , when tumor volume was approximately 100 mm . saline ( diamonds ) , nontargeted l1-davbh ( triangles ) , and targeted crl - l1-davbh ( squares ) conjugates were administered intraperitoneally following a tiw schedule for 3 weeks . excised tumors from mice treated with ( a ) saline or ( b ) crl - l1-davbh were sectioned and stained with hematoxylin and eosin . arrows show the abundance of cancer cells in panel a and a reduced number of cancer cells in panel b. because complete tumor remission was not achieved at a dose of 2 g / kg with crl - l1-davbh , we elected to develop a more potent cck2r - targeted conjugate . for this purpose , tubulysin b hydrazide ( tubbh ) , a microtubule inhibitor with 10 the potency of davbh , was conjugated to crl via the same l1 linker ( figure 1 , si figures 2 and 3 ) . as shown in figure 5a , free tubulysin b hydrazide was found to be very potent in vitro , exhibiting an ic50 of 2.7 nm on hek 293-cck2r cells . similarly , the targeted crl - l1-tubbh conjugate yielded an ic50 of 2 nm , whereas nontargeted l1-tubbh displayed the anticipated significant drop in potency ( ic50 of 310 nm ) due to its membrane impermeability and lack of targeting . to ensure that the cytotoxicity of crl - l1-tubbh was receptor mediated , cells were incubated with crl - l1-tubbh in the presence of 100-fold excess crl - l1 . as expected , the ic50 value of the competed crl - l1-tubbh was nearly identical to that of the nontargeted l1-tubbh conjugate ( ic50 of 340 and 310 nm , respectively ) . in vitro cytotoxicity of tubbh derivatives . the cytotoxicity of free tubbh ( circles ) , the nontargeted l1-tubbh ( triangles ) , the targeted crl - l1-tubbh ( squares ) , and the targeted crl - l1-tubbh in the presence of 100-fold greater concentration competing crl - l1 ( diamonds ) conjugates in hek 293 cells transfected with cck2r when pulsed for 2 h at 37 c , washed with culture medium three times , and then incubated for an additional 66 h. cells were incubated with h - thymidine for 4 h and washed three times . after the final wash , the cells were dissolved in 0.5 ml of 0.25 m naoh . viability of the cells was assessed by analyzing the incorporation of h - thymidine . error bars represent standard deviation . to determine the efficacy of crl - l1-tubbh in vivo , we followed the same treatment regimen used for davbh conjugates except that tubbh conjugates were injected intravenously . crl - l1-tubbh showed efficient antitumor activity , eliminating all detectable malignant lesions from all five mice ( figure 6a ) and prolonged survival ( si , figure 9 ) . as expected , nontargeted l1-tubbh showed no difference from the saline - treated control group when an identical concentration as that of the crl - l1-tubbh targeted conjugate was tested . moreover , when crl - l1-tubbh was competed with 100-fold excess of crl - l1 , the antitumor effect of the targeted tubbh was negated ( figure 6a ) . this indicates that the antitumor activity observed in the crl - l1-tubbh group was entirely receptor mediated . importantly , the crl - l1-tubbh - treated mice displayed no visible signs of gross toxicity , and no weight loss was observed ( figure 6c ) . in vivo therapeutic efficacy and gross toxicity of tubbh conjugates . ( a , b ) volume of tumors and ( c , d ) percent weight change in nu / nu mice subcutaneously injected with 5.0 10 cck2r - transfected hek 293 cells ( cck2r(+ ) cells ) in 50% hc matrigel ( a , c ) or 1.0 10 kb cells ( cck2r( ) cells ) in 50% hc matrigel on day 0 . mice were randomized to different treatment groups ( n = 5 ) and began treatment on day 17 , when the tumor volume was 100 mm . saline ( diamonds ) , nontargeted l1-tubbh ( triangles ) , targeted crl - l1-tubbh ( squares ) , and targeted crl - l1-tubbh in the presence of 100-fold greater concentration of competing crl - l1 ( circles ) conjugates were administered via lateral vein injection following a tiw schedule for 3 weeks . finally , to more thoroughly establish the necessity of receptor - mediated targeting for crl - l1-tubbh efficacy , the targeted conjugate was tested using the same protocol in a cck2r - negative kb xenograft model . in vitro crl - l1-tubbh conjugate yielded an ic50 value of 269 nm ( si , figure 9 ) , similar to the ic50 values observed with the nontargeted and competed targeted conjugates in the cck2r - expressing hek cells . as shown in figure 6b , crl - l1-tubbh showed no difference in tumor growth from the vehicle control . because free tubulyisn b hydrazide is known to be cytotoxic against kb cells , the lack of reduction in tumor volume in the kb xenograft must derive from the absence of cck2r and not from any intrinsic resistance to tubbh . again , none of the mice in this study exhibited any signs of gross toxicity , and no substantial weight loss was observed ( figure 6d ) . conjugation of cytotoxic drugs to ligands that selectively target cancer cells has emerged as a promising method to both improve drug efficacy and reduce drug toxicity . although only a few low molecular weight ligand - targeted cancer drugs have been tested in vivo to date , results suggest that the potential to design such targeted therapeutics for most human malignancies is promising . folate receptor - targeted chemotherapeutic agents have demonstrated the capacity to treat malignancies of the ovary , lung , kidney , breast , and endometrium , and psma - targeted drugs have shown promise for treating cancers of the prostate . lhrh receptor- and v3-targeted therapeutics can expand this list to include cancers of the ovary and breast , but to date , few ligands are available to target cancers of the gi tract . in this study , we attempted to obtain a ligand that would deliver attached drugs to gastroinstestinal malignancies . on the basis of literature demonstrating overexpression of cck2r in gastrointestinal stromal tumors and colon , stomach , esophageal , and pancreatic cancers , the crl conjugates described in this paper should have the potential to address this deficiency . one of the more interesting results to emerge from this study was the high specificity displayed by both crl - l1-davbh and crl - l1-tubbh for cck2r - expressing cancer cells . crl , davbh , and tubbh were all found to be very hydrophobic , predicting that any conjugate of the ligand to a drug would be extremely lipophilic , resulting in a strong proclivity to associate nonspecifically with all cell membranes . thus , to increase hydrophilicity , a water - soluble linker ( l1 ) was utilized to conjugate crl to the cytotoxic drugs . cck2r - dependent binding of crl - l1-davbh and crl - l1-tubbh was established by the absence of toxicity when ( i ) crl was deleted , ( ii ) conjugate binding was competed with excess ligand , or ( iii ) cck2r was absent from the targeted cancer cell . drug conjugates can be rendered tumor specific by the judicious choice of a hydrophilic linker due to the linker impeding the diffusion of the hydrophobic drug through the cell membranes , thus reducing nonspecific activity . however , this effect is somewhat concentration dependent as some conjugates can diffuse into cells , and therefore at high concentrations , nonreceptor mediated activity can occur . in the case of linking crl to davbh and tubbh , the insertion of a bulky water - soluble linker between the ligand and drug converted an otherwise nonspecific drug conjugate into a highly receptor - targeted drug conjugate at physiologically relevant concentrations . our crl - tubbh conjugates were also tested against xenograft tumors similar to those exposed to the davbh conjugates , except an additional treatment group was included where crl - l1-tubbh was competed with excess crl - l1 . similar to the davbh conjugates , the nontargeted l1-tubbh conjugate did not appear to have any effect on tumor growth . the lack of efficacy of l1-conjugated cytotoxic compounds in vivo is most likely due to the hydrophilic linker , which does not allow for the cytotoxic compound to readily diffuse through the cell membrane , whereas the targeted crl - l1-davbh and crl - l1-tubbh both showed efficacy in vivo . somewhat surprisingly , the crl - l1-davbh conjugate did not regress tumor volume even though similar concentrations of davbh conjugated to folate or dupa did exhibit tumor regression . this result is most likely due to a lower number of cck2r receptors present on the cell surface as compared to folate or psma . this resulting lack of tumor regression in vivo encouraged the testing of a more potent cytotoxic agent with a similar mechanism of action . when the more potent crl - l1-tubbh conjugate was tested , not only do it halt tumor growth , but it even regressed the tumor to an indistinguishable level . importantly , the crl - l1-tubbh treatment group that was competed with crl - l1 showed no effect on inhibiting tumor growth , similar to both the vehicle control and the nontargeted conjugate . to further assess the necessity of receptor - mediated targeting , we tested xenografts of the receptor - negative kb cell line with the targeted crl - l1-tubbh conjugate . unlike the cck2r - positive hek 293 xenografts , the kb xenografts showed neither regression nor a delay of tumor growth when treated with the conjugate . taken together , these results strongly support the necessity of receptor - mediated targeting with these conjugates for any in vivo efficacy . in summary , the current study demonstrates that cck2r - targeting ligands can be used to deliver drugs selectively to cck2r - positive tumors to generate a dramatic receptor - specific antitumor effect without observable gross toxicity to healthy tissues . because current treatments for colon , lung , pancreatic , and related cancers commonly cause hair loss , bone marrow suppression , weight loss , and so forth , the prospects for developing a less toxic treatment for these malignant diseases offer some optimism for the management of these cancers in the future .

objective . we aimed to examine the independent contributions of physical health and cognitive function to disability among chinese older adults living in two asian metropolises and explore the potential influences of environment . design and participants . cross - sectional analysis based on data from two population - based studies : the shanghai survey of alzheimer 's disease and dementia ( n = 4639 ) and the singapore longitudinal ageing study ( n = 2397 ) . disability was defined as needing help in at least one activity of daily living . results . the prevalence of functional disability was higher in shanghai sample ( 5% ) than that in singapore sample ( 1.8% ) . number of chronic diseases , self - rated health status , cognitive function ( measured by the mini - mental state examination ) , and environment ( singapore versus shanghai ) significantly contributed to functional disability independent of each other . the adjusted odds ratio was 1.35 ( 95%ci 1.221.50 ) , 2.85 ( 95% ci 2.363.43 ) , 0.89 ( 95% ci 0.850.94 ) , and 0.68 ( 95% ci 0.480.96 ) , respectively . the strength of associations between health variables and disability appeared to be influenced by environment . conclusion . physical health and cognitive function independently contributed to functional disability . the associations are modulated by environmental factors . as the delivery of selectively targeted cytotoxic agents via antibodies or small molecule ligands to malignancies has begun to show promise in the clinic , the need to identify and validate additional cellular targets for specific therapeutic delivery is critical . although a multitude of cancers have been targeted using the folate receptor , psma , bombesin receptor , somatostatin receptor , lhrh , and v3 , there is a notable lack of specific small molecule ligand / receptor pairs to cellular targets found within cancers of the gi tract . because of the selective gi tract expression of the cholecystokinin 2 receptor ( cck2r ) , we undertook the creation of conjugates that would deliver microtubule - disrupting drugs to malignancies through the specific targeting of cck2r via a high affinity small molecule ligand . the cytotoxic activity of these conjugates were shown to be receptor mediated in vitro and in vivo with xenograft mouse models exhibiting delayed growth or regression of tumors that expressed cck2r . overall , this work demonstrates that ligands to cck2r can be used to create selectively targeted therapeutic conjugates .

often , a new physics faculty member is faced with the duty of renovating the introductory physics labs . we will provide a list of experiments and equipment needed to convert about half of the traditional labs on a 1-year introductory physics lab into microcomputer - based laboratories ( mbls ) . our student body consists mostly of science majors that take the algebra - based course . but , the lab renovation described here could be used for the calculus - based group as well . we would suggest adjusting the lab manuals . nowadays , mbls are usually the choice when thinking of a renovation . they have effectively demonstrated an advantage to the learning process over the years.[14 ] moreover , for our science students , the labs are important in reinforcing the concepts learned in class . it is very common to hear , during the lab sections , students commenting on their grasp of the concept learned in class due to the experiment being performed . the overall 1-year lab experience follows the guidelines provided by the american association of physics teachers . it contains a brief theoretical description and the procedures to be followed on the day of the lab . on the lab day , the students should come prepared and ready to start without additional instructions . the instructor circles around the stations to guide and answer appropriate questions if needed . on the lab day , the students are handed the lab report . it contains a data analysis part , some discovery questions , and ends with a summary and conclusion part . the report is completed by the student during the session . we did not find an appropriate version for the other half to meet our overall goal . therefore , we kept a few traditional labs . the list of experiments is chosen based on the lecture material . one of the concerns was to always be able to cover the theory before the lab was performed by the student . one - year introductory microcomputer - based laboratory experiment list understanding motion , free fall , projectile motion , atwood 's machine , boyle 's law , electrical equivalent of heat , heat transfer , electrostatic charge , ohm 's law , rc circuit , and magnetic induction . a suggestion for the beginner is to try all the experiments before hand until you get really familiar with the sensors and software and how they work . most of the time , it is a lack of understanding of the use of the apparatus , assuming it is not defective of course . we will list here the total equipment needed per station to implement the 1-year lab described above . it is expected that the laboratory will have a printer that can be shared among all groups . each station consists of a laptop and the science workshop 750 interface ( ci-7650 ) with the datastudio software ( ci-6870 g ) . lab station : science workshop interface , laptop and motion sensor sensors to be used with the interface for data measurement : motion sensor ( ci-6742a ) , photogate and pulley system ( me-6838 ) , accessory photogate ( me-9204b ) , time - of - flight accessory ( me-6810 ) , pressure sensor ( ci-6532a ) , temperature sensor ( ci6605a ) , power amplifier ( ci-6552a ) , charge sensor ( ci-6555 ) , voltage sensor ( ci-6503 ) , photogate head ( me-9498a ) . figure 2 displays a few sensors . from left to right : pressure sensor , charge sensor , and photogate head the datastudio software collects and analyzes the data . it has an easy - to - use interface , allowing the students to explore the data . for instance , the left screenshot in ure 3 displays a graph of voltage versus time . the data are collected using a voltage sensor when a magnet is dropped through a coil . the students can select a region on the graph and the software calculates the area under the curve . the screenshot on the right in figure 3 displays the curve - fitting feature of datastudio . left screenshot : induction lab using graph and area under the curve calculation . the pressure column will be filled as the measurements are taken using the pressure sensor . boyle 's law lab using a table display the additional equipment needed from pasco to perform the experiments are : picket fence ( me-9377a ) , projectile mini launcher ( me-6825a ) , photogate mounting bracket ( me-6821a ) , extension cable ( pi-8117 ) , thermodynamics kit ( ci-6514a ) , charge producers ( ci-6555 ) , faraday ice pail ( es-9057b ) , ac / dc electronics lab ( em-8656 ) , and bar magnet ( em-8620 ) . general lab supplies needed include a pair of scissors and goggles , one digital balance ohaus ( sp-601 ) , one meterstick , one thermometer , tongs ( handling hot bottles ) , gloves ( handle hot containers ) , braided physics string ( se-8050 ) , 500 ml glass container ( 90c water ) , banana plug cord red and black ( 5 on set ) ( se-9750 ) or ( se-975 ) , masses and hanger set ( me-8979 ) , universal table clamp ( me-9376b ) , calorimetry cups ( td-8825a ) , and hot plates ( se-8830 ) . oftentimes , this task is hard to accomplish for a solo faculty in a small institution . we provided here a list of the experiments and equipments needed to upgrade about half the experiments to mbls on a 1-year introductory physics lab . we would like to add that although we used pasco , there are other comparable systems in the market . the intention of this paper is to help others with their own lab renovation . in order to better fulfill this purpose ,

nowadays , data acquisition software and sensors are being widely used in introductory physics laboratories . this allows the student to spend more time exploring the data that is collected by the computer hence focusing more on the physical concept . very often , a faculty is faced with the challenge of updating or introducing a microcomputer - based laboratory ( mbl ) at his or her institution . this article will provide a list of experiments and equipment needed to convert about half of the traditional labs on a 1-year introductory physics lab into mbls . octane - enhancing constituents of gasoline pose a number of health hazards . this paper considers the relative risks of metallic ( lead , manganese ) , aromatic ( e.g. , benzene ) , and oxygenated additives in both industrialized and developing countries . technological advances , particularly in industrialized countries , have allowed the progressive removal of lead from gasoline and the increased control of exhaust emissions . the developing world , by contrast , has relatively lax environmental standards and faces serious public health problems from vehicle exhaust and the rapid increase in automobile use . financial obstacles to the modernization of refineries and vehicle fleets compound this problem and the developing world continues to import large quantities of lead additives and other hazardous materials . progress in decreasing environmental health problems depends both on the adoption of international public health standards as well as efforts to decrease dependence on the private automobile for urban transport.imagesfigure 1.figure 2 .

type 2 diabetes mellitus ( t2 dm ) is a growing epidemic : in the united states of america alone , nearly 25.8 million people ( approx 8.3% of the total population ) have the disease.1 estimates have placed the global prevalence of the disease at around 217 million.2 the consequence to the individual of prolonged exposure to hyperglycemia is a marked increase in the risk of mortality and morbidity , with an associated reduction in life expectancy of around 1213 years.3 diagnosis usually occurs some time after development of the disease and they have often already experienced occult pathology by the time patients receive a diagnosis . early adoption of an aggressive approach to disease management improves patient outcome , with marked reductions in morbidity and mortality.46 the first line approach to treatment is through lifestyle modification.7 however , t2 dm is progressive in nature , and lifestyle changes sufficient to halt the disease are difficult to achieve . intense efforts by patients and the healthcare team frequently fail . similarly , most conventional antidiabetic drugs ( eg , metformin , sulfonylureas etc . ) often fail to slow the progression of t2 dm , despite the availability of a broad range of agents employing different mechanisms of action.79 the progressive nature of the disease and how it overwhelms available therapies was highlighted in the ukpds study ; suggesting that fewer than half of all patients actually achieve adequate levels of disease control.4,5 there is a recognized need for new treatment options for t2 dm . characterization of the mechanisms facilitating glucose resorption by the kidney has raised the possibility of a novel treatment for diabetes : inhibition of the type 2 sodium - glucose transporter ( sglt2 ) , a 672 amino - acid , high capacity , low affinity transmembrane protein that promotes reabsorption of glucose as the glomerular filtrate passes down the nephrons.10 several candidate molecules are currently in development and may soon be available for use in the treatment of diabetes . we provide a brief review of sglt2 inhibitors and their possible role in the treatment of t2 dm . most ( > 99% ) of the plasma glucose entering the kidney filters into the nephrons though the glomeruli . under normal circumstances , the reabsorptive capacity of the early part of the nephron , the proximal tubule , is sufficient to clear the filtered glucose load from the luminal fluid before it enters the loop of henl . in normal individuals , around 180 g of glucose passes into the proximal tubules each day , from where it is almost completely reabsorbed.11,12 as plasma glucose concentrations increase , the filtered glucose load increases in a linear manner . when the rate of glucose entering the nephron rises above 260350 mg / min/1.73 m , for example in patients with diabetes , the excess glucose outstrips resorptive capacity and appears in the urine ( glycosuria).13 in a healthy adult , this equates to a blood glucose concentration of approximately 11 mmol / l ( 200 mg / dl).14 as much as 90% of the filtered glucose load is extracted in the s1 segment , and the remaining 10% is removed in the distal straight tubules ( s2 and s3 segments ) ( figure 1 ) . until recently , the mechanisms behind glucose reabsorption were poorly understood , although it was proposed as early as 1960 that glucose transmembrane flux could be achieved through the coupling of glucose transport with that of sodium.15figure 1summary of glucose reabsorption from the lumen of the nephrons . glucose concentration in the glomerular filtrate reflects plasma concentration . in most normal healthy subjects , the majority of glucose reabsorption is believed to occur in the early part of the proximal tubule . glucose concentration in the glomerular filtrate reflects plasma concentration . in most normal healthy subjects , the majority of glucose reabsorption is believed to occur in the early part of the proximal tubule . since the start of the 20 century , phlorizin , a toxic 2-glucoside of phloretin , has been known to increase glycosuria , and has been used in the study of renal function.16,17 during the 1930s , phlorizin was used in non - invasive human experiments that revealed some of the fundamental mechanisms of renal hemodynamics and metabolic transport.18 in the 1950s , studies delineated phlorizin s mechanism of action on inhibition of glucose transport in the kidney and small intestine at the cellular and molecular levels . renal micropuncture studies conducted with phlorizin in the 1970s showed that the transporter was located in the brush border of the proximal tubule , and that sodium was required for the renal absorption of glucose.11,19,20 studies performed since then confirmed that phlorizin is a competitive inhibitor of glucose transport , with a binding affinity for the transporter that is 1000- to 3000-fold greater than that of glucose.21 the rabbit homolog of the human type 1 sodium - glucose transporter ( sglt1 ) , which is coded by the slc5a gene , was the first mammalian cotransporter carrier protein to be identified , cloned , and sequenced.22 a family of slc5a gene sodium - dependent transporters has since been sequenced and identified in a broad range of tissues.23,24 sglt1 and sglt2 are , perhaps , the slc5a family members that have received greatest coverage within the literature . the high affinity , low capacity slgt1 is the main gastrointestinal glucose transporter the relatively widespread distribution of sglt1 is contrasted by the almost exclusive expression on the luminal surface of proximal tubules ( mainly in the renal cortex ) of the low glucose affinity , high capacity sglt2 , responsible for most renal tubular glucose reabsorption.2226 cellular glucose and sodium uptake occurs in a 1:1 ratio ( figure 2 ) . the sodium : potassium adenosine triphosphatase ( atp ) pump transports sodium across the basolateral surface into the intracellular fluid , maintaining the physiological levels of sodium in the cell . cellular glucose concentrations are maintained by facilitative glucose outflow through transporters in the basolateral membrane of the cell . after binding intracellular glucose the transporters undergo a conformational change that subsequently moderates the movement of glucose ( down its concentration gradient ) back into the blood ( figure 3 ) . figure 2representation of the 1:1 transport of sodium and glucose across the luminal membrane of the epithelial cells of the early part of the proximal tubule facilitated by sglt2figure 3summary of the glucose transport cascade in the brush border epithelial cells of the proximal tubule demonstrating how the process is driven by the sodium gradient maintained by the na - k atpase of the basal membrane representation of the 1:1 transport of sodium and glucose across the luminal membrane of the epithelial cells of the early part of the proximal tubule facilitated by sglt2 summary of the glucose transport cascade in the brush border epithelial cells of the proximal tubule demonstrating how the process is driven by the sodium gradient maintained by the na - k atpase of the basal membrane the antidiabetic properties of phlorizin were investigated in the 1980s . in partially pancreatectomized rats , phlorizin increased glucose secretion in urine and this was associated with a normalizing of plasma glucose , without inducing hypoglycemia.17 despite its promising in vitro properties , phlorizin does not fit the profile that we have come to expect from a modern therapeutic agent . phlorizin is also potentially toxic and is non - selective , inhibiting both sglt1 and sglt2 transporters . in the last decade , several alternative candidate molecules , targeted to specifically inhibit sglt2 , have been investigated in both pre - clinical and clinical settings.27 the aim has been to take advantage of the potential for turning - off glucose reabsorption as a new therapeutic target for the treatment of t2 dm . first reports of devised sglt2 inhibitors started to emerge in the scientific literature in the second half of the 1990s . developed with a view to overcoming the shortcomings of phlorizin , sglt2 inhibitors represented a new mechanism to manage hyperglycemia that acted independently of insulin and irrespective of patients glycemic status . first indications suggest that the mechanism of action , which is independent of insulin , further reduces glycemia when used in combination with traditional antidiabetic treatments . results with early compounds were promising in terms of specificity for the transporter : the compound t-1095 has inhibitory capacity for sglt2 that is 4-fold greater than for sglt1.25 pharmacodynamic studies demonstrated attenuated hyperinsulinemia and hypertriglyceridemia in kk rats following oral administration of t-1095.26 lowering of insulin resistance and hba1c levels along with normalized hepatic glucose production and glucose utilization rate were also observed in streptozotocin - induced diabetic rats26,28 and zucker diabetic fatty rats28,29 following oral administration of t-1095 . long - term administration of t-1095 restored impaired insulin secretion from pancreatic -cells in goto - kakizaki ( gk ) rats30 and suppressed diabetic complications in both c57bl / ksj - db / db mice and gk rats.31,32 however , retained co - inhibition of sglt1 by t-1095 led to development of the compound being discontinued in 2003 , having reached phase ii clinical trials . various sglt2 inhibitors based on the glucoside structure of phlorizin have since been proposed ( table 1 ) , and narratives of the discovery pathway of the different inhibitors have recently been published.27,33 the glucoside moiety of phlorizin binds to sglt2 transporters and the o-linked phenolic distal ring is responsible for its inhibitory properties.34 structure - activity analysis of the parent molecule shows that addition of lipophilic groups to the distal ring augments the inhibition of the sglt2 transporter , and increases selectivity for sglt2 over sglt1.25 however , the o - linkage is a metabolic target for -glucosidase enzymes that can curtail the activity of sglt2 inhibitors in vivo . to address this possible limitation to therapeutic utility , candidate sglt2 inhibitors both the o- and c - glucosides appear to bind to a single site on the sglt2 transporter . the aromatic and heteroaromatic c - glucosides are metabolically more stable than o - glucosides , due to their relative resistance to hydrolysis . alternative candidate sglt2 inhibitors that have also been considered include modified sugar rings , n - glucosides and , more recently , a bridged ketal ring.35table 1candidate sglt2 inhibitorsmanufacturers : johnson & johnson / tanabe seiyaku co. ltd ( japan ) ( t-1095 ) ; sanofi - aventis ( ave-2268 ) ; glaxosmithkline ( remogliflozin / kgt 1681/sergliflozin ) ; wyeth ( way-123783 ) ; astellas pharma inc ( asp 1941/ym-543 ) ; boehringer ingelheim gmbh ( bi-10773/bi-44847 ) ; boehringer ingelheim , gmbh / ajinomoto ( canagliflozin ) ; johnson & johnson / tanabe mitsubishi ( ta-7284/jnj 28431754 ) ; bristol - myers squibb co / astrazeneca ( dapagliflozin ) ; lexicon ( lx 4211 ) ; isis pharmaceuticals ( isis 388626 ) ; roche / chugai ( r7201/csg452 ) ; pfizer ( pf-04971729 ) . candidate molecules have also been registered by kissei , taisho , theracos , and daiichi sankyo . candidate sglt2 inhibitors manufacturers : johnson & johnson / tanabe seiyaku co. ltd ( japan ) ( t-1095 ) ; sanofi - aventis ( ave-2268 ) ; glaxosmithkline ( remogliflozin / kgt 1681/sergliflozin ) ; wyeth ( way-123783 ) ; astellas pharma inc ( asp 1941/ym-543 ) ; boehringer ingelheim gmbh ( bi-10773/bi-44847 ) ; boehringer ingelheim , gmbh / ajinomoto ( canagliflozin ) ; johnson & johnson / tanabe mitsubishi ( ta-7284/jnj 28431754 ) ; bristol - myers squibb co / astrazeneca ( dapagliflozin ) ; lexicon ( lx 4211 ) ; isis pharmaceuticals ( isis 388626 ) ; roche / chugai ( r7201/csg452 ) ; pfizer ( pf-04971729 ) . candidate molecules have also been registered by kissei , taisho , theracos , and daiichi sankyo . administration of synthesized strands of nucleic acid targeted to specifically bind to sglt2 messenger rna blocks the transporter s translation , protein production , and expression in the cells of the proximal tubule . a summary of the status of inhibitor development is provided in table 2.3654table 2clinical status of sglt2 moleculescompoundphasecompanyclintrial.gov studies completed / totalliterature citationsc - glucosidesdapagliflozin ( bms-512148)iiibristol - myers squibb co./astrazeneca38/47 canagliflozin ( ta-7284 , jnj 28431754)iiijohnson & johnson / tanabe mitsubishi7/26 asp-1941iiiastellas pharma inc./kotobuki18/27 bi-10773iiiboehringer ingelheim16/31-lx-4211iilexicon pharmaceuticals4/4-dsp-3235 ( kga-3235 , gsk 1614235 , 1614235)iglaxosmithkline / dainippon sumitomo ( under license from kissei pharmaceuticals)1/1-o - glucosidessergliflozin ( gw869682)iiglaxosmithkline3/3 remogliflozin ( kgt 1681)iiglaxosmithkline / kissei sanofi - aventis16/16 ave-2268-sanofi - aventis1/1-ym-543iiaastellas pharmaceutical inc / kotobuki1/1-otherisis - sglt2rx ( isis-388626)iisis pharmaceuticals1/1-pf04971729iipfizer7/8- _ clinical status of sglt2 molecules as the above discussion suggests , there are several hypothetical reasons why the sglt2 transporter represents an opportune target for managing blood glucose . however , the challenge is to establish therapeutic utility while demonstrating an acceptable safety profile . a detailed summary of clinical findings has recently been published.55 the mechanism of action of sglt2 inhibitors predicts a beneficial effect , but the long - term glucose lowering capacity in a clinical setting may not impart significant reductions in hba1c . modest hba1c lowering in the region of 0.5%0.9% , that may be predicted from early clinical studies , would be comparable to that achieved with other currently marketed oral agents.55 it remains to be seen whether promoting glucose excretion will result in long - term benefits for the patient in terms of returning metabolic balance , or even weight loss . clearly , blocking glucose reabsorption permits the clearance of glucose from the body , and thus must eventually serve to reduce levels of plasma glucose . the amount of glucose available for excretion is dependent on the amount entering the nephrons , which , in turn , depends on blood glucose concentration at the glomerulus . thus , the amount of glucose excreted is greater when the blood plasma glucose concentrations are highest . in effect , glucose removal might be expected only to be greatest at times when it is most needed ( when glucose levels are highest ) , such as during post - prandial hyperglycemia . the benefit to those patients in whom treatment has provided mild - to - moderate glycemic control might be questioned , as the potential for glucose excretion would be relatively low . nevertheless , patients who achieve moderate glycemic control may be exposed to clinically relevant post - prandial glucose excursions that can impart disproportionate effects on hba1c and possibly the morbidity and mortality associated with t2dm.56 in such a patient population , sglt2 inhibitors might attenuate the impact of post - prandial glucose spikes . nevertheless , clinical experience with agents , such as the meglitinides , that target post - prandial glucose control , suggest that the clinical benefit of this approach is disappointing . treatments targeting post - prandial glucose levels provide little more than modest improvements in hba1c with little evidence of long - term outcome benefits for patients.57 as sglt2 may be responsible for as much as 90% of glucose reabsorption by the kidney , there is the clinical potential for as much as 160 g of glucose to be excreted each day following effective sglt2 inhibition.23 however , it appears that the actual glucose loss achieved in clinical studies is only about half that predicted.38 it is not clear whether this is a consequence of compensating mechanisms undertaking tubular reabsorption or incomplete inhibition of the transporter . thus far , the safety profile of sglt2 inhibitors reported from clinical studies appears to fulfill expectations.33,34,55,40,58 sglt2 inhibitors are designed to target a highly specific membrane transporter that is almost exclusively expressed within the renal tubules . clearly , compared with less specific molecules , the potential for it is also unlikely that sglt2 inhibitors will induce hypoglycemia , since when plasma glucose levels are low the amount of glucose excreted will also be low.59 this prediction appears to be confirmed by clinical studies reported thus far , which show no apparent increases in hypoglycemic episodes with sglt2 inhibitors.50,60 even when sglt2 is blocked completely , a degree of renal glucose recovery is maintained via the relatively unhindered sglt1 transporter . one aspect of sglt2 inhibition that has been raised as a potential issue of safety concern is that of glycosuria , which could predispose patients to increased urinary tract infections ( uti ) . there have been some reports of infection in clinical studies.60,61 however , a study that reviewed risk factors for developing utis in women with diabetes observed that glucosuria was not a significant contributing factor.62 interestingly , there is a rare group of individuals who do not express the sglt2 transporter or in which its functionality has been partially or completely lost due to a genetic mutation for which both an autosomal recessive and dominant pattern of inheritance has been reported . these people do not appear to suffer any ill consequences , suggesting that blockade of the transporter per se in t2 dm patients would offer no immediate risk . patients expressing these mutations have decreased renal tubular reabsortion of glucose from the lumen in the absence of hyperglycemia , or any other signs of tubular dysfunction . it is not clear whether familial renal glucosuria ( due to sglt2 mutation ) protects against t2 dm ; although sglt2 deletion in animal models appears to improve glucose homeostasis and preserve pancreatic -cell function.63 we did not find any recorded evidence of an increased disposition to urinary tract or vulvovaginal infections , although identification and study of these subjects is difficult due to the rarity of the disease . clearly , clinical development programs will need to address the concern of a possible increased risk of uti . increased glucose content in the urine following sglt2 inhibition will likely serve to increase urinary flow as a consequence of the osmotic diuretic effect in the lumen of the nephron . this could result in modest , possibly beneficial , reductions in blood pressure , but raises additional safety concerns associated with possible loss of fluid and solutes . this may be of particular concern in elderly patients or those who do not have the capacity to maintain their fluid balance . however , it should be noted that the effect is considerably lower than that seen with frequently used loop diuretics and there is no apparent change in glomerular filtration rate that would be indicative of a direct effect on renal function . simple instructions on maintaining a state of hydration with regular drinks may serve to overcome the concerns over both urinary infection and fluid imbalance . the mechanism of action of sglt2 inhibitors predicts a beneficial effect , but the long - term glucose lowering capacity in a clinical setting may not impart significant reductions in hba1c . modest hba1c lowering in the region of 0.5%0.9% , that may be predicted from early clinical studies , would be comparable to that achieved with other currently marketed oral agents.55 it remains to be seen whether promoting glucose excretion will result in long - term benefits for the patient in terms of returning metabolic balance , or even weight loss . clearly , blocking glucose reabsorption permits the clearance of glucose from the body , and thus must eventually serve to reduce levels of plasma glucose . the amount of glucose available for excretion is dependent on the amount entering the nephrons , which , in turn , depends on blood glucose concentration at the glomerulus . thus , the amount of glucose excreted is greater when the blood plasma glucose concentrations are highest . in effect , glucose removal might be expected only to be greatest at times when it is most needed ( when glucose levels are highest ) , such as during post - prandial hyperglycemia . the benefit to those patients in whom treatment has provided mild - to - moderate glycemic control might be questioned , as the potential for glucose excretion would be relatively low . nevertheless , patients who achieve moderate glycemic control may be exposed to clinically relevant post - prandial glucose excursions that can impart disproportionate effects on hba1c and possibly the morbidity and mortality associated with t2dm.56 in such a patient population , sglt2 inhibitors might attenuate the impact of post - prandial glucose spikes . nevertheless , clinical experience with agents , such as the meglitinides , that target post - prandial glucose control , suggest that the clinical benefit of this approach is disappointing . treatments targeting post - prandial glucose levels provide little more than modest improvements in hba1c with little evidence of long - term outcome benefits for patients.57 as sglt2 may be responsible for as much as 90% of glucose reabsorption by the kidney , there is the clinical potential for as much as 160 g of glucose to be excreted each day following effective sglt2 inhibition.23 however , it appears that the actual glucose loss achieved in clinical studies is only about half that predicted.38 it is not clear whether this is a consequence of compensating mechanisms undertaking tubular reabsorption or incomplete inhibition of the transporter . thus far , the safety profile of sglt2 inhibitors reported from clinical studies appears to fulfill expectations.33,34,55,40,58 sglt2 inhibitors are designed to target a highly specific membrane transporter that is almost exclusively expressed within the renal tubules . clearly , compared with less specific molecules , the potential for it is also unlikely that sglt2 inhibitors will induce hypoglycemia , since when plasma glucose levels are low the amount of glucose excreted will also be low.59 this prediction appears to be confirmed by clinical studies reported thus far , which show no apparent increases in hypoglycemic episodes with sglt2 inhibitors.50,60 even when sglt2 is blocked completely , a degree of renal glucose recovery is maintained via the relatively unhindered sglt1 transporter . one aspect of sglt2 inhibition that has been raised as a potential issue of safety concern is that of glycosuria , which could predispose patients to increased urinary tract infections ( uti ) . there have been some reports of infection in clinical studies.60,61 however , a study that reviewed risk factors for developing utis in women with diabetes observed that glucosuria was not a significant contributing factor.62 interestingly , there is a rare group of individuals who do not express the sglt2 transporter or in which its functionality has been partially or completely lost due to a genetic mutation for which both an autosomal recessive and dominant pattern of inheritance has been reported . these people do not appear to suffer any ill consequences , suggesting that blockade of the transporter per se in t2 dm patients would offer no immediate risk . patients expressing these mutations have decreased renal tubular reabsortion of glucose from the lumen in the absence of hyperglycemia , or any other signs of tubular dysfunction . it is not clear whether familial renal glucosuria ( due to sglt2 mutation ) protects against t2 dm ; although sglt2 deletion in animal models appears to improve glucose homeostasis and preserve pancreatic -cell function.63 we did not find any recorded evidence of an increased disposition to urinary tract or vulvovaginal infections , although identification and study of these subjects is difficult due to the rarity of the disease . clearly , clinical development programs will need to address the concern of a possible increased risk of uti . increased glucose content in the urine following sglt2 inhibition will likely serve to increase urinary flow as a consequence of the osmotic diuretic effect in the lumen of the nephron . this could result in modest , possibly beneficial , reductions in blood pressure , but raises additional safety concerns associated with possible loss of fluid and solutes . this may be of particular concern in elderly patients or those who do not have the capacity to maintain their fluid balance . however , it should be noted that the effect is considerably lower than that seen with frequently used loop diuretics and there is no apparent change in glomerular filtration rate that would be indicative of a direct effect on renal function . simple instructions on maintaining a state of hydration with regular drinks may serve to overcome the concerns over both urinary infection and fluid imbalance . the question arises as to where sglt2 inhibitors might fit in the current cascade of treatments for the management of t2 dm . while treatment of t2 dm follows prescribed guidelines , there are many approaches and permutations to their application in clinical practice . although the sglt2 inhibitors mechanism of action would make them suitable for initial monotherapy in patients with early stage t2 dm , it is unlikely they would be considered at this stage . most treatments are currently initiated with metformin , which is relatively inexpensive , has a good historical safety profile , and is efficacious . clearly , when they are first launched , the sglt2 inhibitors will not be able to compete with metformin , purely on the issue of cost . however , as explained earlier , the advantage of this may be minimal in patients achieving a degree of glycemic control . by promoting an escape mechanism for glucose , sglt2 inhibitors introduce a new mode to the control of t2 dm . with the exception of -glucosidase inhibitors , which block glucose uptake from the gut , all currently available antidiabetic therapies directly or indirectly modulate insulin to manipulate endogenous glucose utilization . despite the modest effect on hba1c predicted for sglt2 inhibitors , the introduction of a novel means of reducing hyperglycemia increases the treatment options available to physicians for a disease that frequently requires the use of multiple agents to achieve control targets.57 the expected favorable safety profile and insulin - independent mechanism of action appear to support the use of sglt2 inhibitors in combination with other antidiabetic drugs . insulin - dependent therapies become less effective with the development of insulin resistance and/or deterioration of -cell function ; particularly in patients with low insulin resistance ( high glucose ) or poorly controlled disease . the insulin - independent action of sglt2 inhibitors suggests potential for a synergistic effect in such scenarios . the insulin - independent action of sglt2 inhibitors also means that they may be of use in type 1 diabetes , perhaps as a means of moderating post - prandial glucose excursions . by increasing excretion of glucose , sglt2 inhibitors offer an opportunity to increase calorie loss in t2 dm patients , most of whom are overweight . the continual loss of 8090 g of glucose per day ( 300400 calories ) is a significant loss of calories that should work synergistically with weight reduction programs.64 short - term studies in both animals and man appear to confirm the predicted weight reducing property . this contrasts with several drug therapies , including sulfonylureas , insulin , and thiazolidinediones , which are generally associated with weight gain.65,66 but there are currently no data to confirm whether the rate of calorie loss continues with chronic therapy . in a clinical setting it would be easy to overcome any benefits that patients may derive from sglt2 inhibition if they perceived the drug as a means of acclimatized to higher levels of plasma glucose would experience a marked change in appetite with substantial loss of glucose / calories.67 one final issue is that for these drugs to work , they need to be delivered to the epithelial luminal surface of the nephron , requiring them to be cleared by the glomerulus . consequently , their efficacy may be affected in those instances when the functioning of the kidney is impaired , for example in diabetic nephropathy . most patients with t2 dm eventually succumb to the progressive nature of the disease and will , at some point , require multiple therapies to attain treatment targets . only half the patients with t2 dm achieve the hba1c target of less than 7% , despite the many different treatment options currently available . with sglt2 inhibitors introducing an alternative means of managing glucose , we gain a further treatment option that may increase our ability to control t2 dm . experience will lead to better understanding of which patients are likely to respond best , and under what circumstances . the goal of defibrillation ( df ) testing is to verify the detection of ventricular tachyarrhythmias by implantable cardioverter - defibrillators ( icds ) , and to ascertain df efficacy . all clinical trials , which demonstrated the benefit of icd therapy , have included some kind of df testing . according to the food and drug administration , the instructions for use of icds include the recommendation with df testing at the time of implantation . the absence of df testing may have medico - legal implications whether a device fails to terminate ventricular fibrillation ( vf ) during follow - up . nevertheless , df testing has been deliberately omitted in the majority of patients enrolled in contemporary icd studies . despite the stochastic nature of df , ineffective shocks during vf induction result in system modifications in up to 5% of patients . system modifications , such as repositioning of the electrode , reversal of polarity , use of a high - energy device , use of a dual instead of a single - coil electrode , or the implantation of an additional subcutaneous electrode , may be unnecessary or even harmful since many patients have a successful retesting with the original system configuration . no single system modification in itself has ever been shown to improve outcome . with current devices and programming , an inadequate safety margin occurs infrequently , and the incidence of ventricular tachyarrhythmias requiring shocks is low at 46% per year . while df testing has never been shown to improve survival , it is associated with an increased , albeit small risk of major adverse events of 0.4% . by omitting routine df testing , the approach to icd implantation will likely change considerably , as the procedures will be shorter and performed without technical support and anaesthesia . similar to the simple trial , we hypothesized that , with current high - energy icd devices , the omission of df testing during implantation will not result in an inferior shock efficacy during follow - up when compared with standard df testing . as opposed to the simple trial , which used the composite outcome of arrhythmic death or failed appropriate shock , the primary end point of our study was the shock efficacy of all true ventricular tachycardia ( vt ) and vf episodes occurring in any patient during follow - up . the nordic icd trial ( nct01282918 ) was a prospective , randomized , parallel group , multi - centre non - inferiority trial conducted at 48 centres in five european countries , and designed to investigate the effect of df testing at the time of icd implantation on first shock efficacy ( fse ) during follow - up . briefly , patients with an indication for icd implantation according to the current european society of cardiology guidelines were included . patients with hypertrophic or arrhythmogenic right ventricular cardiomyopathy were excluded because of special considerations necessary during icd implantation . one thousand and seventy - seven patients were randomized ( 1 : 1 ) to first time icd implantation with or without df testing during the procedure . patients were followed for at least 12 months through regular on - site visits , and remotely via home monitoring ( hm ; biotronik se & co. kg , berlin , germany ) . all true ventricular tachyarrhythmias treated with an icd shock in any patient were included in the primary analysis . the study was conducted in accordance with the declaration of helsinki , and country - specific regulatory requirements . the protocol was approved by the institutional review board or ethics committee of each participating centre . a standard df testing protocol was used by all participating centres , as described previously . briefly , the initial shock for df was programmed to 15 j. if successful , df testing was terminated . if unsuccessful , a second shock programmed to 24 j was delivered , and had to be confirmed . if unsuccessful , a system revision was recommended , and the df testing procedure was repeated . the primary end point was the fse to terminate all true episodes of vt / vf during follow - up . a blinded two - member clinical event committee reviewed the diagnostic information of the vt / vf treated with icd shocks . safety end points included procedural serious adverse events , vt / vf conversion rate , and all - cause , cardiac , or arrhythmic mortality during follow - up . all adverse events were reviewed and adjudicated by a data and safety monitoring board ( dsmb ) , consisting of three members . all patient deaths were adjudicated by the dsmb according to a classification originally described by epstein et al . the goal of the nordic icd trial was to demonstrate the non - inferiority of no df testing when compared with df testing with respect to the primary endpoint of fse . this margin was chosen because the variation of fse in published trials was in the range of 8392% . after a pre - specified blinded re - assessment , the sample size was increased to 1080 patients recruited over a period of 28 months , with a follow - up of at least 12 months . the intention - to - treat ( itt ) population which included all randomized patients , the evaluated - for - safety population which included randomized patients who received an icd , and the per protocol ( pps ) population which included randomized patients who received an icd and did not have a major protocol violation . however , for time - dependent outcomes , these patients were included up to the date of the protocol violation . the primary analysis was performed in the per protocol episode data set ( p - pps ) , which included all sufficiently documented shocked episodes in pps patients . baseline characteristics are presented group wise for the itt population as mean ( sd ) or frequency ( % ) . to compensate for the dependence structure induced by recurrent episodes with similar outcome in some of the patients , a random effects logit model with patients as a random factor and group as a fixed factor was used for calculation of a two - sided 95% confidence interval ( ci ) of the difference fsenodftest fsedftest . to state non - inferiority , this ci had to lie completely above the pre - specified non - inferiority margin of 10% . without the use of a model that takes the correlation structure into account , the few patients with an extreme numbers of shocks would have dominated the results . procedural and safety outcomes were compared with a student 's t test , or test or fisher 's exact test , whichever was appropriate . hazard rate reduction was assessed using a cox proportional hazards model . a two - tailed p - value < 0.05 was considered to be statistically significant . data were analysed using sas ( version 9.4 ) and stata ( version 13.1 ) by an independent statistical institute ( department of medical biometry and epidemiology , university medical centre hamburg eppendorf , germany ) ( a.s . , k.w . ) . the first draft of the manuscript was written by the first author , and revised by all co - authors . the authors take full responsibility for the accuracy and completeness of the findings , as well as for the fidelity to the study protocol . the nordic icd trial ( nct01282918 ) was a prospective , randomized , parallel group , multi - centre non - inferiority trial conducted at 48 centres in five european countries , and designed to investigate the effect of df testing at the time of icd implantation on first shock efficacy ( fse ) during follow - up . briefly , patients with an indication for icd implantation according to the current european society of cardiology guidelines were included . patients with hypertrophic or arrhythmogenic right ventricular cardiomyopathy were excluded because of special considerations necessary during icd implantation . one thousand and seventy - seven patients were randomized ( 1 : 1 ) to first time icd implantation with or without df testing during the procedure . patients were followed for at least 12 months through regular on - site visits , and remotely via home monitoring ( hm ; biotronik se & co. kg , berlin , germany ) . all true ventricular tachyarrhythmias treated with an icd shock in any patient were included in the primary analysis . the study was conducted in accordance with the declaration of helsinki , and country - specific regulatory requirements . the protocol was approved by the institutional review board or ethics committee of each participating centre . a standard df testing protocol was used by all participating centres , as described previously . briefly , the initial shock for df was programmed to 15 j. if successful , df testing was terminated . if unsuccessful , a second shock programmed to 24 j was delivered , and had to be confirmed . if the shocks were successful twice , df testing was terminated . if unsuccessful , a system revision was recommended , and the df testing procedure was repeated . the primary end point was the fse to terminate all true episodes of vt / vf during follow - up . a blinded two - member clinical event committee reviewed the diagnostic information of the vt / vf treated with icd shocks . safety end points included procedural serious adverse events , vt / vf conversion rate , and all - cause , cardiac , or arrhythmic mortality during follow - up . all adverse events were reviewed and adjudicated by a data and safety monitoring board ( dsmb ) , consisting of three members . all patient deaths were adjudicated by the dsmb according to a classification originally described by epstein et al . the goal of the nordic icd trial was to demonstrate the non - inferiority of no df testing when compared with df testing with respect to the primary endpoint of fse . this margin was chosen because the variation of fse in published trials was in the range of 8392% . after a pre - specified blinded re - assessment , the sample size was increased to 1080 patients recruited over a period of 28 months , with a follow - up of at least 12 months . the intention - to - treat ( itt ) population which included all randomized patients , the evaluated - for - safety population which included randomized patients who received an icd , and the per protocol ( pps ) population which included randomized patients who received an icd and did not have a major protocol violation . however , for time - dependent outcomes , these patients were included up to the date of the protocol violation . the primary analysis was performed in the per protocol episode data set ( p - pps ) , which included all sufficiently documented shocked episodes in pps patients . baseline characteristics are presented group wise for the itt population as mean ( sd ) or frequency ( % ) . to compensate for the dependence structure induced by recurrent episodes with similar outcome in some of the patients , a random effects logit model with patients as a random factor and group as a fixed factor was used for calculation of a two - sided 95% confidence interval ( ci ) of the difference fsenodftest fsedftest . to state non - inferiority , this ci had to lie completely above the pre - specified non - inferiority margin of 10% . without the use of a model that takes the correlation structure into account , the few patients with an extreme numbers of shocks would have dominated the results . procedural and safety outcomes were compared with a student 's t test , or test or fisher 's exact test , whichever was appropriate . mortality was analysed in the itt population using the kaplan meier approach and the log - rank test . hazard rate reduction was assessed using a cox proportional hazards model . a two - tailed p - value < 0.05 was considered to be statistically significant . data were analysed using sas ( version 9.4 ) and stata ( version 13.1 ) by an independent statistical institute ( department of medical biometry and epidemiology , university medical centre hamburg eppendorf , germany ) ( a.s . , k.w . ) . the first draft of the manuscript was written by the first author , and revised by all co - authors . the authors take full responsibility for the accuracy and completeness of the findings , as well as for the fidelity to the study protocol . the baseline clinical characteristics and cardiovascular medications of the 1077 patients comprising the intention - to - treat population ( without df = 537 and with df = 540 ) are presented in table 1 . seven hundred and one patients ( 65.1% ) had an ischaemic cardiomyopathy and 873 ( 81.8% ) received the icd for primary prevention of sudden cardiac death . ten patients ( six in the df group and four in the without df group ) did not receive an icd , leaving 1067 patients for the safety evaluation ( figure 1 ) . both groups were well matched for baseline demographics and clinical characteristics , as well as for the use of dual ( 51.6% ) and a single coil ( 48.4% ) electrode ( table 2 ) . table 1baseline demographic and clinical characteristics of subjects by treatment group ( intention to treat)characteristictotal ( n = 1077)without df test ( n = 537)with df test ( n = 540)age ( year)64.8 ( 10.9)64.7 ( 11.2)64.9 ( 10.6)male sex , n ( % ) 873 ( 81.1)430 ( 80.1)443 ( 82.0)ischaemic disease , n ( % ) 701 ( 65.1)341 ( 63.5)360 ( 66.7)hypertension , n / total n ( % ) 509/700 ( 72.7)249/349 ( 71.3)260/351 ( 74.1)diabetes mellitus , n ( % ) 368 ( 34.2)183 ( 34.1)185 ( 34.3)renal insufficiency , n ( % ) 303 ( 28.1)144 ( 26.8)159 ( 29.4)nyha class , n ( % ) i59 ( 5.5)31 ( 5.8)28 ( 5.2 ) ii464 ( 43.1)226 ( 42.1)238 ( 44.1 ) iii487 ( 45.2)254 ( 47.3)233 ( 43.1 ) iv14 ( 1.3)5 ( 0.9)9 ( 1.7 ) unknown53 ( 4.9)21 ( 3.9)32 ( 5.9)nyha class , n ( % ) ii523 ( 48.6)257 ( 47.9)266 ( 49.3 ) iii501 ( 46.5)259 ( 48.2)242 ( 44.8 ) unknown53 ( 4.9)21 ( 3.9)32 ( 5.9)estimated bmi ( n = 1054)28.2 ( 4.9)28.1 ( 4.8)28.2 ( 5.1)lvef , n ( % ) < 20%101 ( 9.4)53 ( 9.9)48 ( 8.9 ) 2030%566 ( 52.6)278 ( 51.8)288 ( 53.3 ) > 30%404 ( 37.5)204 ( 38.0)200 ( 37.0 ) not done6 ( 0.6)2 ( 0.4)4 ( 0.7)af at enrolment , n ( % ) 85 ( 7.9)45 ( 8.4)40 ( 7.4)indication for implantation , n / total n ( % ) primary prevention873/1067 ( 81.8)434/531 ( 81.7)439/536 ( 81.9 ) secondary prevention194/1067 ( 18.2)97/531 ( 18.3)97/536 ( 18.1)hospital stay ( days ) ( n = 1066)4.0 ( 4.0)4.0 ( 3.6)4.0 ( 3.8)medication , n ( % ) ace inhibitors / at receptor blockers , n ( % ) 986 ( 91.6)501 ( 93.3)485 ( 89.8 ) -blockers , n ( % ) 1007 ( 93.5)500 ( 93.1)507 ( 93.9 ) ca antagonists , n ( % ) 140 ( 13.0)74 ( 13.8)66 ( 12.2 ) spironolactones , n ( % ) 618 ( 57.4)302 ( 56.2)316 ( 58.5 ) ( other ) diuretics , n ( % ) 794 ( 73.7)395 ( 73.6)399 ( 73.9 ) nitrates , n ( % ) 75 ( 7.0)38 ( 7.1)37 ( 6.9 ) digitalis , n ( % ) 105 ( 9.7)54 ( 10.1)51 ( 9.4 ) lipid - lowering agents , n ( % ) 763 ( 70.8)377 ( 70.2)386 ( 71.5 ) amiodarone , n ( % ) 116 ( 10.8)61 ( 11.4)55 ( 10.2 ) dronedarone , n ( % ) 5 ( 0.5)4 ( 0.7)1 ( 0.2 ) sotalol , n ( % ) 9 ( 0.8)5 ( 0.9)4 ( 0.7 ) ( other ) anti - arrhythmics , n ( % ) 22 ( 2.0)9 ( 1.7)13 ( 2.4 ) platelet aggregation inhibitor , n ( % ) 753 ( 69.9)370 ( 68.9)383 ( 70.9 ) anti - coagulants , n ( % ) 350 ( 32.5)183 ( 34.1)167 ( 30.9 ) other cardiovascular medication , n ( % ) 255 ( 23.7)121 ( 22.5)134 ( 24.8)values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate.ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . table 2procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - safety)characteristictotal ( n = 1067)without df test ( n = 533)with df test ( n = 534)p - valueimplanted lead type , n ( % ) single coil516 ( 48.36)262 ( 49.16)254 ( 47.57)0.603 dual coil551 ( 51.64)271 ( 50.84)280 ( 52.43)type , n ( % ) single chamber icd466 ( 43.67)236 ( 44.28)230 ( 43.07)0.648 dual chamber icd245 ( 22.96)116 ( 21.76)129 ( 24.16 ) crt - d356 ( 33.36)181 ( 33.96)175 ( 32.77)left position , n ( % ) 1058 ( 99.16)527 ( 98.87)531 ( 99.44)0.341 ( f)vegm signal amplitude ( mv ) ( n = 1057)12.42 ( 5.30)12.72 ( 5.37)12.12 ( 5.21)0.065pacing threshold ( v ) at pulse duration of 0.4 ms ( n = 527)0.51 ( 0.20)0.49 ( 0.17)0.53 ( 0.22)0.040pacing threshold ( v ) at pulse duration of 0.5 ms ( n = 533)0.45 ( 0.23)0.45 ( 0.27)0.44 ( 0.17)0.613patients received df test , n ( % ) 527 ( 49.39)7 ( 1.31)520 ( 97.38)delivered icd shocks per patient0.71 ( 1.16)0.01 ( 0.11)1.41 ( 1.31)<0.001df energy at final position ( j ) ( n = 526)16.74 ( 4.47)16.67 ( 4.38)patients with intra - procedural system revisions and icd re - programming , n / total n ( % ) 25/527 ( 4.74)25/520 ( 4.81)patients with electrode repositionings , n / total n ( % ) 8/527 ( 1.52)8/520 ( 1.54)patients with changes of shock polarity , n / total n ( % ) 20/527 ( 3.80)20/520 ( 3.85)patients with modification of lead system ( shock pathway ) , n / total n ( % ) 5/527 ( 0.95)5/520 ( 0.96)patients with other revisions / re - programming , n / total n ( % ) 7/527 ( 1.33)7/520 ( 1.35)procedure duration ( min ) ( n = 1060)64.54 ( 39.88)63.11 ( 40.84)65.97 ( 38.88)0.243fluoroscopy exposure duration ( min ) ( n = 1056)8.02 ( 11.43)8.00 ( 11.44)8.05 ( 11.43)0.943crt - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter-defibrillator.one patient was not inducible . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . baseline demographic and clinical characteristics of subjects by treatment group ( intention to treat ) values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate . ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - safety ) crt - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter - defibrillator . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . of the 534 patients randomized to the df arm with a successful first icd implantation ( figure 1 ) , testing was performed in 97.4% . in one patient , vt / vf was not inducible . four hundred and ninety - four of 519 inducible patients ( 95.2% ) passed the test without any system reconfiguration , and 440 patients ( 84.8% ) had their vt / vf terminated with a 15 j shock . in 25 patients , the initial df test was not successful , and system reconfigurations were performed once in 20 and more than once in 5 ( table 2 ) . all patients subsequently passed the test successfully , but still with a higher energy level at the final position ( 22.2 7.4 j vs. 16.4 4.0 j , p < 0.001 ) and with significantly more shocks ( 5.7 3.0 vs. 1.2 0.7 , p < 0.001 ) than the 494 patients who passed the test immediately . in addition , the duration of the procedure ( 90.2 56.2 min vs. 63.5 36.1 min , p < 0.001 ) and fluoroscopy exposure ( 11.6 14.2 min vs. 7.5 10.7 min , p = 0.064 ) was increased . during a median follow - up of 22.7 and 22.9 months , respectively , 211 true vt / vf episodes with at least one appropriate shock occurred in 8.6% of the per protocol patients with df testing , while 218 true vt / vf episodes occurred in 8.8% of patients without df testing ( table 3 , figure 1 ) . all true vt / vf episodes were terminated with any appropriate icd shock in the without df test group , whereas 96.7% of all vt / vf were terminated in the group with df test . thus , the conversion efficacy was 100.0% without df testing and 96.7% with df testing . the model - based shock efficacy was 97.1% without df testing and 94.1% with df testing . the difference in fse was 3.0% ( ci 3.0% , 9.0% ) in favour of the without df test group , and remained above the pre - defined 10% margin for the primary end point ( pnon - inferiority < 0.001 , table 3 , figure 2 ) . table 3primary analysis shock efficacy ( per protocol episode data set)totalwithout df testwith df testall patients with at least one delivered appropriate shock for a true vt / vf episode , n / total n ( % ) 91/1046 ( 8.7)46/523 ( 8.8)45/523 ( 8.6)all true vt / vf episodes with at least one delivered appropriate icd shock ( vt / vf ) , n429218211delivered appropriate shocks per true vt / vf episode ( mean ( sd))1.18 ( 0.69)1.09 ( 0.37)1.28 ( 0.90)true vt / vf episodes terminated with first appropriate icd shock , n / total n ( % ) 384/429 ( 89.5)204/218 ( 93.6)180/211 ( 85.3)true vt / vf episodes terminated with any appropriate icd shock , n / total n ( % ) 422/429 ( 98.4)218/218 ( 100.0)204/211 ( 96.7)true vt / vf episodes not terminated with any appropriate icd shock , n / total n ( % ) 7/429 ( 1.6)0/218 ( 0.0)7/211 ( 3.3)conversion efficacy [ proportion ( 95% ci)]0.98 ( 0.97;0.99)1.00 ( 0.98;1.00)0.97 ( 0.93;0.99)first shock efficacy model based [ proportion ( 95% ci)]0.96 ( 0.89;0.98)0.97 ( 0.91;0.99)0.94 ( 0.83;0.98))difference between the groups ( without df testing vs. with df testing ) : 0.03 ( 0.03;0.09).df , defibrillation ; icd , implantable cardioverter - defibrillator ; p - pps , per protocol episode data set ; vt / vf , ventricular tachycardia / ventricular fibrillation . primary analysis shock efficacy ( per protocol episode data set ) difference between the groups ( without df testing vs. with df testing ) : 0.03 ( 0.03;0.09 ) . df , defibrillation ; icd , implantable cardioverter - defibrillator ; p - pps , per protocol episode data set ; vt / vf , ventricular tachycardia / ventricular fibrillation . a total of 201 serious adverse events related to the study procedure occurred in 168 patients ( 15.7% ) , of which 89 events occurred in 74 patients without df testing and 112 events occurred in 94 patients with df testing within 30 days of follow - up ( p = 0.095 ) . the only significant difference in single serious adverse events was intra - operative hypotension , which manifested more often in patient with df testing ( 1.7% ) than those without df testing ( 0.0% ) ( p = 0.004 ) . lead - related complications occurred in 15 patients without df testing ( 2.8% ) compared with 24 events in 21 patients with df testing ( 3.9% ) ( p = 0.311 ) . one sudden death occurred in each study group within 30 days of follow - up , and the mean length of hospitalization was 4.0 days in both study groups ( table 1 ) . the proportion of patients with inappropriate shocks during follow - up was low at 3.7% , and similar between groups . forty - four and 52 patients without and with df testing died , respectively ( p = 0.377 , table 4 ) . differences with respect to non - cardiac and cardiac mortality were not statistically significant . six deaths ( 5 with df and 1 without df testing ) were adjudicated arrhythmic and due to repetitive ventricular tachyarrhythmias / vt clusters ( p = 0.100 , table 4 ) . table 4mortality ( intention - to - treat)outcomeeventsincidencelog rank95% ciwithout df test ( n = 537)with df test ( n = 540)without df testwith df testp - valuehrlowerupperall - cause mortality44 ( 8.2%)52 ( 9.6%)0.0440.0530.3770.8350.5591.248non - cardiac mortality24 ( 4.5%)23 ( 4.3%)0.0240.0230.9071.0350.5841.833unknown ( primary organ cause)6 ( 1.1%)10 ( 1.9%)0.0060.010.3060.5930.2161.632cardiac mortality14 ( 2.6%)19 ( 3.5%)0.0140.0190.3500.7210.3611.438arrhythmic mortality1 ( 0.2%)5 ( 0.9%)0.0010.0050.1000.1980.0231.694non - arrhythmic mortality13 ( 2.4%)13 ( 2.4%)0.0130.0130.9480.9750.4522.103unknown cardiac mortality0 ( 0.0%)1 ( 0.2%)00.001df , defibrillation ; hr , hazard ratio ; itt , intention to treat.incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . mortality ( intention - to - treat ) df , defibrillation ; hr , hazard ratio ; itt , intention to treat . incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . the baseline clinical characteristics and cardiovascular medications of the 1077 patients comprising the intention - to - treat population ( without df = 537 and with df = 540 ) are presented in table 1 . seven hundred and one patients ( 65.1% ) had an ischaemic cardiomyopathy and 873 ( 81.8% ) received the icd for primary prevention of sudden cardiac death . ten patients ( six in the df group and four in the without df group ) did not receive an icd , leaving 1067 patients for the safety evaluation ( figure 1 ) . both groups were well matched for baseline demographics and clinical characteristics , as well as for the use of dual ( 51.6% ) and a single coil ( 48.4% ) electrode ( table 2 ) . table 1baseline demographic and clinical characteristics of subjects by treatment group ( intention to treat)characteristictotal ( n = 1077)without df test ( n = 537)with df test ( n = 540)age ( year)64.8 ( 10.9)64.7 ( 11.2)64.9 ( 10.6)male sex , n ( % ) 873 ( 81.1)430 ( 80.1)443 ( 82.0)ischaemic disease , n ( % ) 701 ( 65.1)341 ( 63.5)360 ( 66.7)hypertension , n / total n ( % ) 509/700 ( 72.7)249/349 ( 71.3)260/351 ( 74.1)diabetes mellitus , n ( % ) 368 ( 34.2)183 ( 34.1)185 ( 34.3)renal insufficiency , n ( % ) 303 ( 28.1)144 ( 26.8)159 ( 29.4)nyha class , n ( % ) i59 ( 5.5)31 ( 5.8)28 ( 5.2 ) ii464 ( 43.1)226 ( 42.1)238 ( 44.1 ) iii487 ( 45.2)254 ( 47.3)233 ( 43.1 ) iv14 ( 1.3)5 ( 0.9)9 ( 1.7 ) unknown53 ( 4.9)21 ( 3.9)32 ( 5.9)nyha class , n ( % ) ii523 ( 48.6)257 ( 47.9)266 ( 49.3 ) iii501 ( 46.5)259 ( 48.2)242 ( 44.8 ) unknown53 ( 4.9)21 ( 3.9)32 ( 5.9)estimated bmi ( n = 1054)28.2 ( 4.9)28.1 ( 4.8)28.2 ( 5.1)lvef , n ( % ) < 20%101 ( 9.4)53 ( 9.9)48 ( 8.9 ) 2030%566 ( 52.6)278 ( 51.8)288 ( 53.3 ) > 30%404 ( 37.5)204 ( 38.0)200 ( 37.0 ) not done6 ( 0.6)2 ( 0.4)4 ( 0.7)af at enrolment , n ( % ) 85 ( 7.9)45 ( 8.4)40 ( 7.4)indication for implantation , n / total n ( % ) primary prevention873/1067 ( 81.8)434/531 ( 81.7)439/536 ( 81.9 ) secondary prevention194/1067 ( 18.2)97/531 ( 18.3)97/536 ( 18.1)hospital stay ( days ) ( n = 1066)4.0 ( 4.0)4.0 ( 3.6)4.0 ( 3.8)medication , n ( % ) ace inhibitors / at receptor blockers , n ( % ) 986 ( 91.6)501 ( 93.3)485 ( 89.8 ) -blockers , n ( % ) 1007 ( 93.5)500 ( 93.1)507 ( 93.9 ) ca antagonists , n ( % ) 140 ( 13.0)74 ( 13.8)66 ( 12.2 ) spironolactones , n ( % ) 618 ( 57.4)302 ( 56.2)316 ( 58.5 ) ( other ) diuretics , n ( % ) 794 ( 73.7)395 ( 73.6)399 ( 73.9 ) nitrates , n ( % ) 75 ( 7.0)38 ( 7.1)37 ( 6.9 ) digitalis , n ( % ) 105 ( 9.7)54 ( 10.1)51 ( 9.4 ) lipid - lowering agents , n ( % ) 763 ( 70.8)377 ( 70.2)386 ( 71.5 ) amiodarone , n ( % ) 116 ( 10.8)61 ( 11.4)55 ( 10.2 ) dronedarone , n ( % ) 5 ( 0.5)4 ( 0.7)1 ( 0.2 ) sotalol , n ( % ) 9 ( 0.8)5 ( 0.9)4 ( 0.7 ) ( other ) anti - arrhythmics , n ( % ) 22 ( 2.0)9 ( 1.7)13 ( 2.4 ) platelet aggregation inhibitor , n ( % ) 753 ( 69.9)370 ( 68.9)383 ( 70.9 ) anti - coagulants , n ( % ) 350 ( 32.5)183 ( 34.1)167 ( 30.9 ) other cardiovascular medication , n ( % ) 255 ( 23.7)121 ( 22.5)134 ( 24.8)values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate.ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . table 2procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - safety)characteristictotal ( n = 1067)without df test ( n = 533)with df test ( n = 534)p - valueimplanted lead type , n ( % ) single coil516 ( 48.36)262 ( 49.16)254 ( 47.57)0.603 dual coil551 ( 51.64)271 ( 50.84)280 ( 52.43)type , n ( % ) single chamber icd466 ( 43.67)236 ( 44.28)230 ( 43.07)0.648 dual chamber icd245 ( 22.96)116 ( 21.76)129 ( 24.16 ) crt - d356 ( 33.36)181 ( 33.96)175 ( 32.77)left position , n ( % ) 1058 ( 99.16)527 ( 98.87)531 ( 99.44)0.341 ( f)vegm signal amplitude ( mv ) ( n = 1057)12.42 ( 5.30)12.72 ( 5.37)12.12 ( 5.21)0.065pacing threshold ( v ) at pulse duration of 0.4 ms ( n = 527)0.51 ( 0.20)0.49 ( 0.17)0.53 ( 0.22)0.040pacing threshold ( v ) at pulse duration of 0.5 ms ( n = 533)0.45 ( 0.23)0.45 ( 0.27)0.44 ( 0.17)0.613patients received df test , n ( % ) 527 ( 49.39)7 ( 1.31)520 ( 97.38)delivered icd shocks per patient0.71 ( 1.16)0.01 ( 0.11)1.41 ( 1.31)<0.001df energy at final position ( j ) ( n = 526)16.74 ( 4.47)16.67 ( 4.38)patients with intra - procedural system revisions and icd re - programming , n / total n ( % ) 25/527 ( 4.74)25/520 ( 4.81)patients with electrode repositionings , n / total n ( % ) 8/527 ( 1.52)8/520 ( 1.54)patients with changes of shock polarity , n / total n ( % ) 20/527 ( 3.80)20/520 ( 3.85)patients with modification of lead system ( shock pathway ) , n / total n ( % ) 5/527 ( 0.95)5/520 ( 0.96)patients with other revisions / re - programming , n / total n ( % ) 7/527 ( 1.33)7/520 ( 1.35)procedure duration ( min ) ( n = 1060)64.54 ( 39.88)63.11 ( 40.84)65.97 ( 38.88)0.243fluoroscopy exposure duration ( min ) ( n = 1056)8.02 ( 11.43)8.00 ( 11.44)8.05 ( 11.43)0.943crt - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter-defibrillator.one patient was not inducible . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . baseline demographic and clinical characteristics of subjects by treatment group ( intention to treat ) values are means sd . there were no significant differences at p < 0.05 between groups , except for ace inhibitors p = 0.039 . t - test for metric variables , -test for categorical , or fisher 's exact test ( f ) if is not appropriate . ace inhibitors , angiotensin converting enzyme inhibitors ; af , atrial fibrillation ; at receptor blockers , angiotensin receptor blockers ; bmi , body mass index ; df , defibrillation ; nyha , new york heart association ; itt , intention - to - treat ; lvef , left ventricular ejection fraction . procedural characteristics according to treatment group ( successful primary implantable cardioverter - defibrillator implantation ; evaluated - for - safety ) crt - d , cardiac - resynchronization therapy with defibrillator ; df , defibrillation ; efs , evaluated for safety ; vegm , ventricular electrogram ; icd , implantable cardioverter - defibrillator . itt , full analysis set ( all patients as randomized ) ; efs , evaluated for safety set ( all patients randomized , who had a successful primary icd implantation ) ; pps , per protocol set ( all patients randomized , who are compliant with the study protocol ) ; p - pps , all randomized patients at least one day at risk before protocol deviation ( includes all sufficiently documented shocked episodes until time point of protocol deviation ) . of the 534 patients randomized to the df arm with a successful first icd implantation ( figure 1 ) , testing was performed in 97.4% . in one patient , vt / vf was not inducible . four hundred and ninety - four of 519 inducible patients ( 95.2% ) passed the test without any system reconfiguration , and 440 patients ( 84.8% ) had their vt / vf terminated with a 15 j shock . in 25 patients , the initial df test was not successful , and system reconfigurations were performed once in 20 and more than once in 5 ( table 2 ) . all patients subsequently passed the test successfully , but still with a higher energy level at the final position ( 22.2 7.4 j vs. 16.4 4.0 j , p < 0.001 ) and with significantly more shocks ( 5.7 3.0 vs. 1.2 0.7 , p < 0.001 ) than the 494 patients who passed the test immediately . in addition , the duration of the procedure ( 90.2 56.2 min vs. 63.5 36.1 min , p < 0.001 ) and fluoroscopy exposure ( 11.6 14.2 min vs. 7.5 10.7 min , p = 0.064 ) was increased . during a median follow - up of 22.7 and 22.9 months , respectively , 211 true vt / vf episodes with at least one appropriate shock occurred in 8.6% of the per protocol patients with df testing , while 218 true vt / vf episodes occurred in 8.8% of patients without df testing ( table 3 , figure 1 ) . all true vt / vf episodes were terminated with any appropriate icd shock in the without df test group , whereas 96.7% of all vt / vf were terminated in the group with df test . thus , the conversion efficacy was 100.0% without df testing and 96.7% with df testing . the model - based shock efficacy was 97.1% without df testing and 94.1% with df testing . the difference in fse was 3.0% ( ci 3.0% , 9.0% ) in favour of the without df test group , and remained above the pre - defined 10% margin for the primary end point ( pnon - inferiority < 0.001 , table 3 , figure 2 ) . table 3primary analysis shock efficacy ( per protocol episode data set)totalwithout df testwith df testall patients with at least one delivered appropriate shock for a true vt / vf episode , n / total n ( % ) 91/1046 ( 8.7)46/523 ( 8.8)45/523 ( 8.6)all true vt / vf episodes with at least one delivered appropriate icd shock ( vt / vf ) , n429218211delivered appropriate shocks per true vt / vf episode ( mean ( sd))1.18 ( 0.69)1.09 ( 0.37)1.28 ( 0.90)true vt / vf episodes terminated with first appropriate icd shock , n / total n ( % ) 384/429 ( 89.5)204/218 ( 93.6)180/211 ( 85.3)true vt / vf episodes terminated with any appropriate icd shock , n / total n ( % ) 422/429 ( 98.4)218/218 ( 100.0)204/211 ( 96.7)true vt / vf episodes not terminated with any appropriate icd shock , n / total n ( % ) 7/429 ( 1.6)0/218 ( 0.0)7/211 ( 3.3)conversion efficacy [ proportion ( 95% ci)]0.98 ( 0.97;0.99)1.00 ( 0.98;1.00)0.97 ( 0.93;0.99)first shock efficacy model based [ proportion ( 95% ci)]0.96 ( 0.89;0.98)0.97 ( 0.91;0.99)0.94 ( 0.83;0.98))difference between the groups ( without df testing vs. with df testing ) : 0.03 ( 0.03;0.09).df , defibrillation ; icd , implantable cardioverter - defibrillator ; p - pps , per protocol episode data set ; vt / vf , ventricular tachycardia / ventricular fibrillation . figure 2primary outcome in the per protocol population . primary analysis shock efficacy ( per protocol episode data set ) difference between the groups ( without df testing vs. with df testing ) : 0.03 ( 0.03;0.09 ) . df , defibrillation ; icd , implantable cardioverter - defibrillator ; p - pps , per protocol episode data set ; vt / vf , ventricular tachycardia / ventricular fibrillation . a total of 201 serious adverse events related to the study procedure occurred in 168 patients ( 15.7% ) , of which 89 events occurred in 74 patients without df testing and 112 events occurred in 94 patients with df testing within 30 days of follow - up ( p = 0.095 ) . the only significant difference in single serious adverse events was intra - operative hypotension , which manifested more often in patient with df testing ( 1.7% ) than those without df testing ( 0.0% ) ( p = 0.004 ) . lead - related complications occurred in 15 patients without df testing ( 2.8% ) compared with 24 events in 21 patients with df testing ( 3.9% ) ( p = 0.311 ) . one sudden death occurred in each study group within 30 days of follow - up , and the mean length of hospitalization was 4.0 days in both study groups ( table 1 ) . the proportion of patients with inappropriate shocks during follow - up was low at 3.7% , and similar between groups . forty - four and 52 patients without and with df testing died , respectively ( p = 0.377 , table 4 ) . differences with respect to non - cardiac and cardiac mortality were not statistically significant . six deaths ( 5 with df and 1 without df testing ) were adjudicated arrhythmic and due to repetitive ventricular tachyarrhythmias / vt clusters ( p = 0.100 , table 4 ) . table 4mortality ( intention - to - treat)outcomeeventsincidencelog rank95% ciwithout df test ( n = 537)with df test ( n = 540)without df testwith df testp - valuehrlowerupperall - cause mortality44 ( 8.2%)52 ( 9.6%)0.0440.0530.3770.8350.5591.248non - cardiac mortality24 ( 4.5%)23 ( 4.3%)0.0240.0230.9071.0350.5841.833unknown ( primary organ cause)6 ( 1.1%)10 ( 1.9%)0.0060.010.3060.5930.2161.632cardiac mortality14 ( 2.6%)19 ( 3.5%)0.0140.0190.3500.7210.3611.438arrhythmic mortality1 ( 0.2%)5 ( 0.9%)0.0010.0050.1000.1980.0231.694non - arrhythmic mortality13 ( 2.4%)13 ( 2.4%)0.0130.0130.9480.9750.4522.103unknown cardiac mortality0 ( 0.0%)1 ( 0.2%)00.001df , defibrillation ; hr , hazard ratio ; itt , intention to treat.incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . mortality ( intention - to - treat ) df , defibrillation ; hr , hazard ratio ; itt , intention to treat . incidence referring to person - years without df test ( 1001.5 ) and with df test ( 985.9 ) . defibrillation testing during icd implantation has been a matter of debate for the last two decades . to the best of our knowledge , the nordic icd trial is the first prospective randomized trial , which investigated the effect of df testing on the actual fse during follow - up as the single primary end point . this is in contrast with the recently published simple trial , which used the composite outcome of arrhythmic death or failed appropriate shock to terminate a spontaneous episode of vt / vf to determine the non - inferiority of the no testing approach . nordic icd also provides detailed information about intra - operative df testing outcome . in 9196% of icd implants , system modifications were reported in 2.212.0% of patients . with the high - energy devices used in the nordic icd study , only 4.8% of all inducible patients had an inadequate safety margin , which required one or two system reconfigurations in 3.8 and 1.0% , respectively . this is in agreement with the results of the sudden cardiac death in heart failure trial ( scd - heft ) , in which 97.8% of patients had a df threshold 20 j. following the system revisions , all patients passed the test successfully , but with a significantly higher energy level than for those who did not require revision ( 22.2 j vs. 16.4 j , p < 0.001 ) . this is in agreement with a canadian survey in which an insufficient safety margin with the initial system configuration could be improved in only 56% of patients . while the device revisions provided no benefit to our patients during follow - up , they significantly increased the total procedure time by 30 min and the fluoroscopy exposure by 40% . the mean number of shocks needed to find an acceptable safety margin was also increased more than four - fold . however , in a trial with variable use of devices , the shock efficacy is similar to that observed in the nordic icd trial . the observed lower rate of arrhythmic death for patients without df testing ( 0.2 vs. 0.9% ) corresponds to the sudden cardiac death rate reported in another trial ( 0.9 and 1.2% in patients without and with df testing ) during a similar follow - up period . because the simple trial did not use a specific model for multiple shock events in the same patient , the shock efficacy can only be compared at the level of raw shock efficacy . in the nordic icd and the simple trials , a higher raw shock efficacy was observed in the group without df vs. with df testing ( nordic icd : 93.6 vs. 85.3% and simple : 92.0 vs. 88.5% ) . on the other hand , the higher first shock energy we used ( 40 j vs. 31 j in simple ) was not associated with a higher fse irrespective of testing . the high level of fse is consistent with the relatively low mean df energy ( 16.7 j ) during testing , with > 95% of the inducible patients undergoing successful testing without any system reconfiguration . a 15.7% serious adverse events rate ( 17.6% with testing and 13.9% without testing , p = 0.095 ) was found within 30 days following successful icd implantation . this is much higher than what has been described in recent reports , in which the complication rates ranged between 2.3 and 2.7% in patients with and without an adequate safety margins during df testing or compared with the simple trial results regarding adverse events included in the pre - defined as the primary safety outcome ( 5.6% without df testing vs. 6.5% with df testing ) . it is noteworthy that the nordic icd trial not only collected data on protocol - specified complications but also on all procedure- and patient - related adverse events which might explain our higher event rate . the 30-day procedure - related mortality rate was 0.2% , and consisted of two sudden deaths which is in the range of recent reports . the stroke rate was 0.8% for patients without and 0.2% for patients with df testing ( p = 0.217 ) , which is in agreement with the 0.4% rate reported in the credit registry . the two types of adverse events that were increased in patients with df testing were intra - operative hypotension ( 1.7% vs. 0.0% , p = 0.004 ) and lead - related complications ( 3.9 vs. 2.8% , p = 0.311 ) . a recent article summarized implant - related complications that were directly or indirectly related to the df testing procedure . the rate of haemodynamic complications was 1% , and lead dislodgement was listed as the most frequent single complication with rates between 1 and 6% . both the nordic icd and simple trials showed a trend towards lower procedural complication rates in patients without df testing despite the different definitions and methods used for analysing these events ( nordic icd : 13.9 vs.17.6% , p = 0.095 , simple : 5.6 vs. 6.5% , p = 0.33 ) . the rates of all - cause , cardiac , and non - cardiac mortality were similar between patients with and without df testing , which is in agreement with most non - randomized trials reported . in accordance with the simple trial , no statistical difference in all - cause mortality between patients with and without df testing however , the annual mortality rates of nordic icd were lower when compared with other large randomized icd trials , and also lower than those of the simple trial . the nordic icd trial supports the hypothesis that if 40 j devices are used , df testing does not improve df efficacy during follow - up . it suggests that df testing or the various measures to improve df efficacy lengthen the procedure , and may even be harmful . the nordic icd trial is one of two large prospective randomized trials , which do not support the routine use of df testing during left - sided first time icd implantation . this work was supported by biotronik se & co. kg ( berlin , germany ) . funding to pay reports receiving grants and personal fees form medtronic , st jude medical , biosense , biotronik , and boston scientific during the conduct of the study , and grants and personal fees from biosense and biotronik outside of the submitted work . reports receiving grants , personal fees , and non - financial support from biotronik during the conduct of the study , and personal fees and non - financial support from boehringer ingelheim outside the submitted work . reports personal fees from biotronik during the conduct of the study , and grants from st jude medical and personal fees from boston scientific outside the submitted work .

there is a recognized need for new treatment options for type 2 diabetes mellitus ( t2 dm ) . recovery of glucose from the glomerular filtrate represents an important mechanism in maintaining glucose homeostasis and represents a novel target for the management of t2 dm . recovery of glucose from the glomerular filtrate is executed principally by the type 2 sodium - glucose cotransporter ( sglt2 ) . inhibition of sglt2 promotes glucose excretion and normalizes glycemia in animal models . first reports of specifically designed sglt2 inhibitors began to appear in the second half of the 1990s . several candidate sglt2 inhibitors are currently under development , with four in the later stages of clinical testing . the safety profile of sglt2 inhibitors is expected to be good , as their target is a highly specific membrane transporter expressed almost exclusively within the renal tubules . one safety concern is that of glycosuria , which could predispose patients to increased urinary tract infections . so far the reported safety profile of sglt2 inhibitors in clinical studies appears to confirm that the class is well tolerated . where sglt2 inhibitors will fit in the current cascade of treatments for t2 dm has yet to be established . the expected favorable safety profile and insulin - independent mechanism of action appear to support their use in combination with other antidiabetic drugs . promotion of glucose excretion introduces the opportunity to clear calories ( 8090 g [ 300400 calories ] of glucose per day ) in patients that are generally overweight , and is expected to work synergistically with weight reduction programs . experience will most likely lead to better understanding of which patients are likely to respond best to sglt2 inhibitors , and under what circumstances . aimsthis trial was designed to test the hypothesis that shock efficacy during follow - up is not impaired in patients implanted without defibrillation ( df ) testing during first implantable cardioverter - defibrillator ( icd ) implantation.methods and resultsbetween february 2011 and july 2013 , 1077 patients were randomly assigned ( 1 : 1 ) to first time icd implantation with ( n = 540 ) or without ( n = 537 ) df testing . the intra - operative df testing was standardized across all participating centres , and all icd shocks were programmed to 40 j irrespective of df test results . the primary end point was the average first shock efficacy ( fse ) for all true ventricular tachycardia and fibrillation ( vt / vf ) episodes during follow - up . the secondary end points included procedural data , serious adverse events , and mortality . during a median follow - up of 22.8 months , the model - based fse was found to be non - inferior in patients with an icd implanted without a df test , with a difference in fse of 3.0% in favour of the no df test [ confidence interval ( ci ) 3.0 to 9.0% , pnon - inferiority < 0.001 for the pre - defined non - inferiority margin of 10% ) . a total of 112 procedure - related serious adverse events occurred within 30 days in 94 patients ( 17.6% ) tested compared with 89 events in 74 patients ( 13.9% ) not tested ( p = 0.095).conclusiondefibrillation efficacy during follow - up is not inferior in patients with a 40 j icd implanted without df testing . defibrillation testing during first time icd implantation should no longer be recommended for routine left - sided icd implantation .

bladder cancer is the seventh most common malignancy in the world in men and the third most common in poland . in women , this cancer occurs less frequently ( 17 in the world and 15 in poland ) , but in contrast to men , an increasing trend of morbidity can be observed [ 15 ] . in poland in 2010 , 6296 incidents of bladder cancer were noticed ( 4919 men , 1377 women ) . mortality of this cancer is high , reaching 50.2% for men and 46.5% for women . approximately 1520% of bladder cancers infiltrate the bladder muscle layer ; in such cases , the treatment of choice is radical cystectomy , which connects with the necessity of urine diversion after bladder removal . in 2010 , 1260 patients required urinary diversion . in europe , each year 140 000 new cases of bladder cancer are noticed , which gives 25 000 patients for urinary diversion . urinary diversion can be divided into incontinent and continent ( formed orthotopically or non orthotopically ) . incontinent ileocutaneostomy is most commonly used among surgeons and this type of urinary diversion has the greatest chances to be constructed using tissue engineering methods . the use of an ileal segment , beside advantages such as maintaining ureter continuity together with urine outflow using autologous material with well developed blood supply , has number of disadvantages especially in the long term follow up [ 1012 ] . this method is also associated with the necessity of additional surgical procedure conduction on the ileum . the use of tissue engineering techniques gives opportunities to construct artificial conduits de novo in the laboratory without affecting the ileum . the first attempt at incontinent urinary diversion was carried out in 1851 in london by john simon , who conducted two ureterosigmoidal fistulas using a specially designed silver catheter . ernst kster performed the first time radical cystetctomy in a patient suffering from localized urinary bladder cancer . robert coffey developed a new method of ureter implantation into the bowel wall which was used in clinical practice by charles mayo in 1912 ( coffey mayo technique ) . this method used a bowel submucosal tunnel which protects against urine outflow ( reflux ) . with some modifications , frank hinman and henry m. weyrauch analyzed 740 urinary diversion procedures performed using 60 different methods . the most effective incontinent urinary diversion method with the highest survival rate in these years was cuteneous ureterostomy . the first attempt at a unilateral cuteneous ureterostomy was performed in 1889 by jean f. a. le dentu . in 1892 , ludwik rydygier performed the first bilateral cuteneous ureterostomy . in 1905 , rowsing modified rydygier 's technique by constructing a nipple after ureter exteriorization , over which a sliver urine reservoir was placed . other incontinent urinary diversion techniques included ureter anastomosis with the urethral groove ( eduard sonnenburg 1881 ) or ureter implantation into the vagina ( karl pawlick 1888 ) . the use of an ileal segment as a conduit for urine diversion was performed for the first time in 1911 by zaayer on two patients . the first patient died because of cancer and the second because of peritonitis . in 1950 , eugene m. bricker presented 307 incontinent urinary diversion procedures using an ileal segment reaching 12.4% mortality ; only 3.4% directly involved urinary diversion . this method was considered a gold standard for 35 years and with some modification is used even now . over the past 65 years the first attempt at artificial urinary conduit construction using tissue engineering methods was performed by drewa in 2007 . in this experiment , small intestine submucosa ( sis ) , acellular or seeded , with 3t3 fibroblast cell line was used on rat models . the experiment was performed on 6 animals ; patent conduits were observed in three rats at the end of follow up . no differences in cell layer regeneration were observed in seeded and unseeded groups . additionally , acellular matrices induced less severe inflammatory responses . five years later , two other groups conducted urinary conduit construction on a porcine model . animals were divided into 2 groups : acellular matrices ( n = 4 ) and matrices seeded with urothelial cells ( n = 6 ) . patent urostomy was obtained in 5 animals , with no differences between seeded and unseeded matrices . they used polyglicolic acid coated with poly(lactide co glicolide ) scaffold ( pga / plga ) unseeded or seeded with smooth muscle cells derived from different origin ( bladder , adipose tissue , blood ) . in contrast to previously presented experiments , in this study , the bladder was removed and both ureters were transplanted to the conduit . obtained results showed that the use of smooth muscle cells from different origins led to regeneration of a neo organ resembling native bladder tissue composed of urothelium and smooth muscle layers . the use of acellular scaffold resulted in fibrous connective tissue development with a small number of smooth muscle cells . acellular matrices were used in 6 animals and scaffolds seeded with urothelial cells were used in 24 rabbits . in this study , the bladder was also removed and both ureters were transplanted to the conduit . in the group where matrices were seeded , up , conduit lumen was covered with multilayer urothelium and no severe complications were observed . in the unseeded group , 4 animals died one month before the end of follow up and the two remaining animals had fistulas and lack of urothelium regeneration . scaffold seeded with urothelial cells was protected against scar and kidney stone formation , atresia and hydronephrosis . in our study , we compared two acellular matrices form different origins : autologous naturally derived acellular aortic arch and synthetic policaprolactone ( plcl ) produced using the electrospining method . the experiment was conducted on 12 wistar rats divided into 2 equal groups , six rats for each tested scaffold . obtained results indicated that acellular aortic arch is an unsuitable scaffold for urinary conduit construction in a rat model . in all animals with acellular aortic arch conduit , in which plcl scaffold was used in 3 cases , constructed conduits were patent at the end of follow up ( 4 weeks ) , but only in one case intense urine flow , without the presence of pus in the urinary tract , was observed . autologous acellular aortic arch , despite easy accessibility and proper extracellular matrix composition , is unsuitable for urinary conduit construction because of its small diameter and too elastic structure , which leads to atresion about one week after the surgical procedure . plcl is more rigid and its diameter can be regulated during the electrospining method , which is why better results were obtained using this scaffold . unsatisfactory results with plcl were probably caused by open urinary tract during follow up , as use of urostomy bags on rat models is impossible . such complications were not observed when a ureter segment was regenerated using this same scaffolds . use of plcl resulted in ureter segment reconstruction in 4 cases ( n = 6 ) , which was confirmed by urography . in that group , all currently performed attempts of experimental artificial urinary conduit construction using tissue engineering techniques are presented in figure 1 . different approaches currently used in experimental urinary conduit construction using tissue engineering techniques . a experiment carried out only on one ureter ; b two ureters were anastomosed with artificial conduit ; urinary bladder was removed [ 21 , 22 , 23 ] . plcl caprolactone ) ; pga / plga polyglicolic acid coated with poly ( lactide co glicolide ) scaffold ; sis small intestine submucosa ; bam bladder acellular matrix . in literature there is still a small number of works describing the use of tissue engineering for urinary conduit construction . in the paper by sloff et al . , all experiments describing the use of tissue engineering in urinary diversion construction conducted so far were evaluated . they analyzed 8 works , of which 5 were related to urinary conduit and 3 to neo bladder construction . this shows how little is known about ureter segment regeneration including artificial urinary conduit construction , which continues to be an unresolved problem . synthetic polymers have promising properties because these materials can be produced de novo and different shapes , porosity and degradation time can be obtained . the best solution for a patient would be the use of an acellular scaffold without the necessity of cell seeding . such an approach will eliminate invasive collection of tissue for cell isolation and long in vitro culture ( about 2 weeks ) . construction of an artificial urinary conduit without cell seeding makes it the ideal off the shelf product which could be purchased from the pharmacy directly before the surgical procedure . scaffold which protects seeded cells from the toxic influence of urine is an important issue . in an in vitro culture study , urine acted as a cytotoxic agent against urothelial cells and bone marrow mesenchymal stem cells [ 26 , 27 ] . urothelial cells build the inner layer of the ureter , protecting against urine components reabsorption . urothelium can self regenerate on a scaffold surface after transplantation by migration from surrounding tissue [ 2830 ] . epithelial regeneration should be potentially easier in the case of ureter segment regeneration in which urothelial cells can migrate from two edges , compared to urinary conduit . data available in literature gives contrary results about the use of urothelial cells in tissue engineering applications . some studies showed no differences in seeded and unseeded group ; on the other hand , other researches obtained positive results only when scaffold was seeded with urothelial cells [ 22 , 23 ] . they concluded that after 4 week follow up , the epithelial layer regenerated only on 0.5 cm scaffold , which is not a clinically important segment . on longer scaffolds , regeneration was observed only at the anastomotic edges with dense fibrosis throughout the grafts . in previous experiments , only one scaffold type was analyzed . in our work , we compared two scaffold types : natural and synthetic . obtained results showed better regeneration of urothelium on synthetic biodegradable polymer compared to natural derived acellular blood vessel matrix . it should be noted that material for artificial urinary conduit construction should be produced from components that will protect from fibrosis and calcification of urine substrates on the scaffold surface . the crucial point is regeneration of the smooth muscle layer and restoration of peristaltic waves on the reconstructed segment . the urinary conduit must be constructed from rigid material in order to protect from scaffold occlusion at the site of anastomosis with skin . such properties can result in peristaltic wave arrest at the site of ureter anastomosis with scaffold , which can lead to urine reflux and development of hydronephrosis . quick regeneration of smooth muscle layer should prevent the development of the side effects mentioned above . despite the fact that acellular scaffold might be the best solution , some papers indicate that scaffold pre seeded with cells ( autologous form bladder biopsy or mesenchymal stem cells from different origin ) showed better smooth muscle layer regeneration compared to unseeded controls . the best type of cells seems to be mesenchymal stem cells from fat tissue or bone marrow and from promising sources like amniotic fluid or hair follicles [ 13 , 33 ] . use of differentiated autologous cells from bladder biopsy is limited due to the risk of cancer development in the case of bladder cancer patients , who are the main candidates for urinary conduit construction . the number of cells seeded on scaffold is also very important and the conception seems to be simple : the more cells on cm of scaffold , the better the results that can be achieved . the achievement of such large cell numbers is challenging issue because the average bladder cancer patient is 65 years old and mesenchymal stem cells proliferation capacity decreases with the age of the patient and with increasing passage numbers [ 36 , 37 ] . on the other hand , some authors suggested that stem cell proliferation capacity is not dependent on the donors age , which increase the chances of tissue engineering therapy use in artificial urinary conduit construction . despite that , such a large number of cells necessary for regeneration is hard to obtain and very costly because of the price of culture media containing appropriate growth factors . that is why an efficient cell culture method has to be developed to provide success of this procedure . for many years , atypical smooth muscle cells ( asmcs ) localized in proximal regions of the renal pelvis were considered a peacemaker of peristaltic waves responsible for urine passage into the bladder . recent studies indicated that interstitial cells of cajal like cells ( icc lc ) expressing the c kit gene , which are sparsely distributed within the lamina propria and muscle layer of upper urinary tract , play an important role in promoting pyeloureteric peristalsis . contraction waves generated in the renal pelvis are probably propagated , coordinated and modulated in the upper urinary tract by icc lc [ 41 , 42 ] . iccs , thanks to their automatism , are able to replace discontinued atypical smcs impulses and maintain peristaltis of lower ureter parts [ 40 , 43 ] . in vitro isolation and culture of icc lc from the urinary tract has not yet been established , but taking into consideration previous works and our experience on urinary conduit construction , addition of these cells or coculture of them with smooth muscle cells can potentially accelerate the restoration of peristaltic waves on the reconstructed segment , which could prevent development of hydronephrosis . the first attempt at incontinent urinary diversion was carried out in 1851 in london by john simon , who conducted two ureterosigmoidal fistulas using a specially designed silver catheter . the patient died one year after the procedure due to peritonitis . in 1891 , ernst kster performed the first time radical cystetctomy in a patient suffering from localized urinary bladder cancer . robert coffey developed a new method of ureter implantation into the bowel wall which was used in clinical practice by charles mayo in 1912 ( coffey mayo technique ) . this method used a bowel submucosal tunnel which protects against urine outflow ( reflux ) . with some modifications , frank hinman and henry m. weyrauch analyzed 740 urinary diversion procedures performed using 60 different methods . the most effective incontinent urinary diversion method with the highest survival rate in these years was cuteneous ureterostomy . the first attempt at a unilateral cuteneous ureterostomy was performed in 1889 by jean f. a. le dentu . in 1892 , ludwik rydygier performed the first bilateral cuteneous ureterostomy . in 1905 , rowsing modified rydygier 's technique by constructing a nipple after ureter exteriorization , over which a sliver urine reservoir was placed . other incontinent urinary diversion techniques included ureter anastomosis with the urethral groove ( eduard sonnenburg 1881 ) or ureter implantation into the vagina ( karl pawlick 1888 ) . the use of an ileal segment as a conduit for urine diversion was performed for the first time in 1911 by zaayer on two patients . the first patient died because of cancer and the second because of peritonitis . in 1950 , eugene m. bricker presented 307 incontinent urinary diversion procedures using an ileal segment reaching 12.4% mortality ; only 3.4% directly involved urinary diversion . this method was considered a gold standard for 35 years and with some modification is used even now . over the past 65 years , a more effective method of incontinent urinary diversion was not developed . the first attempt at artificial urinary conduit construction using tissue engineering methods was performed by drewa in 2007 . in this experiment , small intestine submucosa ( sis ) , acellular or seeded , with 3t3 fibroblast cell line was used on rat models . the experiment was performed on 6 animals ; patent conduits were observed in three rats at the end of follow up . five years later , two other groups conducted urinary conduit construction on a porcine model . animals were divided into 2 groups : acellular matrices ( n = 4 ) and matrices seeded with urothelial cells ( n = 6 ) . patent urostomy was obtained in 5 animals , with no differences between seeded and unseeded matrices . they used polyglicolic acid coated with poly(lactide co glicolide ) scaffold ( pga / plga ) unseeded or seeded with smooth muscle cells derived from different origin ( bladder , adipose tissue , blood ) . in contrast to previously presented experiments , in this study , the bladder was removed and both ureters were transplanted to the conduit . obtained results showed that the use of smooth muscle cells from different origins led to regeneration of a neo organ resembling native bladder tissue composed of urothelium and smooth muscle layers . the use of acellular scaffold resulted in fibrous connective tissue development with a small number of smooth muscle cells . acellular matrices were used in 6 animals and scaffolds seeded with urothelial cells were used in 24 rabbits . in this study , the bladder was also removed and both ureters were transplanted to the conduit . in the group where matrices were seeded , up , conduit lumen was covered with multilayer urothelium and no severe complications were observed . in the unseeded group , 4 animals died one month before the end of follow up and the two remaining animals had fistulas and lack of urothelium regeneration . scaffold seeded with urothelial cells was protected against scar and kidney stone formation , atresia and hydronephrosis . in our study , we compared two acellular matrices form different origins : autologous naturally derived acellular aortic arch and synthetic policaprolactone ( plcl ) produced using the electrospining method . the experiment was conducted on 12 wistar rats divided into 2 equal groups , six rats for each tested scaffold . obtained results indicated that acellular aortic arch is an unsuitable scaffold for urinary conduit construction in a rat model . in all animals with acellular aortic arch conduit , atresion was observed . in a second group , in which plcl scaffold was used in 3 cases , constructed conduits were patent at the end of follow up ( 4 weeks ) , but only in one case intense urine flow , without the presence of pus in the urinary tract , was observed . autologous acellular aortic arch , despite easy accessibility and proper extracellular matrix composition , is unsuitable for urinary conduit construction because of its small diameter and too elastic structure , which leads to atresion about one week after the surgical procedure . plcl is more rigid and its diameter can be regulated during the electrospining method , which is why better results were obtained using this scaffold . unsatisfactory results with plcl were probably caused by open urinary tract during follow up , as use of urostomy bags on rat models is impossible . such complications were not observed when a ureter segment was regenerated using this same scaffolds . use of plcl resulted in ureter segment reconstruction in 4 cases ( n = 6 ) , which was confirmed by urography . in that group , all currently performed attempts of experimental artificial urinary conduit construction using tissue engineering techniques are presented in figure 1 . different approaches currently used in experimental urinary conduit construction using tissue engineering techniques . a experiment carried out only on one ureter b two ureters were anastomosed with artificial conduit ; urinary bladder was removed [ 21 , 22 , 23 ] . poly ( l lactide co caprolactone ) ; pga / plga polyglicolic acid coated with poly ( lactide co glicolide ) scaffold ; sis small intestine submucosa ; bam bladder acellular matrix . in literature there is still a small number of works describing the use of tissue engineering for urinary conduit construction . in the paper by sloff et al . , all experiments describing the use of tissue engineering in urinary diversion construction conducted so far were evaluated . they analyzed 8 works , of which 5 were related to urinary conduit and 3 to neo bladder construction . this shows how little is known about ureter segment regeneration including artificial urinary conduit construction , which continues to be an unresolved problem . synthetic polymers have promising properties because these materials can be produced de novo and different shapes , porosity and degradation time can be obtained . the best solution for a patient would be the use of an acellular scaffold without the necessity of cell seeding . such an approach will eliminate invasive collection of tissue for cell isolation and long in vitro culture ( about 2 weeks ) . construction of an artificial urinary conduit without cell seeding makes it the ideal off the shelf product which could be purchased from the pharmacy directly before the surgical procedure . scaffold which protects seeded cells from the toxic influence of urine is an important issue . in an in vitro culture study , urine acted as a cytotoxic agent against urothelial cells and bone marrow mesenchymal stem cells [ 26 , 27 ] . urothelial cells build the inner layer of the ureter , protecting against urine components reabsorption . urothelium can self regenerate on a scaffold surface after transplantation by migration from surrounding tissue [ 2830 ] . epithelial regeneration should be potentially easier in the case of ureter segment regeneration in which urothelial cells can migrate from two edges , compared to urinary conduit . data available in literature gives contrary results about the use of urothelial cells in tissue engineering applications . some studies showed no differences in seeded and unseeded group ; on the other hand , other researches obtained positive results only when scaffold was seeded with urothelial cells [ 22 , 23 ] . they concluded that after 4 week follow up , the epithelial layer regenerated only on 0.5 cm scaffold , which is not a clinically important segment . on longer scaffolds , regeneration was observed only at the anastomotic edges with dense fibrosis throughout the grafts . in previous experiments , only one scaffold type was analyzed . in our work , we compared two scaffold types : natural and synthetic . obtained results showed better regeneration of urothelium on synthetic biodegradable polymer compared to natural derived acellular blood vessel matrix . it should be noted that material for artificial urinary conduit construction should be produced from components that will protect from fibrosis and calcification of urine substrates on the scaffold surface . the crucial point is regeneration of the smooth muscle layer and restoration of peristaltic waves on the reconstructed segment . the urinary conduit must be constructed from rigid material in order to protect from scaffold occlusion at the site of anastomosis with skin . such properties can result in peristaltic wave arrest at the site of ureter anastomosis with scaffold , which can lead to urine reflux and development of hydronephrosis . quick regeneration of smooth muscle layer should prevent the development of the side effects mentioned above . despite the fact that acellular scaffold might be the best solution , some papers indicate that scaffold pre seeded with cells ( autologous form bladder biopsy or mesenchymal stem cells from different origin ) showed better smooth muscle layer regeneration compared to unseeded controls . the best type of cells seems to be mesenchymal stem cells from fat tissue or bone marrow and from promising sources like amniotic fluid or hair follicles [ 13 , 33 ] . use of differentiated autologous cells from bladder biopsy is limited due to the risk of cancer development in the case of bladder cancer patients , who are the main candidates for urinary conduit construction . the number of cells seeded on scaffold is also very important and the conception seems to be simple : the more cells on cm of scaffold , the better the results that can be achieved . the achievement of such large cell numbers is challenging issue because the average bladder cancer patient is 65 years old and mesenchymal stem cells proliferation capacity decreases with the age of the patient and with increasing passage numbers [ 36 , 37 ] . on the other hand , some authors suggested that stem cell proliferation capacity is not dependent on the donors age , which increase the chances of tissue engineering therapy use in artificial urinary conduit construction . despite that , such a large number of cells necessary for regeneration is hard to obtain and very costly because of the price of culture media containing appropriate growth factors . that is why an efficient cell culture method has to be developed to provide success of this procedure . for many years , atypical smooth muscle cells ( asmcs ) localized in proximal regions of the renal pelvis were considered a peacemaker of peristaltic waves responsible for urine passage into the bladder . recent studies indicated that interstitial cells of cajal like cells ( icc lc ) expressing the c kit gene , which are sparsely distributed within the lamina propria and muscle layer of upper urinary tract , play an important role in promoting pyeloureteric peristalsis . contraction waves generated in the renal pelvis are probably propagated , coordinated and modulated in the upper urinary tract by icc lc [ 41 , 42 ] . iccs , thanks to their automatism , are able to replace discontinued atypical smcs impulses and maintain peristaltis of lower ureter parts [ 40 , 43 ] . in vitro isolation and culture of icc lc from the urinary tract has not yet been established , but taking into consideration previous works and our experience on urinary conduit construction , addition of these cells or coculture of them with smooth muscle cells can potentially accelerate the restoration of peristaltic waves on the reconstructed segment , which could prevent development of hydronephrosis . in conclusion , success in ureter conduit construction using tissue engineering techniques depends on finding the proper scaffold . we believe that implementation of this procedure is possible and urinary conduit should be the first commercially available product in clinical practice constructed using regenerative medicine . confirmation of this opinion is registered in a clinicaltrial.gov study about incontinent urinary conduit construction using pga / plga scaffold seeded with autologous smooth muscle cells derived from adipose tissue biopsy . renal replacement treatment , in the form of kidney transplantation or dialysis , is a life - supporting therapy for patients with end - stage renal disease . at the end of 2010 , ~1.81 million chronic kidney disease patients globally were undergoing hemodialysis ( hd ) treatment , with an estimated 90,000 ( 5% ) thereof being treated with hemodiafiltration ( hdf).1 online hdf has been suggested as a possible cost - effective alternative compared to standard hd , mainly due to its superior middle molecule clearance resulting from the combination of diffusion and convection solute removal mechanisms . several studies suggest that hdf may lead to better clinical results than high - flux hd ( hf - hd).2,3 according to a recent survey , hdf was started to prevent dialysis complications , increase the efficiency of dialysis for uremic accumulates , treat dialysis - related hypotension and dialysis - related amyloidosis , and for intractable pruritus , restless leg syndrome and arthralgia not related to dialysis - related amyloidosis.4 however , to date , randomized studies have not conclusively supported the superiority of hdf regarding clinical outcomes.57 in addition , concerns have been raised about its cost - utility,8 leading investigators to conduct cost analyses to assess the differential expenditures between the two therapies.9 in a recent editorial , vanholder et al concluded that more expensive therapies should be reimbursed only when evidence of their cost - utility , defined as the ratio of treatment cost to treatment outcome , taking into consideration also the quality of life ( qol ) , is sufficiently robust.10 the cost of hdf per se has decreased in recent years due to 1 ) increased market penetration and the subsequent economy of scale in the production of equipment and disposables , 2 ) the pressure on health care costs due to the economic crises prevailing since 2008 , and 3 ) the safety and reliability of online method that have reduced demand for microbiological testing and monitoring.11,12 furthermore , after the publication of the study of canaud et al,2 hdf treatment effectiveness has increasingly been understood to be positively associated with the magnitude of the convection volume used , resulting in trends toward the application of higher hdf convection volumes than were applied in earlier cost analyses . accordingly , there is a need for a new cost - effectiveness evaluation . the aim of this study is to assess the cost - effectiveness of hdf compared to hd by a simulation using existing study data . these data facilitate an understanding of whether longer survival and better health - related quality of life ( hrqol ) are able to offset the possibly higher costs of hdf , and thus whether it is worth investing in this more innovative therapy . the analysis comprised a simulation1315 based on results from combined previous observational studies , randomized clinical trials , and a meta - analysis with the following steps : 1 ) estimation of a ) the survival function of hf - hd patients from the membrane permeability outcome study16 dataset and b ) estimation of the survival function of hdf patients using the risk reduction estimates due to the treatment effect from the meta - analysis of mostovaya et al17 ( even though it includes also studies comparing hdf to low - flux hd , it was considered the best proxy with respect to other alternative meta - analyses available in the literature ) ; 2 ) simulation of the survival of the same sample of patients as if allocated to hf - hd or hdf using three - state markov models ; 3 ) application of state - specific hrqol coefficients and differential costs ( materials , testing , and consumption of water ) derived from the literature . in addition , in a secondary subgroup analysis we considered the eshol5 estimations related to diabetics and nondiabetics . this was done because among all the studies considered the eshol study provides results related to these particular subgroups of patients . two mirror - image markov models ( figure 1 ) were developed with the aim to generate a simulation comparing the survival of the same sample of patients treated with hf - hd and hdf . three possible states were considered : alive and under therapy , dead due to the disease under therapy , and dead for other cause ( ie , considering the overall mortality , based on age- and sex - adjusted life tables ) . the probabilities defining the transition of patients between each of these states were calculated from the previously estimated hazard functions and were specified over a defined time frame ( cycle ) . the model was run for a specified number of cycles to see how the hypothetical cohort of patients moved between states . the length of the cycle was fixed to 1 year and the number of cycles to ten , for a total of 10 years . accordingly , total dialysis costs can be divided into : 1 ) direct health care costs , such as staff , material , vascular access , routine diagnostics , hospitalization , drugs , and medications ; 2 ) direct nonhealth care costs , such as cost of transport and cost of informal care ; and 3 ) indirect nonhealth care costs , productivity losses due to disability or premature death . it is reasonable to assume that direct nonhealth care costs and indirect nonhealth care costs are the same for the two therapies , and also that most of the direct health care costs do not vary by treatment . as a consequence , the comparison on the incremental costs of hdf with respect to hf - hd focused only on the costs of equipment , disposables , ultrapure water testing , and water consumption.18 in order to obtain more generalizable results , we analyzed two alternative cost settings , involving or not involving the use of ultrapure water . the first was based on the assumption that , since dialyzers used to perform both hdf and hf - hd are designed to have better solute clearances on a wider range of uremic toxins and higher hydraulic permeability , it is reasonable to argue that the same water and dialysis fluid microbiological purity were necessary for both therapies due to backfiltration phenomena , leading to similar costs of water testing . as a consequence , and because differences in total water consumption are negligible , it was possible to limit the analysis to the costs of disposables ( blood lines , dialyzers ) and equipment only . in the prospective observational study by oates et al,9 some patients on hf - hd were switched to hdf and others remained on hf - hd . the additional cost of disposables related to hdf was of 1.32 per session ( 207 per annum [ p.a . ] ) in the standard case , that is , with the use of a cuvette for relative blood volume monitoring . the alternative cost setting was based on the analysis by lebourg et al12 where , in addition to a cost variability due to disposables and monitors , the difference in cost between the two different therapies caused by water analysis and water consumption was considered . the additional hdf cost per session ranged from a minimum value of 1.29 ( 202 p.a . ) to a maximum value of 4.86 ( 730 p.a . ) . once the relevant costs were determined , the corresponding estimated and discounted costs ( or a differential cost ) taken from the literature were allocated to each state of the model . contradicting results have been reported on the difference in qol related to hf - hd and hdf . few studies addressed the qol evaluation , and none provided hrqol coefficients for the specific comparison of hf - hd and hdf , fundamental for the purpose of this analysis . mazairac et al8 estimated higher scores with euroqol 5d for patients on hdf compared with hd . this preference - based measure yields a set of weights on which quality - adjusted life years ( qalys ) calculations can be based . in this study as there are no other data about qol differences between hdf and hf - hd in the literature , these coefficients have been used in our simulation . in particular , the hrqol coefficients linked to age were selected for the model because otherwise a fundamental component of the benefit measurement would have been neglected . as recommended by the economic analysis guidelines,19 an annual discount rate of 3.5% for both costs and benefits was selected , and changes in results as a function of different values of these parameters were considered . parameter uncertainty was included in the model through a probabilistic sensitivity analysis , taking the intrinsic imprecision of the estimate of the parameters of the survival model into account . consequently , the quality and quantity of information available can be reflected in the probability distributions assigned to each input parameter in the model.20 several monte carlo simulations were performed , also for patients with different risk profiles by age ( patients aged 40 , 50 , and 60 years ) , sex , and diabetic status.21,22 for the sake of simplicity , the cohort of simulated 50-year - old male patients were selected as the reference population for the analysis . as discussed in the next section , we found similar results in all age subgroups , with identical conclusions in terms of cost - effectiveness ; therefore , we used this cohort to briefly recap the main results of our analysis . the characteristics of this cohort are coherent with the results from the edta registry reported by van de luijtgaarden et al:23 in the last 20 years indeed the mean age of patients who started with hd treatment was 5560 years , and on average > 60% of patients were male . output analyses included scatter plots of simulations in the cost - effectiveness plane , the estimation of incremental cost - effectiveness ratios ( icers ) and the computation of cost - effectiveness acceptability curves ( ceacs ) . the results of the 1,000 monte carlo simulations for the cohort of 50-year - old male patients are shown in the cost - effectiveness scatter plot in figure 2 . from the mean values of all the simulations in this specific cohort of patients , the icer was 6,982/qaly . on the basis of these simulations it was possible to derive the probability of cost - effectiveness given different thresholds , as shown in figure 3 . the ceac corresponding to hdf starts from 0 , meaning that there is no possibility that this alternative therapy is cost - saving . the threshold must be at least 2,000 per qaly to have a probability of cost - effectiveness > 0 . the probability of cost - effectiveness increased to 50% with a threshold of 7,000/qaly , to 70% with a threshold of 15,000/qaly and to 81% with the commonly accepted threshold of 40,000/qaly . the probability of cost - effectiveness always stayed below 84% for a threshold of over 40,000/qaly . this asymptotic value results from the fact that a fraction of the simulations represent cases where the alternative therapy ( hdf ) causes higher costs and provided fewer benefits . this means that even with any budget constraint , there is some probability that the alternative therapy is not cost - effective and the traditional one ( hf - hd ) is preferred . the same analyses were carried out for the subgroups of 40- , 50- , and 60-year - old male and female patients ( table 1 ) : hdf appears to be more cost - effective for younger patients . this message is reinforced in figure 4 , which shows that a given threshold value is associated to a higher probability of hdf being cost - effective for the 40- and 50-year - old patients than for the 60-year - old ones . moreover , for the 60-year - old groups there is a probability of around 28% for both females and males that hdf is not cost - effective even at extreme values of the threshold . regarding the discount rates for costs and benefits , results were robust even after considering alternative scenarios with lower or higher discount rates . by applying a 0% or a 5% discount rate for both costs and effects , 50-year - old male patient icer decreased to 6,676/qaly and increased to 6,960/qaly , respectively , confirming the robustness of the estimations . a further analysis was performed to investigate the importance of the qualitative component of the qalys ( qol ) compared to the quantitative one ( life years ) . the effect of using overall hrqol coefficients ( rather than the hrqol coefficients linked to patient age ) on the cohort of 50-year - old male patients is shown in figure 5 . the mean icer increased to 19,423/qaly but , more importantly , almost 37% of the simulations were on the left of the qaly zero value ( less effectiveness , more costs ) and , accordingly , the uncertainty around the decision whether to invest in the new therapy was much higher . even an infinite value of the threshold would not lead to a probability of cost - effectiveness > 65% ( data not shown ) . this is caused by the uncertain values of the overall hrqol coefficients , leading to some cases where one period spent under hf - hd is worth even more than the same period under hdf . this shows that it is crucial to consider the role of qol is in such evaluations . previously , cost inputs were point estimates , whereas now costs vary over the range assessed by lebourg et al.12 the ceac for the subgroup of 50-year - old male patients shows that 21.5% of the simulations are cost - saving ( figure 6 ) . the probability of being cost - effective reaches 80% at the commonly accepted threshold of 40,000/qaly , and it increases only by 2.4 percentage points even with a threshold of 70,000/qaly . , hdf seems a little more expensive : for instance , the icers of the younger patients rise from 5,878/qaly and 5,732/qaly to 7,748/qaly and 7,724/qaly for female and male patients , respectively ( table 2 ) . this subgroup analysis confirmed the previous results : the icer increases with the age of the cohort . diabetic patients follow a different survival function and are subject to a different treatment effects of the alternative therapy compared to nondiabetic patients . nevertheless , the comparison between a cohort of 50-year - old diabetic male patients and a cohort of 50-year - old nondiabetic male patients did not reveal significant differences ( figure 7 ) . given the commonly accepted threshold of 40,000/qaly , this study demonstrated that , compared to hf - hd , hdf is a cost - effective treatment for patients on dialysis . this is particularly true for patients < 60 years , irrespective of sex or diabetic status . either way , the variability in the results due to different characteristics of the patients does not affect the conclusions about the cost - effectiveness of hdf . these results are quite different from those reported by mazairac et al8 in their cost - utility analysis of hdf versus conventional low - flux hd based on the unadjusted results for convective volume of the convective transport study ( contrast).7 their estimated incremental cost per quality - adjusted life year of hdf compared to low - flux hd was 287,679 and certainly not below 140,000 under the most favorable assumptions ( eg , applying a convection volume > 20.3 l ) . they argued that the hdf is characterized by higher cost and better health and was not cost - effective given the currently accepted cost - utility thresholds . however , it has to be mentioned that the same analysis performed on patients treated in montreal ( canada ) according to the same protocol yielded different , positive , results.24 our analysis , on the other hand , reports that hdf is cost - effective with a probability of ~81% . this conclusion is based on what is considered the commonly accepted threshold for health policy decisions,25 which is ~40,000 ( or 30,000 gbp , or 50,000 usd ) per qaly . even if this threshold is difficult to quantify and is often based on a rule of thumb , it is commonly applied as an estimate of the forgone health resulting from services displaced to accommodate the additional costs of the new technology . of note , the magnitude of the difference between the two studies may be related to the different comparison groups , which is low - flux hd in the contrast study and hf - hd in our model . the icer estimates show a good value for money , but some doubts about the cost - effectiveness of hdf over hf - hd remain due to the residual ~20% probability of costs being higher and/or benefits being lower with hdf . this line of reasoning is also shared by mcbrien and manns26 in their commentary of the mazairac study , where they argued that hdf is characterized by higher procedural costs and has no proven health benefit . our study specifies that the survival difference is not the only important factor ( especially considering the lack of agreement on this aspect ) , but that differences in terms of qol are of fundamental importance and must be measured . this was the rationale for conducting a value - based study incorporating hrqol as a main outcome of hdf versus hf - hd . convective treatments as hdf indeed reduce intradialytic hypotension ( which affects qol ) , and possibly limit the poor appetite and nutritional state in patients with chronic kidney disease . however , we believe that more research is warranted to assess the impact of hdf specifically on the nutritional state of end - stage renal disease patients . therefore , this study recommends prioritizing the research which evaluates the qol related to hdf . it must be stressed that the purpose itself of an economic decision model is to synthesize all the relevant evidence in order to make a sense of it and inform the decision about the adoption of a new technology in an uncertain context.15 we have indeed identified the relevant studies involving the therapies under assessment , and we have included all the uncertainty in our parameter estimates , without neglecting any relevant information in the literature . our result is therefore based on published data , but its accuracy is sensitive to some decisions that we were forced to take in the model construction phase . the first decision was to use survival estimates from the hf - hd arm of the membrane permeability outcome16 randomized clinical trial . this trial was based on incident patients , whereas other studies on hdf were mainly performed on prevalent european dialysis patients . additionally , the low crude mortality rate observed in the membrane permeability outcome study may produce results not completely transferrable to ordinary ( nontrial participating ) patients . indeed , as stressed by palmer et al,20 relative risk reductions applied to low baseline risks produce low absolute reduction in event rates and low gains in health . it is possible that this low overall mortality rate affected the lack of difference between diabetic and nondiabetic patients . as previously stressed , one of the main requirements for an economic evaluation is to include all the relevant evidence . with regard to the effectiveness data , this concept does not change , and meta - analysis is the usual technique employed to synthesize all the available evidence . to date five meta - analyses aggregating the results of the main convective dialysis therapy studies17,2730 have been published . however , a huge heterogeneity of interventions was included in these meta - analyses , and the meta - analysis by mostovaya et al17 was the only one which focused on trials on hdf as convective therapy with appropriate definition of convective volume . nevertheless , there is still a great variety of patients and treatment modalities characteristics among the studies . indeed , for instance , both high- and low - flux modalities were included in the comparison . as a consequence , a relative risk based on a random effects model has been used , which makes the estimate of the pooled effect more conservative , and takes into account the clinical heterogeneity among the single studies . of note , this meta - analysis includes also the contrast study , and our cost - effectiveness analysis does include all the relevant randomized clinical trials . on the contrary , for instance , the economic evaluation by mazairac is based only on the contrast study ; this is a subjective decision , while a complete cost - effectiveness analysis should instead take into consideration all the relevant available evidence , as previously pointed out . with regard to the subanalysis of the diabetic patients , the eshol5 estimates of risk reduction have been used . as the eshol study provided the most positive results in favor of hdf , concerns about the overestimation of the reduction in mortality could be raised . nevertheless , the model did not show any difference between the two groups of patients , and this result highlights again that the impact on mortality is less important than the effect on the qol . the estimations used for this study were mainly derived from a meta - analysis with basically 23 years follow - up time , but a more appropriate time horizon would be the patient s lifetime . this is especially true in the case of treatment of chronic diseases where the initiation of an intervention in middle - aged patients may have cost and effect implications on the rest of their lives.31 an important role of a decision model , therefore , is to bridge the gap between what has been observed in trials and what would be expected to happen over the long - term , forcing one to make assumptions about the long - term effects and consequences of treatment modalities . additionally , the lack of qol coefficients related to hf - hd forced the use of data from a comparison of hdf with low - flux hd.8 uncertainty around results is high , but this is typical in this kind of analyses : neglecting some available evidence only because of its uncertainty would contrast with the comprehensive nature of an economic model . this study contributes also to show the scarcity and heterogeneity of data available in the literature that jeopardize the economic evaluation of hdf when compared to hf - hd . new studies are needed to assess more precisely the differential benefits between the therapies : value - of - information analysis might help to identify more specifically the parameters which would be worth to be further investigated . a more comprehensive evaluation performed in a randomized clinical trial is required in order to include other relevant cost components ( eg , pharmaceuticals , hospitalizations , prevention of long - term dialysis - related complications , beta-2 microglobulin - amyloidosis , and transplantation access ) in the analysis . possible savings resulting from a reduction in the cost of ancillary pharmacological therapy and hospitalization have not been considered in the current analysis .

introductionincontinent urinary diversion using an ileal conduit is the most popular method used by urologists after bladder cystectomy resulting from muscle invasive bladder cancer . the use of gastrointestinal tissue is related to a series of complications with the necessity of surgical procedure extension which increases the time of surgery . regenerative medicine together with tissue engineering techniques gives hope for artificial urinary conduit construction de novo without affecting the ileum.material and methodsin this review we analyzed history of urinary diversion together with current attempts in urinary conduit construction using tissue engineering methods . based on literature and our own experience we presented future perspectives related to the artificial urinary conduit construction.resultsa small number of papers in the field of tissue engineered urinary conduit construction indicates that this topic requires more attention . three main factors can be distinguished to resolve this topic : proper scaffold construction along with proper regeneration of both the urothelium and smooth muscle layers.conclusionsartificial urinary conduit has a great chance to become the first commercially available product in urology constructed by regenerative medicine methods . backgroundclinical studies suggest that hemodiafiltration ( hdf ) may lead to better clinical outcomes than high - flux hemodialysis ( hf - hd ) , but concerns have been raised about the cost - effectiveness of hdf versus hf - hd . aim of this study was to investigate whether clinical benefits , in terms of longer survival and better health - related quality of life , are worth the possibly higher costs of hdf compared to hf-hd.methodsthe analysis comprised a simulation based on the combined results of previous published studies , with the following steps : 1 ) estimation of the survival function of hf - hd patients from a clinical trial and of hdf patients using the risk reduction estimated in a meta - analysis ; 2 ) simulation of the survival of the same sample of patients as if allocated to hf - hd or hdf using three - state markov models ; and 3 ) application of state - specific health - related quality of life coefficients and differential costs derived from the literature . several monte carlo simulations were performed , including simulations for patients with different risk profiles , for example , by age ( patients aged 40 , 50 , and 60 years ) , sex , and diabetic status . scatter plots of simulations in the cost - effectiveness plane were produced , incremental cost - effectiveness ratios were estimated , and cost - effectiveness acceptability curves were computed.resultsan incremental cost - effectiveness ratio of 6,982/quality - adjusted life years ( qaly ) was estimated for the baseline cohort of 50-year - old male patients . given the commonly accepted threshold of 40,000/qaly , hdf is cost - effective . the probabilistic sensitivity analysis showed that hdf is cost - effective with a probability of ~81% at a threshold of 40,000/qaly . it is fundamental to measure the outcome also in terms of quality of life . hdf is more cost - effective for younger patients.conclusionhdf can be considered cost - effective compared to hf - hd .

alzheimer s disease ( ad ) is the most frequent neurodegenerative disease ( ferri et al . , 2005 ) and is characterized by a progressive dementia that occurs in middle or late life . the neuropathological hallmark of ad is the presence of cortical intracellular neurofibrillary tangles ( nft ) and extracellular -amyloid ( a ) plaques ( braak and braak , 1991 ) , which leads to synapse dysfunction , neuronal cell loss and subsequent brain atrophy ( ballard et al . , 2011 ) . in the past few decades the knowledge of the key pathogenic mechanisms of the disease has improved , but they are still not completely understood ( querfurth and laferla , 2010 ) . the natural history of ad could be divided in three different phases : the pre - clinical phase , where the pathogenic mechanisms of the disease have started but no symptoms can be identified ; the prodromal phase , where mild cognitive symptoms appear , but there are not severe enough to meet dementia criteria ; and the dementia phase ( dubois et al . , 2007 ) . on the other hand , mild cognitive impairment ( mci ) is a clinical construct created to capture patients with subtle cognitive symptoms at risk for ad ( petersen et al . , 2009 ) . however , some subjects develop other types of dementias , do not progress , or even revert to normal cognition ( ganguli et al . , 2004 ; brooks and loewenstein , 2010 ) . therefore , it is difficult to label mci patients as having prodromal ad . currently , the most frequently used clinical diagnostic criteria for ad are the diagnostic and statistical manual of mental disorders ( dsm - iv - tr ; apa , 2000 ) and the national institute of neurological disorders and stroke - alzheimer disease and related disorders ( nincds adrda ; mckhann et al . , 1984 ) working group . first , the dementia syndrome is established and then , criteria based on the clinical features of the ad type dementia are applied . the dsm - iv - tr criteria require the presence of impairment in multiple cognitive domains of sufficient severity to interfere with the individuals activities of daily living to diagnose dementia . hence , the clinical diagnosis of ad is not possible until a patient reach the dementia phase of the disease . on the other hand , these criteria have shown to have good sensitivity and specificity to distinguish between patients with ad type dementia and non - demented subjects , but are less accurate to differentiate ad dementia from other primary dementias ( knopman et al . in fact , up to 20% of patients clinically diagnosed with ad do not have ad pathology at autopsy ( mayeux et al . a more accurate and earlier diagnosis of ad ( e.g. , in mci patients that will progress to the ad dementia phase or ideally those subjects in the pre - clinical phase ) could enable the administration of potential disease - modifying drugs that would have a great impact on patients life and profound implications for public health ( brookmeyer et al . , 1998 ) . in this context , there is a need of specific biological markers for ad diagnosis in the earliest stages . a biomarker is defined as a measureable feature that can be used to diagnose a physiologic or pathologic condition . the ideal biomarker of ad would be : ( 1 ) directed at the fundamental pathophysiology of the disease , ( 2 ) a marker of the presence of disease itself , ( 3 ) efficacious at prodromal , and even pre - clinical stages of ad , ( 4 ) an indicator of disease s severity , ( 5 ) a marker of treatment effectiveness , and ( 6 ) inexpensive and non - invasive ( klunk , 1998 ) . in the case of ad , different biomarkers have been described using diverse approaches . the development of cerebrospinal fluid ( csf ) assays and neuroimaging techniques that can provide information about the presence of ad pathological changes has been a major step forward in the field . consequently , biomarkers for ad are called to play a central role in the clinical characterization of the disease . more recently , the nincds adrda criteria have been revised by the national institute on aging and the alzheimer s association ( nia aa ) workgroup to include the experience in research over the past 25 years ( mckhann et al . , 2011 ) . the nia aa proposed a series of diagnostic classification from pre - clinical ad to mci to full - blown ad , and introduced csf and neuroimaging biomarkers as supportive features of the disease ( albert et al . , 2011 ; mckhann et al . , 2011 ; sperling et al . , 2011 ) . however , the authors cautioned about the use of biomarkers , and stated that their use should be limited to research and drug trials . nevertheless , the core clinical criteria for ad remained relatively intact in the new criteria . this review will focus on the most widely studied and currently accepted sources of biomarkers in ad : csf , magnetic resonance imaging ( mri ) , and nuclear medicine techniques , including amyloid imaging . typically , the csf better reflects the brain neurochemistry , while biochemical values in plasma or serum may be affected by many non - neurological factors . csf biomarker research has focused mainly on molecules related to the central neuropathological features of ad , such as a , the main component of the senile neuritic plaques ( snp ) , and tau proteins that are part of the nfts . a is a 3643 amino acid peptide originated from proteolysis of the amyloid protein precursor ( querfurth and laferla , 2010 ) . snps are mainly composed by a protein with 40 ( a40 ) or 42 ( a42 ) amino acids , and it seems that a deposition begins with a42 , and during plaque maturation , they acquire a40 ( iwatsubo et al . , 1994 ) . by contrast , nfts are formed by tau proteins , a microtubule - associated stabilizing protein essential for axonal transport . tau is hyperphosphorylated in ad , which tends to aggregate , leading to synaptic and neuronal dysfunction ( lee , 1996 ; blennow et al . , 2010 ; this process of nfts formation starts in the entorhinal cortex , spreading to the hippocampus , and then to the rest of the brain . these neuropathological changes begin early , even decades before the onset of clinical symptoms of dementia ( braak and braak , 1991 ) , but the relative contributions of altered tau and amyloid metabolism to neurodegeneration remain under investigation . nevertheless , nfts are more robust associated with measures of synapse loss and cognitive impairment that snps ( gomez - isla et al . , 1997 ) . typically , ad patients have lower levels of a42 in the csf compared to normal controls , a40 tends to remain constant , and tau ( t - tau ) and phosphorylated tau ( p - tau ) increase ( arai et al . , 1995 ; blennow et al . , 1995 ; , 1995 ; galasko et al . , 1998 ; kurz et al . , 1998 ; shoji et al . , 1998 ; hulstaert et al . , 1999 ; , 2001 ; sunderland et al . , 2003 ; lewczuk et al . , 2004 ; the low csf a42 levels in this entity may represent the retention of the peptide into plaques , resulting in reduced availability to diffuse into the csf . this is supported by the findings that a42 in the csf is inversely correlated with amyloid plaque load at autopsy ( strozyk et al . , 2003 ; tapiola et al . , 2009 ) and as measured in vivo by amyloid imaging ( fagan et al . , 2006 the increase in t - tau and p - tau in the csf of ad patients may stem from release into csf during neurodegeneration ( blennow et al . , 1995 ) , although the exact mechanism is not known . t - tau and p - tau concentrations in the csf have been associated with nft load at autopsy ( buerger et al . 2009 ) , as well as with cognitive decline and brain atrophy ( buerger et al . , 2002 ; low csf a42 and high t - tau or p - tau proteins have shown high accuracy for ad diagnosis ( galasko et al . , 1998 ; andreasen et al . , 2001 ; schoonenboom et al . , 2004 ) . studies that compared ad patients with normal control subjects have demonstrated that low a42 had 78100% sensitivity and 4781% specificity for ad diagnosis ; high csf t - tau levels had 70% sensitivity and 92% specificity and p - tau showed 77% sensitivity and 87% specificity . however , p - tau appears to be better than t - tau in the diagnosis of ad , and it has shown a positive predictive value of 90% , especially p - tau phosphorylated at threonine 181 ( p - tau181 ; hampel et al . figure 1 shows the t - tau levels in ad , frontotemporal dementia ( ftd ) , and normal autopsied subjects , and figure 2 shows the a42 levels in the same population ( schoonenboom et al . , ad , alzheimer s disease ; ftd , frontotemporal dementia . because some studies found that a42 and tau levels alone were not sufficient to differentiate ad from normal controls or other dementias , the a42/tau ratio is used to improve the diagnosis of ad . a meta - analysis showed that the a42/tau ratio had a sensitivity of 71% and specificity of 83% for ad ( hampel et al . , 2004b ) ; and a recent study showed that the signature of ad , based on the csf a42/p - tau181 ratio cutoffs , was present in 90% of the ad patients compared to 36% in the normal control group ( de meyer et al . , 2010 ) . although , these csf biomarkers seem to be useful to diagnose ad , they are not sensitive enough to assess disease progression ( sunderland et al . , 1999 ; vemuri et al . , 2010b ) . nevertheless , higher csf t - tau and lower a42 values have been associated with rapid cognitive decline , no response to cholinesterase inhibitor treatment and a higher mortality in patients with ad dementia ( wallin et al . , 2010 ) . cerebrospinal fluid biomarkers might have a role in the differential diagnosis of ad and other dementias . patients with dementia with lewy bodies ( dlb ) , like ad patients , have lower levels of a42 in the csf compared to normal controls , reflecting that most of these subjects also present ad pathology . nevertheless , shorter amyloid peptides ( a37 , a38 , and a40 ) appear to be slightly more elevated in ad dementia ( bibl et al . , 2006 ) . consequently , the a42/a37 and a42/a38 ratios seemed to better discriminate ad from dlb ( bibl et al . t - tau and p - tau levels are normal or high in dlb , but most studies have shown that these markers levels in dlb are definitely lower than those found in ad . other proposed biomarker for the differentiation of ad from dlb was monomeric soluble -synuclein , but the available studies in dlb patients showed conflicting results . elevated , unchanged and decreased total -synuclein levels have been reported in these patients compared to ad and control subjects ( mukaetova - ladinska et al . , 2010 ) . in addition , there are limited data about oligomeric -synuclein in csf , although it could be potential biomarker for synucleinopathies ( tokuda et al . , 2010 ) . possibly , the combination of all these markers will help to distinguish between ad and dlb in the future . in ftd , csf a42 levels are lower than in control subjects but higher than in ad , while t - tau and p - tau levels are higher than in controls but lower that in ad patients ( hulstaert et al . hence , t - tau / a42 and p - tau / a42 ratios could be a useful tool to distinguish ad from ftd ( bian et al . creutzfeldt jakob disease ( cjd ) is characterized by decreased csf a42 concentrations and very high t - tau levels , reflecting intense neuronal damage , while p - tau levels are normal ( kapaki et al . , 2001 ; current data suggest that p - tau levels could be the most relevant marker to differentiate ad from other types of dementias ( hampel et al . cerebrospinal fluid biomarkers may also be useful in predicting progression to ad in subjects with mci ( andreasen et al . , 1999 ; hampel et al . , 2004c ; ewers et al . , 2007 low csf a42 levels predicted conversion with 6080% sensitivity and 65100% specificity , while t - tau levels had 8386% sensitivity and 5690% specificity , and p - tau had 7384% sensitivity and 4788% specificity ( hampel et al . , 2004b ) . however , the conversion to ad dementia in mci subjects has shown significant variability among different csf studies . ( 2006 ) found high sensitivity ( 95% ) and specificity ( 87% ) for the conversion from mci to ad dementia using a combination of t - tau and a42/p - tau181 ratio cutoff values . by contrast , a multicenter csf study found lower sensitivity ( 83% ) and specificity ( 72% ) for the conversion from mci to ad than single - site studies , with a considerable inter - site assay variability that highlights a need for standardization of analytical techniques and subject selection ( mattsson et al . , 2009 ) . other large multicenter studies have confirmed the high predictive value of these csf biomarkers in the identification of cases with prodromal ad in the context of the mci syndrome ( shaw et al . , 2009 ; visser et al . , 2009 ) . the csf biomarkers have been used to identify the pre - clinical stage of ad . cross - sectional studies in normal control individuals carrying the apoe4 allele showed lower csf a42 and higher p - tau181 levels ( peskind et al . interestingly , csf levels of a42 correlated with brain volume in this group of subjects , while in mci and ad dementia patients , whole - brain volume correlated with csf t - tau and p - tau181 ( fagan et al . , 2009 ) . a longitudinal csf study in normal controls found increased t - tau levels and no change in a42 after a 1-year follow - up ( vemuri a few longitudinal studies have assessed which csf biomarker or combination of biomarkers better predict ad dementia or cognitive decline . fagan et al . ( 2007 ) demonstrated that csf tau / a42 ratios may predict future dementia in cognitively normal older subjects and two other studies showed that low levels of csf a42 predicted cognitive decline , as measured with the mini mental status examination ( mmse ; gustafson et al . although there are studies that showed that csf a42 could predict prodromal ad , there was a wide sd in these subjects , which made difficult to determine individually who will progress to ad . further studies are needed to assess changes in csf markers over time and to determine the best csf markers of change . structural neuroimaging techniques are normally performed during the clinical assessment of patients with dementia , mainly to rule out structural lesions , such as brain tumors , normal pressure hydrocephalus , or vascular lesions . the mri has shown high sensitivity to detect brain atrophy caused by the neurodegenerative process ( bobinski et al . , 2000 ) . several methods are used to analyze mri data including visual inspection , volumetry , or region of interest ( roi ) analysis ( applying manual tracing , automated , or semi - automated techniques ) and voxel based morphometry ( vbm ; vemuri and jack , 2010 ) . the visual rating of the images and roi analysis depend on the a priori choice of structures , while vbm is a whole - brain operator - independent analysis ( ashburner and friston , 2000 ) . however , vbm analysis only allows comparing groups of patients , and it is not useful to classify individual cases . visual assessment of mri scans is a fast method to assess brain atrophy but is not the most precise ( raji et al . , 2010 ) . volumetry of the medial temporal lobe structures is the most common quantitative method used in ad . the traditional manual tracing is time - consuming , and consequently , automated or semi - automated methods that require no significant manual intervention have been developed . other methods to analyze sequential mris are boundary shift integral ( bsi ) and tensor - based morphometry ( tbm ) . bsi has been developed to quantify the global percentage of change in the brain surfaces between two scans , while tbm provides a three - dimension profile of brain atrophy ( vemuri and jack , 2010 ) . brain mri studies have shown that ad patients have a widespread cortical volume loss , including the frontal lobes , the temporoparietal regions , precuneus , and mesial temporal lobes , with relative sparing of the sensorimotor , visual , and cerebellar regions ( du et al . , 2001 ; good et al . , 2001 ; thompson et al . , 2001 ; karas et al . , 2003 ; testa et al . , 2004 ; ishii et al . , 2005 ; dickerson et al . , 2008 ; burton et al . , 2009 ) longitudinal studies have suggested that atrophy stars in the medial temporal lobes and fusiform gyrus in the prodromal phase of the disease , then spreads out to the posterior temporal and parietal lobes , and by the time of the dementia phase , it involves the medial temporal lobes , temporoparietal cortex , and the frontal lobe ( whitwell et al . , 2007 ) . this pattern of atrophy progression on mri appears to follow the distribution of nft in the different braak and braak ( 1991 ) pathological stages ( whitwell et al . , 2008 ) . in ad , there is also a greater rate of brain atrophy over time compared with healthy elderly subjects ( 1.92.2% per year versus 0.50.7% per year ; obrien et al . , 2001 ; sluimer et al . , hippocampal and whole - brain atrophy in ad correlated well with cognitive performance and measures of disease severity ( jack et al . , 2008a ; ridha et al . , 2008 mri studies show a similar pattern of brain atrophy to that observed in ad ( jack et al . , 2000 ; chetelat et al . , 2002 ; karas et al . , 2004 ; bell - mcginty et al . , 2005 ; whitwell et al . , 2008 ) and rates of whole - brain atrophy are around 1% per year , between ad and control subjects ( jack et al . , 2004 ) . whole - brain atrophy rate in mci is associated with impaired cognitive performance ( evans et al . , 2010 ) and predicts progression to ad dementia ( spulber et al . , 2008 ) . whole - brain techniques have also shown that higher rates of atrophy in specific areas such as the hippocampus , posterior cingulated gyrus , and superior parietal cortex were associated with incident ad in mci cases ( chetelat et al . , 2005 ) , and hippocampal and basal forebrain volumes in normal subjects predicted future development of ad ( hall et al . , 2008 ) . interestingly , a recent study showed that normal subjects with the ad csf profile had increased rates of brain atrophy over time , suggesting they may be in the pre - clinical phase of the neurodegenerative process ( schott et al . , 2010 ) . nevertheless , this observation requires a longer follow - up period to demonstrate that this particular group of subjects has a higher risk to develop ad dementia . because the most important clinical manifestation of ad is memory loss , the majority of the researchers have focused on the study of hippocampal atrophy as a marker of the disease . different neuroimaging studies assessing the mesial temporal lobe structures have consistently shown smaller hippocampal volumes in ad patients compared to controls ( pennanen et al . , 2004 ) . measures of hippocampal atrophy correlate well with the degree of cognitive impairment ( jack et al . , 2000 ; mortimer et al . , 2004 ) and with ad pathology and hippocampal sclerosis in mri - post - mortem studies ( jagust et al . , 2008 ) . both normal aging and ad have gradual volume loss overtime , but as with whole - brain volume is greater in ad ; the annual rate of hippocampal atrophy is 1.41% for healthy controls and several studies have shown that hippocampal atrophy measured by mri predicts conversion from mci to ad ( jack et al . , 1999 ; visser et al . , 1999 , 2002 ; geroldi et al . , 2006 ; devanand et al . , 2007 ; teipel et al . , 2007 ) . a recent meta - analysis showed that this measure could identify mci converters with a sensitivity of 73% and a specificity of 81% ( yuan et al . , 2009 ) . although decreased entorhinal cortical thickness has been proposed as a predictor of conversion from mci to ad , measures of decrease hippocampal volume appeared to be a more robust predictor . there are several limitations for the use of whole - brain or hippocampal volume as a sole biomarker for ad . one of the most important is that small brain volumes can also be seen in normal aging , and consequently , is difficult to separate cases with early ad from normal controls . on the other hand , the rate of change on whole - brain , entorhinal , and hippocampal volumes seemed to be good markers of neurodegeneration and they can be useful in determining the effects of ad therapies , but these techniques are labor intensive and limited to research studies . brain functional neuroimaging techniques positron emission tomography ( pet ) and single photon emission computed tomography ( spect ) allow a broad range of cerebral functions to be assessed in patients currently living with dementia . actually , brain metabolism , brain perfusion , and different neurotransmitter systems can be measured with a great variety of pet and spect tracers . however , brain metabolism with the glucose analog 2-[f]-fluoro-2-deoxy - d - glucose ( f - fdg ) and brain perfusion with technetium-99 m hexamethylpropylamine oxime ( 99mtc - hmpao ) are the two more widely employed techniques in the evaluation of patients with dementia , both in clinical practice and in research . brain spect with 99mtc - hmpao studies provide information of the regional cerebral blood flow , and f - fdg pet estimates the regional brain rate of glucose consumption , therefore providing information about the pattern of neuronal loss or synapse dysfunction in patients with dementia . the pattern of spect hypoperfusion and pet hypometabolism usually seen ad involves the anterior medial temporal lobes , the posterior cingulate and posterior temporoparietal cortex ( kogure et al . , 2000 ; nevertheless , spect seems to be less accurate than pet for the diagnosis of ad . one autopsy study found that this technique alone had a sensitivity of 63% and a specificity of 93% to diagnose ad ( jagust et al . , 2001 ) . on the other hand the sensitivity of this procedure discriminating ad from ftd was 71.5% and the specificity was 78.2% , while its sensitivity and specificity to differentiate ad from vascular dementia were 71.3 and 75.9% , respectively ( dougall et al . , spect has shown that can predict conversion to ad with a test accuracy of 82% ( huang et al . , 2007 ) . positron emission tomography studies in early ad have shown a pattern of reduced metabolism in the posterior cingulate ( minoshima et al . , 1997 ) and mesial temporal cortices . as the disease progresses , there is a greater involvement of the parietotemporal and frontal cortices ( ishii et al . , 2001 ; this progression of the areas of hypometabolism correlated with cognitive deterioration over time as measured by neuropsychological tests ( engler et al . 1984 ; minoshima et al . , 1995 ; silverman et al . , 2001 ; mosconi et al . , 2005 ) . a meta - analysis showed that pooled f - fdg pet sensitivity and specificity to differentiate ad patients from normal subjects were 86% in both cases , but the variability was important between the studies ( sensitivity from 61 to 100% and specificity from 54 to 100% ; patwardhan et al . , 2004 ) . pet also seems to be a good tool to differentiate ad from other forms of dementia . in an autopsy confirmed study , the presence of occipital hypometabolism was able to distinguish dlb from ad with 90% sensitivity and 80% specificity ( minoshima et al . this technique also has a high specificity and sensibility to differentiate ad and ftd ( koeppe et al . , 2005 ; f - fdg pet appears to be a promising instrument to discriminate those patients with mci who will progress to ad dementia . these patients show a medial temporal and posterior cingulate cortices hypometabolism compared with healthy controls ( chetelat et al . has also shown that f - fdg pet performs slightly better than spect and structural mri in identifying the prodromal phase of ad in patients with a clinical diagnosis of mci ( pooled sensitivity of 89% and specificity of 85% ; yuan et al . , 2009 ) . interestingly , similar metabolic reductions in the same regions as those found in mci and ad patients have been seen in asymptomatic subjects at risk of ad , i.e. , in persons with subjective memory complains ( caselli et al . , 2008 ) , carriers of an apolipoprotein e 4 ( apoe4 ) allele ( reiman et al . , 1996 , 2001 , 2004 , 2005 ) , with abnormalities in the csf markers ( petrie et al . , 2009 ) , or with a maternal history of ad ( mosconi et al , 2007 , 2009 ) . furthermore , hippocampal hypometabolism during normal aging predicted cognitive decline years in advance of the clinical diagnosis ( de leon et al . , 2001 ; jagust et al . these data indicate that f - fdg pet has high sensitivity to distinguish ad from controls and from other dementing entities , and also to identify subjects at high ad risk . consequently , the pet ad pattern has been incorporated into the new clinical criteria for prodromal ad ( dubois et al . , 2007 ) , pre - clinical ad ( sperling et al . , 2011 ) , and mci due to ad ( albert et al . , the development of pet radiotracers that bind to brain amyloid has revolutionized the use of neuroimaging techniques in patients with dementia . these molecules allow the localization of ad pathology in vivo , and have helped to further our understanding of the underlying biology of ad . the most actively tested tracers are n - methyl-[c]2-(4-methylaminophenyl)-6-hydroxybenzothiazole ( c - pib ; klunk et al . , 2003 ) , ( e)-4-(2-(6-(2-(2-(2-(18)f - fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-n - methyl benzenamine ( f - av-45 ; choi et al . , 2009 ) and 2-(1 - 96-(2-f - fluoroethyl)(methyl)amino)-2-naphthyl)ethyldene ) malono nitrile ( f - fddnp ; small et al . , 2006 ; shin et al . , 2008 ) , but there are multiple c and f compounds under investigation such as trans-4-(n - methyl - amino)-4-{2-[2-(2-[18f]fluoro - ethoxy)-ethoxy]-ethoxy}-stilbene ( f - bay94 - 9172 ; rowe et al . , 2008 ) , 4-n-[c - methyl]amino-4-hydroxystilbene ( c - sb-13 ; verhoeff et al . , 2004 ) and 2-(2-[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole ( c - bf-227 ; kudo et al . , 2007 ) . while c have better affinity for fibrillar amyloid than f compounds , the former have a very short life , which limits its use to centers with an available cyclotron that can produce them . among these tracers , this thioflavin - t derived binds fibrillar a with high affinity and its brain retention correlates well with levels of amyloid in ad brain tissue ( mathis et al . , 2002 , 2003 ; positron emission tomography studies with c - pib have demonstrated an increased retention of this tracer in frontal and parietotemporal cortices , as well as in the striatum of almost all ad patients compared with controls ( klunk et al . , 2004 ; edison et al . , 2006 ; rowe et al . , 2007 ) . however , it is important to note that a positive c - pib pet scan can also be seen in other entities , often misdiagnosed as ad , such as dlb and cerebral amyloid angiopathy ( edison et al . , 2008 ; gomperts et al . , 2008 ; dierksen et al . , c - pib retention in ad correlates with rates of whole - brain atrophy ( archer et al . , 2006 ) , parietotemporal hypometabolism on f - fdg pet ( klunk et al . , 2004 ; edison et al . , 2006 ; engler et al . , 2006 ) , and decreased csf a42 levels ( fagan et al . , 2006 ; grimmer et al . there are few longitudinal studies using amyloid ligands , and the observation time has been relatively short , because these techniques have only been available for research since 2004 . recent longitudinal studies in ad patients assessing amyloid load and progression of neurodegeneration using structural mri or f - fdg pet found that c - pib retention was relatively stable over time , while neurodegenerative markers worsen in parallel with cognitive decline ( engler et al . , 2006 ; jack et al . , 2009 ; scheinin et al . , 2009 ; kadir et al . , these findings are consistent with the hypothesis that there is dissociation between the amyloid deposition and the neurodegenerative process , where amyloid accumulation precedes the clinical symptoms and reaches its maximum detectable level before the cognitive deficits are evident ( jack et al . , 2010a ) . however , another recent study has shown increases of c - pib retention in mild to moderate ad dementia , questioning this hypothesis and suggesting that a deposition might slow down in the later stages of the disease , but it is still present ( villemagne et al . , 2011 ) . in mci patients , c - pib pet shows a bimodal distribution with some subjects having a pattern of retention similar to ad patients and others similar to control subjects ( pike et al . , 2007 ; rowe et al . in these patients , the a burden is related to episodic memory impairment but not to other cognitive tasks ( pike et al . , 2007 ) . the pib - positive mci subjects are more likely to carry an apoe4 allele and to progress to ad ( pike et al . , 2007 ; factors that seem to influence a shorter time to progression in pib - positive subjects is the presence of hippocampal atrophy , higher c - pib retention values and having an apoe4 allele ( okello et al . , 2009 ; ( 2009 ) rescanned mci subjects after a single year , but only a minimal change has seen in this group of persons 2011 ) have shown small but significant increases in c - pib cortical retention in mci patients from the baseline studies to scans to those performed from 20 months to 3 years later ; other studies have reported similar results ( kadir et al . , 2010 ; koivunen et al . , , approximately 2030% of elderly cognitively normal subjects show some degree of c - pib retention , mainly in the prefrontal cortex , posterior cingulate and precuneus regions ( mintun et al . , 2006 ; pike et al . , 2007 ; rowe et al . , 2007 ; aizenstein et al . , 2008 ) . in fact , the proportion of c - pib - positive healthy control subjects increases with age ( morris et al . , 2010 ) . in these subjects , some studies have reported a strong relationship between impaired episodic memory performance and c - pib binding that could be modified by cognitive reserve ( pike et al . 2010 ) , but some others have not found this association ( mintun et al . , 2006 ; jack et al . , 2008b ; stornadt et al . , 2009 ) . studies directly relating structural mri data to a burden in control subjects has yield contradictory results . 2009 ) , a cortical thinning pattern consistent with early ad ( dickerson et al . , 2008 ; becker et al . , 2010 ) and an increased rate of brain atrophy ( scheinin et al . , 2009 ) , while others have found brain atrophy only in c - pib - positive subjects or in normal individuals with subjective cognitive impairment ( chetelat et al . , there are only two longitudinal studies reporting progression of cognitively normal subjects scanned with c - pib pet to symptomatic ad . in one study , 2009 ) reported that the relative risk of conversion from cognitive normal to ad was increased almost five - fold with a c - pib - positive pet scan . ( 2011 ) performed serial c - pib pet scans in a group of normal subjects and found that 25% of individuals with a positive scan developed mci or ad dementia by 3 years . in addition , the authors also reported an increase in c - pib brain retention in those control subjects with a positive baseline scan , suggesting a slow process of a accumulation in the brain over time ( villemagne et al . these data are consistent with the hypothesis that amyloid imaging can detect a accumulation in advance of the onset of dementia ( jack et al . , 2010a ) , although more longitudinal studies are required to determinate the sequence of pathological events in the process from normalcy to ad . figure 3 shows the spectrum of amyloid deposition in normal controls , mci and ad cases , as measured with c - pib pet ( mathis et al . , 2007 ) . transaxial and sagittal planes of c - pib pet scans in a normal control ( nc ) , a c - pib - positive nc ( nc+ ) , c - pib - negative mci subject ( mci ) , a c - pib - positive mci subject ( mci+ ) , a highly c - pib - positive mci subject ( mci++ ) , and a c - pib - positive ad patient ( ad ) . reprinted from mathis et al . although amyloid imaging is a relatively new technique , we can state that it accurately detects a accumulation in the brain . however , it has been reported the case of a patient clinically diagnosed of mild ad with a negative c - pib pet scan , where low levels of amyloid pathology at autopsy were found ( cairns et al . , 2009 ) . on the other hand , there are not reported cases of subjects with a positive amyloid pet without presence of a at autopsy . these data suggests that c - pib pet is a technique with high specificity and positive predictive value for early ad diagnosis . nevertheless , having an c - pib - positive scan does not mean to be at a pre - dementia stage of ad , and the prognostic value of increased a load on pet has to be established with more extensive longitudinal studies . in this scenario , adding techniques that assess functional brain changes suggesting early ad could be of special interest . definitely , the information obtained with current amyloid tracers will bring to light important issues in the biology of amyloid deposition in the transition from normal to ad . current data suggest that diagnosis of ad can be enhanced by use of these promising biomarkers to increase accuracy and identify early stages of the disease . each of these biomarkers seems to indicate a specific process in ad , so that amyloid imaging and decreased csf a42 are indicators of brain amyloid burden , while csf tau , brain atrophy and brain metabolism are biomarkers of the neurodegenerative process . some authors have proposed a model of disease in which there is timing for the use of different biomarkers ( jack et al . , 2010a ) . the ad pathologic cascade appears to have a sequential two stage process initiated by amyloid accumulation and followed by neuronal pathology . to validate this hypothesis and the right use of biomarkers in the clinical practice , it is necessary to determine the dynamics of the amyloid process with other biomarkers at the different stages of the disease . an early ad diagnosis could enable to administer treatments with potential disease - modifying effect before neurodegeneration becomes severe . 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 . joseph disease , is the most common subtype of sca world - wide , and is caused by a pathologic cag trinucleotide repeat expansion in the atxn3 gene located on chromosome 14q32.12 . the cardinal clinical characteristics of sca3 include gait and stance unsteadiness , limb ataxia , dysarthria , oculomotor dysfunction , sensory disorder , pyramidal and extrapyramidal dysfunction , and so on . sca3 is a slowly progressive and unremitting disease , in which patients generally will become wheelchair - bound and bedridden in the end stage , and the median survival time after disease onset is approximately 21 years . the resulting loss of working ability and reduced survival confer significant disease burden to the patients , their families , and the society . thus , it is of vital importance to explore effective therapeutic options in order to alleviate the symptoms or retard the disease progression in sca3 . nerve growth factor ( ngf ) is the founding member of the neurotrophin family , and is essential for the proper development , patterning , and maintenance of the mammalian nervous system . previous studies have revealed that ngf specifically targets sensory and sympathetic neurons in the peripheral nervous system , as well as basal forebrain cholinergic neurons in the central nervous system . there is also growing evidence to support the role of ngf in the development , differentiation , and maintenance of the human cerebellar connectivity . in this context , ngf and its high - affinity receptor tachykinin receptor antagonist ( tkra ) these data imply that ngf may have neuroprotective effects on cerebellar neurons and hence might serve as a therapeutic candidate of sca3 . therefore , this clinical pilot study was set forth to examine the efficacy of ngf in patients with sca3 . this study was an open - label clinical trial assessing the efficacy of ngf in patients with sca3 ; it was conducted at the first affiliated hospital of zhengzhou university from november 2011 to november 2012 . this study was approved by the ethics committee of first affiliated hospital of zhengzhou university and registered at the chinese clinical trial registry ( www.chictr.org ; chictr - onc-11001954 ) . all study procedures were in accordance with the declaration of helsinki and all recruited subjects have provided written informed consents . ataxia patients with family history were screened at the department of neurology , first affiliated hospital of zhengzhou university and referred for genetic testing at the department of genetic diagnosis . patients who fulfilled the following inclusion criteria : ( 1 ) ataxia patients with family history were checked and diagnosed by two independent doctors , then the genotype sca3 was confirmed by genetic test ; ( 2 ) older than 18 years ; and ( 3 ) willing to give informed consent , will be recruited . the exclusion criteria were as follows : ( 1 ) allergy to neurotrophin ; ( 2 ) with concomitant severe systematic diseases or psychiatric disorders ; ( 3 ) unable to finish the scale for assessment and rating of ataxia ( sara ) score ; ( 4 ) refuse to attend the study , and ( 5 ) ataxias attributed to secondary causes ( such as alcohol or drug abuse and toxic exposure ) . all enrolled patients underwent standard neurological , electrophysiological and neuroimaging examinations , and the sca3 subtype was classified according to these clinical findings . china ) used in this study was extracted and purified from the submandibular gland of the male mouse and has high homology in the amino acid sequence with human ngf . the mngf has been safely used in a series of clinical studies . , and has been approved by china food and drug administration . mngf was administered peripherally by intramuscular injection at the dose of 18 g once daily for 4 weeks consecutively . it has been proven to have good reliability and validity among chinese patients with degenerative cerebellar . the sara evaluates axial ( gait , stance , sitting ) , speech , and appendicular ( finger chase , nose - finger test , fast alternating hand movements ( fahms ) , and heel - shin slide ) functions . the sara sum score ranges from 0 to 40 with 0 indicating no ataxia and 40 the most severe ataxia , thus deterioration or improvement of disease severity is , respectively , represented by an increase or decrease of the sara score . for each patient , chinese version of sara was performed at the baseline , 2 weeks ( midpoint ) and 4 weeks ( endpoint ) after treatment . these sara evaluations were all videotaped and reviewed independently by two other investigators who did not attend the original assessment in a random order . the average of score rated by these two evaluators was denoted as the final sara score . the primary outcome measure was the change of sara score after treatment compared with that at baseline . continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were expressed as frequencies or proportions where appropriate . the observed changes of sara score after treatment from baseline were analyzed with nonparametric wilcoxon signed - rank test . continuous variables were expressed as mean standard deviation ( sd ) , and categorical variables were expressed as frequencies or proportions where appropriate . the observed changes of sara score after treatment from baseline were analyzed with nonparametric wilcoxon signed - rank test . the mean age was 39.14 7.81 years , the mean age of onset was 35.00 6.53 years , the mean disease duration was 4.14 1.90 years , and the mean cag repeats number was 77.57 2.27 . the mean sara score dropped from 8.48 2.40 to 6.94 2.34 ( p < 0.001 ) and 6.30 1.87 ( p < 0.001 ) after 2 and 4 weeks of treatment , respectively [ table 2 ] . significant decrease in subsections sara scores was also observed in stance ( p = 0.008 and 0.003 ) , speech ( p = 0.046 and 0.023 ) , finger - chase ( p = 0.026 and 0.015 ) , fahms ( 0.015 and 0.009 ) , and heel - shin slide ( p = 0.006 and 0.001 ) at 2 and 4 weeks after therapy , respectively . the mean improvement in total sara score was 2.18 1.30 ( ranging from 0 to 5.75 ) in our study . scores of sara and its subsections at baseline , 2 and 4 weeks after ngf therapy * comparisons between midpoint of therapy and baseline ; comparisons between endpoint of therapy and baseline . sara : scale of the assessment of rating of ataxia ; fahm : fast alternating hand movements ; ngf : nerve growth factor . one previous study has shown that insulin - like growth factor-1 , one of the neurotrophic factors , may be effective in reducing the disease progression of sca3 . previous studies have reported that ngf can improve cognitive decline in patients with alzheimer 's disease and may also have potential therapeutic roles in other neurodegenerative diseases . postmortem histopathological study in patients with sca3 has revealed considerable neuronal loss at the cerebellar purkinje cell layer and the four deep cerebellar nuclei . ngf can prevent neuronal death or age - related atrophy in the adult brain by inhibiting apoptosis of cholinergic neurons in the basal forebrain , and it would be reasonable to postulate that it may also inhibit the apoptosis in the cerebellum neurons expressing tyrosine kinase a ( trka ) and serve as a potential therapy for sca3 . such treatment effect is observed as early as 2 weeks after therapy and sustained after 4 weeks . to our knowledge , this study is the first to investigate the efficacy of ngf in sca3 . postmortem study has shown that ngf and its high - affinity receptor tkra are distributed in the neurons of the human cerebellum cortex and its deep nuclei throughout life . these findings support the involvement of ngf in the development , differentiation and maintenance of the cerebellar connectivity . although the blood - brain barrier ( bbb ) has low permeability to large proteins , some autoradiography studies suggested ed that blood - borne ngf and its subunit -ngf can cross the bbb of mice and arrive at the brain parenchyma by direct permeation . second , the therapeutic effect of ngf might also be mediated via the proprioceptive sensation system . it has been reported that most ( 87% ) of the sca3 patients had somatosensory evoked potential abnormalities , especially in the lower limbs , which was due to degenerative lesions in the dorsal column of the spinal cord . hence , ngf therapy may improve stance and heel - knee - shin slide due to improved proprioception . these two proposed mechanisms may explain the observed improvement after therapy and substantiate the use ngf to treat a patient with sca3 . first , it was an open - label study , in which the observed therapeutic efficacy might be contributed by placebo effects . however , in one randomized , double - blind , and placebo - controlled study to evaluate the efficacy of varenicline in sca3 patients , the mean improvements of sara score in the therapeutic group , and the placebo group were 1.97 and 0.86 , respectively . the sara score improvement of 2.18 in our current study is unlikely to be accounted for by placebo effect alone . furthermore , sara is a reliable and valid scale to linearly assess the ataxia symptoms , and changes of sara scores exceeding 1.1 points are considered clinically relevant . nevertheless , our pilot data suggest that ngf may be a promising treatment for patients with sca3 . a large - scale randomized , double - blind placebo - controlled trial would be worthwhile to evaluate the efficacy and tolerability of ngf in sca3 patients .

alzheimer s disease ( ad ) is the most common form of dementia in the elderly , and it is characterized by progressive impairment in multiple cognitive domains of sufficient severity to interfere with individuals daily living activities . historically , the diagnosis of ad has been based on the identification of a clinical syndrome , and accuracy studies of the current clinical criteria conducted in referral clinics have shown high sensitivity for ad . however , the identification of the disease is still not perfect , and there is growing evidence that the use of biomarkers will increase our ability to better indentify the underlying biology of ad , especially in its early stages . these biomarkers will improve the detection of the patients suitable for research studies and drug trials , and they will contribute to a better management of the disease in the clinical practice . in this review , we discuss the most studied biomarkers in ad : cerebrospinal fluid proteins , structural magnetic resonance imaging , functional neuroimaging techniques , and amyloid imaging . background : spinocerebellar ataxia type 3 ( sca3 ) is the most common subtype of sca worldwide , and runs a slowly progressive and unremitting disease course . there is currently no curable treatment available . growing evidence has suggested that nerve growth factor ( ngf ) may have therapeutic effects in neurodegenerative diseases , and possibly also in sca3 . the objective of this study was to test the efficacy of ngf in sca3 patients.methods:we performed an open - label prospective study in genetically confirmed adult ( > 18 years old ) sca3 patients . ngf was administered by intramuscular injection ( 18 g once daily ) for 28 days consecutively . all the patients were evaluated at baseline and 2 and 4 weeks after treatment using the chinese version of the scale for assessment and rating of ataxia ( sara).results : twenty - one sca3 patients ( 10 men and 11 women , mean age 39.14 7.81 years , mean disease duration 4.14 1.90 years , mean cag repeats number 77.57 2.27 ) were enrolled . after 28 days of ngf treatment , the mean total sara score decreased significantly from a baseline of 8.48 2.40 to 6.30 1.87 ( p < 0.001 ) . subsections sara scores also showed significant improvements in stance ( p = 0.003 ) , speech ( p = 0.023 ) , finger chase ( p = 0.015 ) , fast alternating hand movements ( p = 0.009 ) , and heel - shin slide ( p = 0.001).conclusions : our preliminary data suggest that ngf may be effective in treating patients with sca3 .

nanopores are powerful research tools to investigate the structural and dynamic properties of single biomolecules . nanopores have inner diameters of a few nanometers , and following the size - exclusion principle , only individual dna or protein molecules can fit into or pass through a pore . importantly , a passing molecule blocks a pore , leading to a transient change of current . parameters of the current blockade such as duration and amplitude can give information about the length and size of the molecule . one of the most widely published and scientifically attractive subjects is the sensing of dna with nanopores . while an ultimate goal of these studies is to develop an inexpensive method to sequence dna ( dekker 2007 ; branton et al . 2010 ) , a large array of experiments have also shed light on the biophysics of dna or rna translocation through biological pores ( kasianowicz et al . ; healy 2007 ; vercoutere and akeson 2002 ; deamer and branton 2002 ; marziali and akeson 2001 ) as well as inorganic pores ( reviewed in , e.g. , howorka and siwy 2009 ; healy et al . 2007 ; dekker 2007 ) . examined aspects include ( i ) the frequency with which the strands thread into the pore ( henrickson et al . 2000 ; meller and branton 2002 ; nakane et al . 2008 ) , ( ii ) the orientation of strands ( butler et al . 2006 ; wang et al . 2004 ; mathe et al . 2005 ; wanunu et al . 2008 ; wiggin et al . 2008 ; li et al . 2003 ; chen et al . 2004 ; storm et al . 2005 ; fologea et al . 2007 ; steinbock et al . 2010 ) , ( iii ) the speed of dna transport ( meller et al . 2001 ; wanunu et al . 2008 ) , ( iv ) the influence of transmembrane potential ( aksimentiev and schulten 2005 ; mathe et al . 2005 ; kathawalla et al . 1989 ; heng et al . 2005 ; keyser et al . 2006 ) , ( v ) pore geometry ( howorka and bayley 2002 ) as well as ( vi ) interaction with pore walls ( wiggin et al . 2008 ; wanunu et al . 2008 covalently attaching dna to the pore wall is an attractive variation in single - molecule research . in previous studies aimed at dna sensing , one or multiple single stranded dna ( ssdna ) molecules were end - tethered to the pore wall of protein ( howorka et al . complementary or mismatched dna molecules showed that dna duplexes between the free and tethered strands form inside the nanopore . as the perfect and mismatched duplexes , respectively , had different lifetimes , the dna - modified nanopores could be used as biosensor elements to distinguish nucleic acids with single - point mutations ( howorka et al . the covalent attachment of dna strands has also been exploited to control the electronic properties of a pore . in general , artificial pores that exhibit engineered properties such as voltage gating or ion selectivity are attractive model systems for biological voltage - gated ion channels ( hille 2001 ) . ( 2004 ) were the first to demonstrate that dna - modified nanopores can function as ionic switches . a single gold nanotube carrying thiol - terminated dna strands was shown to preferentially transport cations in one direction , while hindering transport in the other . the ion current rectification was explained by assuming that dna molecules deflect by the external electric field and cause voltage - dependent pore opening . the attached dna strands were not localized to a single subnanoscale position but covered the entire pore . additionally , the length of the fully extended dna molecules was smaller than the pore diameter ; hence , it was not possible to completely block the current ( e.g. , 59-nm - diameter gold tube was modified with 30-mer dna ) . with the aim of achieving a greater pore blockade and ion current modulations , this study attempts to tune the ionic pore properties by restricting dna immobilization to a small nanoscale region . single conically shaped nanopores with an opening of 8 or 12 nm were selectively modified at the narrow section with ssdna oligonucleotides ( fig . 1 ) with a length of 30 nucleotides . we compared the current voltage curves of single nanopores before and after dna modification at different kcl concentrations between 1 m and 10 mm . the experimental results indicate that the attached dna either reduced or blocked the ionic current flow through the pore dependent on ionic strength , which also influenced the conformation of the dna strands ( odijk 1977 ; skolnick and fixman 1977 ; kaiser and rant 2010 ) . 1scheme of the surface charge pattern obtained in the process of one - sided modification of a single conically shaped nanopore with point - tethered dna molecules . we estimate the position of the junction between the dna - modified zone and the zone with carboxyl groups to be several tens of nanometers from the small opening of the pore ( vlassiouk and siwy 2007 ) . carboxyl groups are formed in the process of pore preparation scheme of the surface charge pattern obtained in the process of one - sided modification of a single conically shaped nanopore with point - tethered dna molecules . we estimate the position of the junction between the dna - modified zone and the zone with carboxyl groups to be several tens of nanometers from the small opening of the pore ( vlassiouk and siwy 2007 ) . carboxyl groups are formed in the process of pore preparation in addition , the dna - modified conical nanopores presented here function as rectifiers . rectifying systems have a preferential direction of mass flow and , in some cases , can entirely stop the flow in the opposite direction , forming a diode . rectifiers and diodes based on surface charge patterns on the pore walls have previously been created , but these previous embodiments were able to switch the transport of just ions and charged species ( bassignana and reiss 1983 ; mafe and ramirez 1997 ; daiguji et al . 2005 ; vlassiouk and siwy 2007 ; nguyen et al . 2010 ; cheng and guo 2009 ) . as an example , an ionic bipolar diode was built based on nanopores that contained a junction between positive and negative charges on the pore walls ( vlassiouk and siwy 2007 ; cheng and guo 2009 ) . a unipolar diode is formed with a junction between positive ( or negative ) surface charges and a neutral zone ( karnik et al . by contrast , the dna - modified nanopores presented here feature an opening diameter that is tuned by the operation conditions , e.g. , kcl concentration and voltage . the rationally engineered pores can thus be applied to tune and switch the transport of both ionic and neutral species . single conically shaped nanopores were prepared by the track - etching technique according to a published procedure ( fleischer et al . briefly , 12-m - thick films of polyethylene terephthalate ( pet ) were irradiated with single heavy ions ( e.g. , au , u ) with a kinetic energy of ~2 gev . irradiation was performed at the linear accelerator unilac at the institute for heavy ions research ( gsi ) , darmstadt , germany ( spohr 1983 ) . after tracking , foils were etched from one side in 9 m naoh to form conically shaped nanopores ( apel et al . the other side of the membrane was protected against etching by an acidic stopping solution . the preparation was performed in a conductivity cell so that the pore opening could be observed electrochemically . once an etching current of ~200 pa was recorded , the membrane was washed with the stopping medium and kcl solution . the transport properties of single pet nanopores were investigated in kcl solutions buffered to ph 8 with tris buffer . an approximate diameter of the small opening of the conical pore was determined using conductance measurements as described previously ( apel et al . , both sides of a single nanopore membrane were placed in contact with 1 m kcl . ag / agcl electrodes were immersed into each solution , and a current voltage curve was recorded for voltages between + 100 and 100 mv . in this voltage regime the nanopores produce a linear current voltage curve whose slope provides the ionic resistance ( rp ) of the nanopore . the tip diameter ( d ) is related to rp via \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_{p } = { \frac{4\rho l}{\pi dd } } $ $ \end{document } , where l is the membrane thickness , is the specific ionic resistance of the electrolyte and d is the base ( big opening of the cone ) diameter . the diameter d was determined from the so - called bulk - etch rate of a polymer material . for pet and etching conditions of 20c and 9 m naoh , the bulk - etch rate is equal to 2.13 nm / min ( apel et al . 2001 ) . the value of d was then determined from the formula d = 2 2.13 t ( nm ) , where t is time of etching in minutes . the validity of this relation was confirmed by scanning electron microscopy studies ( wharton et al . pore walls and the surface of track - etched pet films contain carboxyl groups at a density of ~1 group / nm ( wolf - reber 2002 ) . the carboxyl groups were the attachment points to covalently couple amino - modified dna using a procedure which had been previously applied for other surface modification purposes . in the procedure , carboxyl groups are activated with 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride ( edc ) and then coupled to the amine , forming an amide ( vlassiouk and siwy 2007 ) . a dna oligonucleotide with a 5-terminal amino c12 spacer and the sequence 5-cgc gag aagtta cat gac ctg tag acg atc-3 was purchased from integrated dna technologies ( coralville , ia ) . the received amount of 1,267 nm was dissolved in 500 l of 0.1 m mes buffer , ph 5.5 . in the amidation protocol , the dna and edc solution was placed in contact with the small opening of the pore , while the large opening of the pore was in contact with 0.1 m 2-(n - morpholino)ethanesulfonic acid ( mes ) solution , ph 5.5 , for 24-h incubation . placing the modifying agent only on one side of the membrane is known to create a very nonhomogeneous distribution of the chemicals along the pore axis ( vlassiouk and siwy 2007 ) . due to the conical pore shape , the concentrations of edc and dna were high at the first tens of nanometers at the narrow tip and decayed hyperbolically along the pore axis . consequently , dna attachment was expected to occur only at the region close to the small opening where the reagents concentration was sufficiently high . it is expected that our nanopore system has a surface chemistry as schematically shown in fig . 1 . four dna - modified nanopores with opening diameters between 4 and 12 nm were studied . the ionic properties of the nanopores were studied in the same conductivity cell in which the etching was performed . two ag / agcl electrodes were used to apply the transmembrane potential and to measure the ion current . we used a two - electrode setup in which the electrode placed at the narrow end of a conical nanopore was grounded , while the other electrode next to the big pore opening was used to apply a given potential difference with respect to the ground electrode . the voltage was swept between 3 and + 3 v for 1 m kcl , and between 4 and + 4 v for lower concentrations , with a voltage step of 100 mv . the presented current voltage curves are averages of typically three sweeps , unless otherwise stated ( figs . the current variations in the averaged signals typically did not exceed 8% ( the current variations were the strongest for negative voltages above 2 v ) . single conically shaped nanopores were prepared by the track - etching technique according to a published procedure ( fleischer et al . briefly , 12-m - thick films of polyethylene terephthalate ( pet ) were irradiated with single heavy ions ( e.g. , au , u ) with a kinetic energy of ~2 gev . irradiation was performed at the linear accelerator unilac at the institute for heavy ions research ( gsi ) , darmstadt , germany ( spohr 1983 ) . after tracking , foils were etched from one side in 9 m naoh to form conically shaped nanopores ( apel et al . the other side of the membrane was protected against etching by an acidic stopping solution . the preparation was performed in a conductivity cell so that the pore opening could be observed electrochemically . once an etching current of ~200 pa was recorded , the membrane was washed with the stopping medium and kcl solution . the transport properties of single pet nanopores were investigated in kcl solutions buffered to ph 8 with tris buffer . an approximate diameter of the small opening of the conical pore was determined using conductance measurements as described previously ( apel et al . , both sides of a single nanopore membrane were placed in contact with 1 m kcl . ag / agcl electrodes were immersed into each solution , and a current voltage curve was recorded for voltages between + 100 and 100 mv . in this voltage regime the nanopores produce a linear current voltage curve whose slope provides the ionic resistance ( rp ) of the nanopore . the tip diameter ( d ) is related to rp via \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_{p } = { \frac{4\rho l}{\pi dd } } $ $ \end{document } , where l is the membrane thickness , is the specific ionic resistance of the electrolyte and d is the base ( big opening of the cone ) diameter . the diameter d was determined from the so - called bulk - etch rate of a polymer material . for pet and etching conditions of 20c and 9 m naoh , the bulk - etch rate is equal to 2.13 nm / min ( apel et al . 2001 ) . the value of d was then determined from the formula d = 2 2.13 t ( nm ) , where t is time of etching in minutes . the validity of this relation was confirmed by scanning electron microscopy studies ( wharton et al . pore walls and the surface of track - etched pet films contain carboxyl groups at a density of ~1 group / nm ( wolf - reber 2002 ) . the carboxyl groups were the attachment points to covalently couple amino - modified dna using a procedure which had been previously applied for other surface modification purposes . in the procedure , carboxyl groups are activated with 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hydrochloride ( edc ) and then coupled to the amine , forming an amide ( vlassiouk and siwy 2007 ) . a dna oligonucleotide with a 5-terminal amino c12 spacer and the sequence 5-cgc gag aagtta cat gac ctg tag acg atc-3 was purchased from integrated dna technologies ( coralville , ia ) . the received amount of 1,267 nm was dissolved in 500 l of 0.1 m mes buffer , ph 5.5 . in the amidation protocol , the dna and edc solution was placed in contact with the small opening of the pore , while the large opening of the pore was in contact with 0.1 m 2-(n - morpholino)ethanesulfonic acid ( mes ) solution , ph 5.5 , for 24-h incubation . placing the modifying agent only on one side of the membrane is known to create a very nonhomogeneous distribution of the chemicals along the pore axis ( vlassiouk and siwy 2007 ) . due to the conical pore shape , the concentrations of edc and dna were high at the first tens of nanometers at the narrow tip and decayed hyperbolically along the pore axis . consequently , dna attachment was expected to occur only at the region close to the small opening where the reagents concentration was sufficiently high . it is expected that our nanopore system has a surface chemistry as schematically shown in fig . 1 . four dna - modified nanopores with opening diameters between 4 and 12 nm were studied . the ionic properties of the nanopores were studied in the same conductivity cell in which the etching was performed . two ag / agcl electrodes were used to apply the transmembrane potential and to measure the ion current . we used a two - electrode setup in which the electrode placed at the narrow end of a conical nanopore was grounded , while the other electrode next to the big pore opening was used to apply a given potential difference with respect to the ground electrode . the voltage was swept between 3 and + 3 v for 1 m kcl , and between 4 and + 4 v for lower concentrations , with a voltage step of 100 mv . the presented current voltage curves are averages of typically three sweeps , unless otherwise stated ( figs . the current variations in the averaged signals typically did not exceed 8% ( the current variations were the strongest for negative voltages above 2 v ) . figure 2a presents a set of current voltage curves of a single conically shaped nanopore with small and big openings of 8 and 400 nm , respectively . a positive voltage in our electrode configuration corresponds to a positively biased electrode placed at the big opening of a conical nanopore , and vice versa for a negative voltage . similar to previous reports , the conical nanopore rectified the ion current due to breaking of the symmetry of the electric potential inside the pore ( siwy and fulinski 2002 ; hanggi and marchesoni 2009 ; cervera et al . 2005 ) . in particular , the broken symmetry affected the interactions between k ions and the negatively charged carboxyl groups on the pore walls . the rectification was quantified by calculating the so - called rectification degree , which is a ratio of currents for voltages of the same absolute value but of opposite polarity , e.g. , i(3 v)/i(+3 v ) . the negatively charged nanopores have transference numbers found from the reversal potential ( hille 2001 ) of at least 0.8 , which indicates that ~80% of the current is carried by potassium ions ( cervera et al . the larger currents for negative voltages correspond to cations moving from the small opening to the large opening of the pore . the experimentally found ion current rectification and selectivity properties of conically shaped nanopores were modeled using the poisson - nernst - planck ( pnp ) equations ( cervera et al . 2005 , 2006 , 2007 ; white and bund 2008 ) as well as monte carlo ( he et al . figure 2b presents average ionic concentrations along the pore axis for + 1 and 1 v , 0.1 m kcl , obtained bynumerically solving the pnp equations , as described ( vlassiouk et al . 2009 ) . at negative voltages , ionic concentrations of both ions , potassium and chloride , are significantly higher than at the corresponding positive voltage , thereby validating that the negative voltage corresponds to the high conductance state . given that the concentration of cations is higher than that of anions for negative and positive potentials , the plot in fig . the modeling furthermore revealed that in the case of conically shaped nanopores , the quantitative estimation of cation selectivity ( based on the reversal potential measurements ) holds true only for low voltages . at high negative potentials 2b ) , indicated by an increase in the concentration of both potassium and chloride ions within the pore ( cervera et al . 2006 2a current voltage curves of a single conically shaped nanopore with openings of ~8 and 400 nm , recorded at ph 8 for three kcl concentrations as indicated . insets show the position of the electrical ground and external voltage relative to the two pore openings . b numerical solutions of the pnp equation for a conically shaped nanopore with surface charge density of 0.5 e / nm showing average ionic concentrations along the pore axis . opening diameters of the modeled nanopore were set at 5 and 500 nm a current voltage curves of a single conically shaped nanopore with openings of ~8 and 400 nm , recorded at ph 8 for three kcl concentrations as indicated . insets show the position of the electrical ground and external voltage relative to the two pore openings . b numerical solutions of the pnp equation for a conically shaped nanopore with surface charge density of 0.5 e / nm showing average ionic concentrations along the pore axis . 2009 ) . opening diameters of the modeled nanopore were set at 5 and 500 nm it is well known that the pores rectify only if the walls have excess surface charge and when the pore diameter is comparable to the thickness of the electrical double - layer ( siwy and fulinski 2002 ; cervera et al . 2006 ) . as a consequence , the rectification degree of the conically shaped nanopores in 1 m kcl was lower than that in 0.1 m and 10 mm kcl . 2a ) 2.7 ( |15.4| na , 5.68 na ) in 1 m , 4.7 ( |8.32| na , 1.75 na ) in 0.1 m kcl and 4.9 ( |4.08| na , 0.828 na ) in 10 mm kcl . the small difference in rectification degrees between 0.1 m and 10 mm kcl is in agreement with predictions based on the pnp equations , which point to a nonlinear dependence of the rectification degree on kcl concentration ( cervera et al . the modeling also showed the optimal ratio between the pore diameter and the screening length that produces the highest rectification degree . for a 6-nm - diameter conical pore , the concentration range at which the pore was predicted to rectify most was between ~0.12 and 0.40 m kcl ( cervera et al . the optimal concentration range is shifted to more diluted solutions ; and in the 8-nm pore studied here , at 10 mm and 0.1 m kcl the pore rectified almost equally well . a very different behavior was observed after modifying the tip of the same nanopore with 30-nucleotide - long ssdna . figure 3 compares current voltage curves of the pore before and after dna modification for 1 m , 0.1 m and 10 mm kcl . for all these concentrations , the dna modification caused a significant decrease of the current , which suggests that the long dna molecules physically occluded the pore and diminished its effective opening diameter.fig . 2 after modification with 30-nucleotide ssdna for a 1 m kcl , b 0.1 m kcl and c 10 mm kcl ( two sweeps from 4 to + 4 v are shown for the dna - modified nanopore ) . red curves in d present three consecutive reverse sweeps of voltages from + 4 to 4 v , showing a big variation between the sweeps and hysteresis compared to the forward sweeps from 4 to + 4 v ( shown in gray ) . in 1 and 0.1 m kcl , no differences between currents recorded for forward and reverse voltage sweeps were observed current voltage curves of the same nanopore studied in fig . 2 after modification with 30-nucleotide ssdna for a 1 m kcl , b 0.1 m kcl and c 10 mm kcl ( two sweeps from 4 to + 4 v are shown for the dna - modified nanopore ) . red curves in d present three consecutive reverse sweeps of voltages from + 4 to 4 v , showing a big variation between the sweeps and hysteresis compared to the forward sweeps from 4 to + 4 v ( shown in gray ) . in 1 and 0.1 m kcl , no differences between currents recorded for forward and reverse voltage sweeps were observed the experimental results suggest that occlusion of the pore by dna becomes more pronounced with lowering the ionic strength . at 1 m kcl , attachment of dna caused a decrease of ~2.5-fold of ion current compared to a non - modified pore ( fig . 3a ) , while at 10 mm kcl , the dna - modified nanopore exhibited at least five times lower current ( fig . any explanation of the observed current change has to consider two effects which were initially used to describe experiments performed with non - attached pore - translocating dna strands ( smeets et al . firstly , each dna molecule excludes a certain volume in the pore so that fewer ions are available for transport . secondly , dna molecules are heavily charged ; thus , their presence brings more counter - ions to the pore lumen . insertion of dna molecules in the pore can therefore cause a decrease or an increase of the current , depending on whether the number of excluded ions is larger or smaller than the number of additionally introduced dna counterions . both effects have been observed in systematic experiments on threading single dna molecules through unmodified nanopores ( smeets et al . 2006 ) . in dependence of the concentration of the background electrolyte , the pore - threaded dna molecules caused a positive or negative spike of the current . in all reported cases , lowering the kcl concentration of the background electrolyte caused the dna counter - ions to become dominant . our observation that a decrease in kcl concentration leads to an increase of the pore blockage suggests that a simple explanation based on volume exclusion and electrostatic effects is insufficient . it is more likely that the transmembrane current is additionally influenced by changes in the conformation of the dna molecules . this effect has recently been observed with dna molecules attached to gold surfaces ( kaiser and rant 2010 ) . in the experiments , the background electrolyte concentration caused the dna molecules to be more extended , and in concentrations ~1 mm the molecules became entirely stretched . the length of the dna strand was determined by applying a negative potential to the gold surface and determining the quenching of a fluorophore which was attached to the other dna end . these results were explained by considering the dependence of the mechanical rigidity of ssdna on the ionic strength of the environment . theories of odijk ( 1977 ) as well as skolnick and fixman ( 1977 ) distinguish bare ( l0 ) and electrostatic ( le ) contributions to the total persistence length of a charged molecule lp : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ l_{p } = l_{0 } + l_{e } $ $ \end{document}. the bare persistence length of ssdna is known to be only 23 nm ( tinland et al . 1997 ) , while the value of le is ionic strength dependent and can be calculated as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ l_{e } = { \frac{{l_{b } } } { 4}}\lambda^{2 } l_{d}^{2 } $ $ \end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ l_{b } = { \frac{{e^{2 } } } { { 4\pi \varepsilon \varepsilon_{0 } k_{b } t } } } $ $ \end{document } is the bjerum length , is the linear density of the polymer and ld stands for the screening length given by the debye - hueckel theory . the parameters kb and t have their usual meaning of the boltzmann constant and temperature , respectively . the length le becomes important only at lower concentrations ; e.g. , at 10 mm kcl it equals ~10 nm . applied to our dna - modified pore , this high length obviously increases the volume of the dna strands and could thereby help explain the high level of occlusion observed for the nanopore which measures 8 nm in diameter ( fig . it is also important to mention that for the lowest studied kcl concentration of 10 mm , the current voltage curves exhibited a strong hysteresis , especially for negative voltages ( fig . when the voltage sweep started at negative voltages , the currents were typically significantly lower than in the case when the sweep started from positive voltages . we think that the hysteresis is caused by the voltage - induced movement of the attached dna strands as well as voltage - dependent ionic concentrations , as discussed above . figure 4 summarizes a model which accounts for the observed hysteresis and high currents for a sweep from positive to negative transmembrane potentials . when a positively biased electrode is placed at the wide opening of the pore , the dna molecules are expected to be deflected away from the narrow opening and toward the wide opening , which does not present any significant steric hindrance for the strands ( fig . 4 , region a ) . due to the conical shape of the pore , this voltage polarity constitutes the lower conductance state of the device . reducing the positive voltage and switching it to the negative values forces the dna strands to deflect toward the small opening of the pore ( fig . 4 , region b ) . it is recalled that at negative voltages concentrations of potassium and chloride ions in the region close to the small pore opening increase at least several times compared to the bulk concentration ( fig . this region with elevated ionic concentration is rather wide and reaches several hundreds of nanometers away from the narrow opening . consequently , the dna strands in the modified pore are in contact with high concentrations of potassium and chloride ions , which in turn likely causes dna to assume a more compact form ( fig . 4 , region b)in line with results from kaiser and rant ( 2010 ) . at moderate negative voltages the currents through the pore are large , leading to high rectification degrees . at negative voltages above a threshold value of ~2 3d , 4 ) , dna molecules might become extended due to the electric field ( heng et al . 2005 ; randall et al . 2006 ) so that the nanopore is again more blocked ( fig . 4 , region c).fig . 3d ) for an 8-nm - diameter nanopore modified with dna , recorded when the voltage was changed from + 4 to 4 v. insets schematically indicate possible configurations of the attached dna molecules as a function of applied voltage two current voltage curves ( the same as shown in fig . 3d ) for an 8-nm - diameter nanopore modified with dna , recorded when the voltage was changed from + 4 to 4 v. insets schematically indicate possible configurations of the attached dna molecules as a function of applied voltage by contrast , when the voltage is swept from negative to positive values , dna molecules have to deflect from the small opening toward the large opening of the pore . we think that this process occurs with significantly more severe steric hindrance because lowering the negative applied voltage decreases the ionic concentrations in the pore , causing the dna to maintain a more elongated form induced by the high voltage , thereby leading to a greater pore blockade . in order to support our general hypothesis on the conformation dependent occlusion of nanopores by dna molecules we expected that in this case the dna would lead to a less extensive pore blockade based on geometric grounds . figure 5 shows the current voltage curves of the 12-nm pore in 0.1 m kcl , ph 8 , before and after dna attachment . 3 ) , the wider structure is characterized by not only a less extensive blockade but also higher negative currents after the dna modification . the larger current after dna modification suggests , according to smeets et al . ( 2006 ) , that the number of dna counter - ions exceeds the number of ions that are excluded from the nanopore . most likely , dna attachment increases the local surface charge density and consequently enhances ionic concentrations at the narrow opening.fig . 5a current voltage curves before and after modification with 30-mer ssdna of a single conically shaped nanopore with a narrow opening of 12 nm . b current voltage curves of 12-nm dna - modified nanopore in 10 mm kcl , ph 8 , for forward bias ( gray squares ) and reverse bias ( red squares ) a current voltage curves before and after modification with 30-mer ssdna of a single conically shaped nanopore with a narrow opening of 12 nm . b current voltage curves of 12-nm dna - modified nanopore in 10 mm kcl , ph 8 , for forward bias ( gray squares ) and reverse bias ( red squares ) the wider nanopore also exhibited an ion current rectification degree in 0.1 m kcl , 4 v equal to almost 17 , which is three times higher than the rectification degree of the 8-nm dna - modified pore ( fig . this observation was at first puzzling to us as one would expect a higher rectification degree for a narrower pore . one possible explanation is that the dna conformational change makes the pore structure less asymmetric and less rectifying . another explanation takes into account the dependence of the rectification degree of a conical nanopore on ionic strength , which we already discussed above . there is an optimal ratio of pore diameter and screening length for a maximal rectification degree ( cervera et al . 2006 ) . for concentrations that are lower than the optimum for a given pore diameter , with dna , the nanopore is effectively smaller so that the pore might be outside its optimal rectifying regime . as expected , for the wider conical nanopore in 10 mm kcl , the hysteresis of ion currents recorded for forward and reverse voltage sweeps was much less pronounced compared to the 8-nm nanopore ( see figs . we have prepared an ionic rectifying system based on single conically shaped nanopores whose small opening was modified with ssdna . in contrast to previously designed bipolar and unipolar diodes ( vlassiouk and siwy 2007 ; karnik et al . 2007 ) containing stationary surface charges , the attached dna molecules are flexible and assume different configurations depending on the operation conditions such as kcl concentration and voltage . our experimental results provide evidence that the level of nanopore blockage and , most likely , the persistence length of tethered dna increase with lower kcl concentration . due to the salt - dependent dna volume , the nanopore changes its effective diameter and thus can be applicable in building systems to control transport of both charged and neutral species . in future research , we will systematically study the transport properties of dna - modified pores as a function of dna length . we expect to be able to tune the rectification properties of the pores as well as the effective pore diameter as a function of external voltage . we will also try to identify a particular ratio of dna length and pore diameter under which dna - modified nanopores exhibit spontaneous openings and closings of the pore similar to biological voltage - gated channels . skin is the largest sensory organ of the body and is densely equipped with highly specialized sensory nerve endings capable of sensing a wide range of sensory stimuli such as light touch , mechanical pressure , temperature , itch , and pain . these cutaneous sensory afferents can be distinguished by many morphological , anatomical , electrophysiological , and molecular criteria ( liu and ma , 2011 , owens and lumpkin , 2014 ) . for example , based on their cell body size , degree of myelination , and axonal conduction velocity , cutaneous fibers can be split into a , a , or c - fibers ( abraira and ginty , 2013 , bessou and perl , 1969 , li et al . , 2011 , smith and lewin , 2009 , zimmermann et al . , 2009 ) . they can be split into free nerve endings , found both in glabrous and hairy skin where they are embedded between the keratinocytes of the epidermis , and a particular population of c - unmyelinated fibers , found exclusively in hairy skin where they form longitudinal lanceolate endings around hair follicles ( delfini et al . , 2013 , li et al . , 2011 ) or terminate in large arborizations similar to c - fiber tactile afferent receptive fields ( liu et al . , 2007 , cutaneous free nerve endings are tuned to respond to itch - inducing compounds and pain - evoking thermal , mechanical , and chemical stimuli ( zimmermann et al . , 2009 ) . mrgprd free nerve endings belong to a subset of non - peptidergic nociceptors that convey noxious mechanical and -alanin - induced itch stimuli ( cavanaugh et al . , 2009 , liu et al . , 2012 ) , whereas c - low - threshold mechanoreceptors ( c - ltmrs ) have been proposed to contribute to light touch under normal conditions ( bessou et al . , 1971 , douglas and ritchie , 1957 , johansson et al . , 1988 , lken et al . , 2009 , maruhashi et al . , 1952 , olausson et al . , 2002 , seal et al . , 2009 , zotterman , 1939 ) and to touch hypersensitivity after injury ( delfini et al . , 2013 , liljencrantz et al . , 2013 , seal et al . , 2009 ) . our knowledge of the molecular contents that contribute to the functional specialization of these diametrically different subpopulations of cutaneous afferents is still at its infancy . in a recent study ( gaillard et al . , 2014 ) , we identified a novel g inhibitory interacting protein ( ginip ) that marks two distinct subsets of non - peptidergic neurons : the cutaneous free nerve endings mrgprd neurons ( dong et al . , 2001 ) and the v - glut3- , th- , and tafa4-expressing c - ltmrs ( delfini et al . , 2013 , li et al . , 2011 , seal et al . , 2009 ) . here , we took advantage of the ginip mouse model in combination with isolectin b4 ( ib4 ) cell surface staining and fluorescence - activated cell sorting ( facs ) . we succeeded to purify three distinct populations of drg neurons and subjected their respective total rna contents to rna deep - sequencing technology ( rna - seq ) . as expected , ginip / ib4 neurons showed a remarkable enrichment in mrgprd transcripts and ginip / ib4 neurons revealed a striking enrichment of the three known markers of c - ltmrs : tafa4 , th , and vglut3 . deeper comparison of the rna - seq data revealed distinct transcriptional signatures between mrgprd - expressing neurons and c - ltmrs that were further confirmed by in situ hybridization analysis of over 100 genes . most importantly , in addition to providing the transcriptional signatures of two categories of primary sensory neurons , our study expands the molecular characterization of c - ltmrs and suggests that this particular subset of primary sensory neurons shares many molecular features of ltmrs . as a functional readout , we used electrophysiological recording to unravel the specific and exclusive functional expression of the low - voltage - gated ca channel cav3.3 in c - ltmrs , where it very likely plays a key role in shaping their functional specialization . in a recent study , we generated a mouse model that expresses the fluorescent protein m - cherry from the ginip locus ( gaillard et al . , 2014 ) . double - labeling experiments using anti - ginip antibody in combination with ib4 staining showed that drg neurons can be split into four distinct categories : ginip / ib4 ; ginip / ib4 ; ginip / ib4 ; and ginip / ib4 neurons . the ginip / ib4 double - positive ( dp ) population corresponds to the cutaneous free nerve endings mrgprd neurons ( figures 1a and 1b ) , the ginip / ib4 population corresponds to tafa4-expressing c - ltmrs ( figures 1a and 1b ) , the ginip / ib4 population contains the 20% remaining ib4 neurons ( figure 1a ) , and the ginip / ib4 double negative ( dn ) neurons represents a heterogeneous population of neurons composed of peptidergic nociceptors , a subset of ret neurons and trkb and trkc neurons ( figure 1a ) . as ginip mouse model allows high - fidelity expression of m - cherry in ginip neurons , we sought to combine live m - cherry fluorescence with ib4 cell surface staining and facs to purify the four categories of drg cells ( figures 1c and 1d ) . neurons with a cell body size above 70 m were eliminated by filtering the suspension , and axonal debris was removed by using a percoll gradient . negative gating was used to exclude dead or dying cells that incorporated sytox blue dye and cells that were autofluorescent in v500 channel ( figures 1c and s1 ) . next , we gated on m - cherry - positive and m - cherry - negative cells that we further subdivided into ib4 and ib4 subsets ( figures 1c and s1 ) . facs - sorted ib4 and ib4 m - cherry - positive neurons gave rise to expected proportions of cells : about two - thirds were ib4 ( corresponding to ginip / ib4 ) and the remaining one - third was ib4 ( corresponding to ginip / ib4 ; figure 1d ) . the m - cherry / ib4 subset was excluded from the transcriptional analysis , because it was highly contaminated by cd31 ( pecam)-expressing endothelial cells , consistent with ib4 binding on this cell type ( data not shown ) . for the remaining three sorted samples ( hereafter called dp for the ginip / ib4 , c - ltmrs for the ginip / ib4 , and dn for ginip / ib4 ) , we first confirmed by rt - pcr the accurate expression of ginip and m - cherry transcripts ( figure s2b ) and then extended the purity evaluation by analyzing the expression of mrgprd , tafa4 , trka , and trpv1 in each sorted sample ( figure s2c ) . as expected , mrgprd transcripts were highly detected in the dp sample but low in the two others , whereas tafa4 expression was high in c - ltmrs . trka and trpv1 transcripts were enriched in the dn sample , consistent with the enrichment in peptidergic nociceptors in the dn fraction ( figure s2c ) . high - quality rna ( figure s2a ) was extracted from the three purified populations of neurons , and their respective transcriptional profiles were generated using rna - seq . after one round of rna amplification , libraries were built in biological duplicate and sequenced in 100-bp paired - end cycles using the illumina hiseq2000 system with sbs technology . image analysis and base calling were performed using the hiseq control software and rta component from illumina . this approach yielded between 40 and 50 millions of reads that were further submitted to sequence mapping on mouse reference genome mm10 with casava 1.8.2 software . after removal of contaminants ( 28s , 18s , and 5s rrna ; mitochondrial chromosomes ; phix ; illumina controls ; and illumina adaptors ) , reads mapping to multiple splicing positions , and reads with no match , we obtained between 32 and 42 millions of reads that were used for the generation of the raw data file ( table s1 ) . among the first - ranked genes in the raw data file , we found genes known to be related to structural organization of neurons such as tuba1a , prph , sncg , and mtap1b ( table s2 ) . all these genes exhibited strong expression in the three sorted samples , as they generated at least 50,000 reads ( table s2 ) . accordingly , they were highly and broadly expressed in drg neurons in allen - brain atlas database . interestingly , genes associated with cellular stress such as hsp90ab1 and fos were also highly expressed in all samples , very likely due to the cell dissociation and facs process ( table s2 ) . most importantly , quick analysis of the reads per kilobase per million of reads ( rpkm ) generated for genes expected to be enriched in each sorted subpopulation revealed high enrichment of mrgprd and p2rx3 in the dp subset , th and tafa4 in c - ltmrs , and cgrp and sp in the dn neurons ( figure 1e ) , providing the first proof of concept of the successful outcome of our experimental paradigm . to identify genes that were preferentially enriched in one sample , we carried a pairwise comparative analysis ( 2-fold enrichment ; p < 0.01 ) after libraries normalization with the trimmed mean of m values method ( tmm ) ( provided by edger statistical package ) . then , for each subset , we extracted the list of genes that were upregulated in one subset as compared to the two others . we found 486 genes enriched in dp subset , 549 in c - ltmrs , and 2,916 in dn subset ( tables s3 , s4 , and s5 ) . these genes encode a variety of molecules including ion channels , receptors , signaling molecules , and previously uncharacterized genes . because not all neuronal subsets were included in our facs strategy and because facs of acutely dissociated neurons may induce cellular stress that impacts on gene expression , the only follow - up strategy that could be employed to validate the specificity of our data sets was in situ hybridization ( ish ) approach on fixed adult drg sections . knowing that we might miss genes that are expressed in subsets of our sorted neuronal subpopulations , we assumed that , below 1,000 number of reads , transcripts detection by ish would be difficult to monitor . therefore , we carried out a second pairwise comparative analysis by considering only the genes with values above 1,000 reads ( figure 2a ) . as a result , we obtained 156 transcripts preferentially expressed in the dp subset , 184 in c - ltmrs , and 784 in the dn population ( figure 2b ; tables s6 , s7 , and s8 ) . we then monitored the expression of 48 dp- , 68 c - ltmrs- , and 13 dn - enriched genes using ish followed by ginip immunostaining and ib4 labeling ( for the dp and c - ltmrs candidate genes ) or ginip and trka immunolabeling ( for the dn candidate genes ) . these triple - labeling experiments provided an unambiguous and accurate expression pattern of each tested gene . the 13 dn - enriched tested genes revealed two different expression patterns : genes mainly expressed in neurons and genes exclusively expressed in non - neuronal cells that harbor satellite and/or glial cell morphology ( figure s3 ) . in agreement with our rna - seq data , genes expressed in neurons were excluded from dp neurons and c - ltmrs . furthermore , we found 6/13 genes expressed in trka peptidergic neurons and others ( 2/13 ) excluded from this neuronal population , revealing the heterogeneity of the dn subset ( figure s3 ) . because the aim of this study consisted at deciphering the transcriptional signatures that confer the functional specializations of mrgprd - expressing cutaneous free nerve ending and c - ltmrs , we analyzed the expression patterns of 48 dp - enriched genes and 68 c - ltmtrs - enriched genes . in line with our rna - seq data sets , the 48 dp - enriched probes were highly expressed in the dp neurons and virtually excluded from c - ltmrs ( http://www.ibdm.univ-mrs.fr/equipes/reynders/dp-ishlibrary.pptx ) . however , the vast majority of the dp - enriched genes displayed an additional expression pattern outside dp neurons . 12 genes showed some expression in few ginip / ib4 neurons , 17 genes showed lower expression in discrete subsets of small ( 3/17 ) or medium - to - large size neurons ( 14/17 ) , and 19 genes were expressed in broader neuronal subsets in addition to intense expression in dp neurons ( figures 2c2e ; dp - ish library ) . the same strategy applied to c - ltmrs - enriched genes showed that all 68 tested genes were expressed in c - ltmrs and massively excluded from dp neurons ( http://www.ibdm.univ-mrs.fr/equipes/reynders/cltmrs-ishlibrary.pptx ) . one striking feature of c - ltmrs - enriched genes is that the vast majority was also expressed in medium - to - large size drg neurons , whereas only two were expressed in small neurons . indeed , out of the 68 tested genes , six were specifically expressed in c - ltmrs , as they only marked a subset of ginip neurons that do not bind ib4 ( figures 3a3f ) . quantitative analyses showed that ceacam10 is expressed in 71.2% 6.3% of c - ltmrs , wfdc2 in 73.4% 1.7% , cacna1i in 89.2% 5.9% , c1ql4 in 70% 1.8% , fbp2 in 19.9% 4.7% , and bok in 62.7% 6.4% of c - ltmrs ( figure 3 g ) , further highlighting our previously suggested molecular heterogeneity of this particular class of neurons ( delfini et al . the remaining 60 genes could be split in three categories : 18 genes exhibited high expression in c - ltmrs but also marked a discrete subset of large - diameter neurons , 23 genes showed equal levels of expression in c - ltmrs and in large - diameter neurons , and 19 genes exhibited a very high enrichment in larger - diameter neurons and a discrete expression in c - ltmrs ( figures 4a4c ; cltmrs - ishlibrary ) . because 88% of c - ltmrs - enriched genes displayed an additional expression preferentially in large - diameter neurons , we sought to unravel the molecular identity of these neurons . to do so , we performed double ish using nf200 antisense probe in combination with selected c - ltmrs - enriched probes followed by trka immunolabeling . this triple - staining experiment showed that all the selected probes were massively excluded from trka peptidergic neurons and expressed in subsets of nf200 neurons ( figure s4 ) . knowing that nf200 is a hallmark of proprioceptors and ltmrs ( bourane et al . , 2009 , li et al . , 2011 , luo et al . , 2009 , wende et al . , 2012 ) , these data suggest that c - ltmrs share some molecular features of ltmrs . mrgprd - expressing neurons and c - ltmrs exhibit fundamental differences both at anatomical and functional levels . however , little is known about the molecular contents that instruct their respective functional specializations . in line with this , we pooled dp and c - ltmrs - enriched genes according to their biological functions by searching for statistically over - represented associations with gene ontology ( go ) terms , using gorillla database ( figures 5 , 6 , and s5 ) . this analysis yielded , respectively , 20 and 33 functional annotations for dp and cltmrs . dp neurons exhibited a high enrichment in g - protein - coupled receptors as well as in transcripts encoding signaling molecules , such as gna14 , gna1 , gnaq , plcb3 , prkca , prkcd , and prkcq ( figures 5a and s5a ; table s9 ) , whereas c - ltmrs exhibited a striking enrichment in transcripts associated with voltage - gated ion channel activity ( figures 6a and s5b ; table s10 ) . detailed analysis of genes associated with g protein receptor binding and signal transducer activity led to the identification of two transcripts encoding for g - protein - coupled receptors , namely the angiotensin receptor type 1a ( agtr1a ) and the prostaglandin receptor d2 ( ptgdr ) ( figure 5a ) . our ish experiments showed that agtr1a is expressed in dp neurons , but not in c - ltmrs or other subsets of drg neurons ( figure 5b ) , whereas ptgdr was found at higher intensity in dp neurons compared to c - ltmrs and large - size neurons ( figure 5c ) . in addition to gpcrs , go analysis revealed a striking enrichment of transcripts encoding signaling molecules in dp neurons , including gna14 , gna1 , gnaq , plcb3 , prkca , prkcd , and prkcq ( table s9 ) . our in situ screen showed that all these transcripts were highly expressed in dp neurons and virtually absent in c - ltmrs ( figures 5d5 g ) . the restricted enrichment of such number of signaling molecules likely endows dp neurons with the molecular machinery required to sense and transduce injury - induced pain hypersensitivity . regarding c - ltmrs , one of the most striking observations with respect to the go analysis was the high enrichment in transcripts associated with voltage - gated ion channel activity . other physiologically pertinent functional annotations encompass substrate - specific channel , ion transmembrane transporter , and g - protein - coupled receptor activities ( figures 6a and s5b ) . voltage - gated ion channels included potassium channels ( kcnq2 , kcnj12 , kcnv1 , kcna2 , and kcna4 ) , sodium channels and associated beta subunits ( scn1a , scn1b , scn3b , scn4b , scn7a , and scn8a ) , and low - voltage activated calcium channels ( cacna1h and cacna1i ; figures 6a and s5b ; table s10 ) . ish screen showed that cacna1i is specifically expressed in c - ltmrs ( figure 3c ) and kcnj12 and cacna1h are expressed in c - ltmrs as well as in some large - diameter neurons but excluded from dp neurons ( figures 6b and 6c ) . scn4b and scn8a exhibited low expression in c - ltmrs , very high expression in myelinated large neurons , and no detectable signal in dp neurons ( c - ltmrs - ish library , slides 67 and 68 ) . the combinatorial enrichment of such channels in c - ltmrs is likely to explain the tonic and repetitive firing properties of this particular class of neurons ( delfini et al . interestingly , substrate - specific channels / ion transmembrane transporters encompassed anoctamin 6 ( ano6 ) and solute carrier 17a2 ( slc17a2 ) . ano6 is part of outwardly rectifying chloride channels ( martins et al . , 2011 ) , and slc12a7 encodes for the k / cl co - transporter kcc4 ( boettger et al . , 2002 ) , suggesting that both channels could be involved in membrane excitability . in situ analysis of ano6 and slc12a7 showed that both transcripts were expressed in c - ltmrs , but not in dp neurons , and exhibited some expression in large drg neurons ( figures 6d and 6e ) . gpcr - activity - related transcripts were also represented in go analysis of c - ltmrs data set , including cholecystokinin receptor a ( cckar ) and purinergic receptor y1 ( p2ry1 ) . our ish data showed that both cckar and p2ry1 transcripts were highly enriched in c - ltmrs , totally excluded from dp neurons , and displayed expression in some large - diameter neurons ( figures 6f , 6 g , and s4b ) . together , our data provide a functional description of sets of genes differentially expressed in dp versus c - ltmrs . the differential expression of such clusters of genes likely contributes to differences in signal transduction , intrinsic membrane excitability , and the firing properties of c - ltmrs and mrgprd neurons . our data also provide a predictive basis for deciphering the mechanisms by which specific external stimuli and downstream - associated signaling pathways fine - tune the activity of these two neuronal subsets . our rna - seq data revealed high enrichment of cacna1h ( cav3.2 ) and cacna1i ( cav3.3 ) in c - ltmrs . our ish showed that transcripts encoding both channels were expressed in c - ltmrs with cav3.3 being exclusively expressed in this neuronal subset ( figures 3e and 7a ) . to explore whether both channels were functional in c - ltmrs , we used whole - cell patch - clamp recordings on primary culture of heterozygous m - cherry - expressing neurons in the presence of alexa 488 ib4 live staining to distinguish between c - ltmrs and dp neurons . in ionic conditions designed to isolate calcium currents , current - voltage relationships showed inward low - voltage - activated ( lva ) and high - voltage - activated ( hva ) currents in all recorded c - ltmrs soma , confirming the systematic presence of t - type currents in these neurons ( figures 7b and 7c ) . in sharp contrast , dp neurons did not express any lva current in agreement with the absence of cav3 transcripts in this population ( figures 7b and 7c ) . to test whether cav3.2 and cav3.3 were functional in c - lmtrs , we took advantage of cav3.2 high sensitivity to nickel ions ( ni ) ( lee et al . , 1999 ) . blockade of t - type calcium currents in c - ltmrs by low concentration of ni ( 30 m ) revealed a ni - insensitive current with slow kinetics of activation and inactivation typical of cav3.3 channel ( figure 7d ; chemin et al . , 2002 ) . in contrast , the ni - sensitive fraction obtained by subtraction revealed faster kinetics with an explicit criss - cross pattern typical of cav3.2 ( figure 7d ) . to date , zinc ( zn ) is the only available pharmacological tool that interferes with the gating kinetics of cav3.3 ( cataldi et al . , 2007 , traboulsie et al . , 2007 ) . after blockade of cav3.2-like fraction by ni , subsequent application of zn ( 100 m ) slowed down the remaining native cav3.3-like current with a prominent slowing of the deactivation ( figure 7e ) . analysis of several c - ltmr soma revealed that cav3.3-like currents account for a third of the total amplitude of c - ltmrs t - type calcium currents ( figure 7f ) . the dramatic slowdown of the deactivation kinetics by zn further shows that cav3.3 is a unique feature in the portfolio of ion channels specific to c - ltmrs ( figure 7 g ) . to test whether these two t - type calcium channel isoforms impact c - ltmr excitability , we performed current - clamp experiments . in neurons , t - type calcium channels are well known to generate rebound low - threshold spikes ( lts ) following a transient hyperpolarization . in c - ltmrs , membrane depolarization triggered small lts when the membrane is held at more and more negative values ( figure 7h ) . inhibition of cav3.2 by ni ( 50 m ) did not significantly alter lts and action potential ( ap ) threshold , whereas potentiation of cav3.3 by zn ( 100 m ) increased both lts amplitude and kinetics ( figure 7h , inset ) . this resulted in an increased excitability with a lowering of ap activation threshold crowning the potentiated lts ( figures 7h and 7i ) . in two cells out of ten , the sole application of zn at the resting potential of c - ltmrs elicited a depolarization sufficient to trigger spontaneous ap trains ( figure 7j ) . application of ni in these conditions did not produce any change in membrane potential ( not shown ) . to exclude trpa1 channel contribution , also activated by zn , these experiments were performed in presence of trpa1 blocker , hc030031 ( 10 m ) . together , our electrophysiological data nicely corroborate the rna - seq and the ish expression data , demonstrating that both cav3.2 and cav3.3 channels are concomitantly expressed in c - ltmrs and that both channels are absent in the free nerve ending mrgprd - expressing neurons . in this study , we used our recently generated mouse model in which the cutaneous mrgprd - expressing neurons and c - ltmrs were genetically marked by the m - cherry protein , in combination with ib4 live staining followed by cell sorting and rna deep sequencing to gene profile three categories of primary sensory neurons : the free nerve ending mrgpd - expressing neurons , c - ltmrs , and a heterogeneous population of drg cells that neither bind ib4 nor are labeled by the m - cherry protein . we provide a digital measure of the presence and prevalence of transcripts from known and previously unknown genes for each of the three neuronal subsets . because the dn population is composed of a variety of neuronal subsets and glial cells , dn data set was used to identify genes specifically enriched in the free nerve ending mrgprd - expressing neurons and those specifically enriched in c - ltmrs . this analysis yielded 156 genes specifically enriched in the dp neurons and 184 in c - ltmrs . these genes encode a variety of molecules including ion channels , receptors , signaling molecules , and previously uncharacterized genes . a major concern with any gene - profiling experiment is the follow - up procedure . which genes should one pursue , and what should be done with these lists of genes ? the answer to this question was motivated by our objectives to expand the molecular characterization of c - ltmrs and to identify the combinatorial expression of the key genes that shape the functional specialization of the free nerve endings mrgprd neurons and c - ltmrs . mrgprd neurons are required for acute and injury - induced mechanical pain as well as for the sensation of itch ( cavanaugh et al . 2012 , shields et al . , 2010 ) , whereas c - ltmrs were postulated to convey light touch under physiological conditions and mechanical and chemical pain after injury ( abraira and ginty , 2013 , delfini et al . , 2013 , our rna - seq data in combination with ish and gene ontology analysis revealed a great number of genes expressed in dp neurons , but not in c - ltmrs , and vice versa , reflecting their anatomical and functional differences . indeed , dp neurons showed very high enrichment in genes encoding gpcrs and signaling molecules in addition to a selective expression of three isoforms of the protein kinase c family . the enrichment of such receptors and signaling molecules is in agreement with the involvement of dp neurons in acute and injury - induced mechanical pain and itch ( cavanaugh et al . we show that dp neurons express high levels of agtr1a and ptgdr , suggesting that the pro - nociceptive effect of their respective agonists , angiotensin ii and prostaglandin d2 , likely occurs through dp neurons activation . pgd2 has been shown to enhance neuronal excitability by increasing the amplitude of ttx - sensitive currents ( ebersberger et al . , 2011 , nakae et al . , 2005 ) , and continuous perfusion of angiotensin ii enhanced tactile and thermal hypersensitivity in the setting of nerve injury via the activation of agtr1a ( pavel et al . , 2013 ) . furthermore , dp neurons exclusively express mrgprd , a gpcr that is activated by -alanine , but not histamine ( liu et al . , 2012 ) , suggesting that dp neurons are part of a histamine - independent itch neural circuit and that these neurons can be targeted for treating clinical itch that is resistant to antihistamines . c - ltmrs on the other hand expressed different gpcrs than those expressed in dp neurons such as cckar and the metabotropic gq p2y1 receptor and showed a striking enrichment in voltage - gated channels including potassium channels ( kcnq2 , kcnj12 , kcnv1 , kcna2 , and kcna4 ) , sodium channels and associated beta subunits ( scn1a , scn1b , scn3b , scn4b , scn7a , and scn8a ) , and the low - voltage - gated calcium channels ( cacna1h and cacna1i ) . it has been shown that cckar was restricted to 10% of neurons that do not overlap with peptidergic cgrp nociceptors ( broberger et al . , 2001 ) . our rna - seq and ish data demonstrate that cckar is expressed in c - ltmrs and excluded from dp neurons . cholecystokinin octapeptide , which in its sulfated form binds the gq/11-coupled cckar , is involved in regulation of a large variety of physiological functions among which opioid - induced anti - nociception ( suh et al . , 1995 ) . electrophysiological studies in rat drg neurons have shown that activation of cckar evokes membrane depolarization , indicating that cckar mediates excitatory responses ( ma et al . , 2006 ) . psychophysiological studies in human using cholecystokinin octapeptide could lend new insights into the role of c - ltmrs in touch sensation under normal and pathological conditions . c - ltmrs also exhibited high expression of the metabotropic gq p2y1 receptor , whereas dp neurons mainly expressed the ionotropic purinergic receptor p2x3 , demonstrating that , in dp neurons , atp / adp compounds are sensed through p2x3 , whereas in c - ltmrs , these signaling intermediates are likely detected though metabotropic gq p2y1 receptor . our data also highlighted the specific and the functional enrichment of the lva t - type channels in c - ltmrs . among the three t - type channels , we found cav3.2 and cav3.3 highly present in c - ltmrs , with cav3.3 being exclusively expressed in this particular class of neurons . t - type currents with slow kinetics and resistance to nickel block have been described in a subset of small - sized rat drg and trigeminal neurons expressing slowly adapting mechano - activated currents ( coste et al . our rna - seq data in combination with ish and electrophysiological recordings provided a clear demonstration that cav3.3 is a specific marker of c - ltmrs and offers a new possible selective pharmacological target to control c - ltmrs excitability in mice . although zinc has plethoric effects , its use to investigate cav3.3 contribution to c - ltmr excitability shows specificity . particularly , the slowing of lts kinetics is highly correlated to the massive changes in cav3.3 currents . in line with this , the depolarizing effects observed result , at least in part , from the persistent depolarizing zinc - modulated cav3.3 current . whereas the contribution of other zinc targets remains possible , trpa1 effect has been eliminated by the use of its specific blocker , and a possible inhibition of two pore potassium task3 channel ( kcnk9 ) is unlikely because the transcript is not present in c - ltmrs ( our study ; usoskin et al . , 2015 ) . mechanistically , our findings suggest a substantial contribution of cav3.3 to rebound firing reminiscent of those observed in nuclear reticularis thalamic neurons that , like c - ltmr , express both cav3.2 and cav3.3 and with a prominent cav3.3 effect on burst firing ( astori et al . , 2011 ) . finally , a recent elegant study using an unbiased large - scale single - cell rna sequencing revealed 11 molecularly distinct subsets of primary sensory neurons , including the th - expressing c - ltmrs population ( usoskin et al . , 2015 ) . very interestingly , genes enriched in c - ltmrs in our study were similar to those upregulated in the th population described in the usoskin and colleagues study . however , our ish experiments showed that the vast majority of c - ltmrs - enriched genes were also expressed in other neurons , preferentially in the nf200 neurons , further supporting our strategy to validate the specific expression of the identified enriched genes using ish approach . given that the majority of c - ltmrs - enriched genes were also expressed in nf200 neurons and that nf200 is a hallmark of proprioceptive and a and a ltmrs ( abraira and ginty , 2013 ) , we postulate that c - ltmrs share many molecular features with some a and a ltmrs . in line with this hypothesis , studies of the last few years demonstrated that c - ltmrs share many functional and anatomical features of some a and a ltmrs ( abraira and ginty , 2013 ) . c - ltmrs have been described to convey low - threshold mechanical stimuli ( bessou et al . , 1971 , douglas and ritchie , 1957 , johansson et al . , 1988 , , 1952 , seal et al . , 2009 , zotterman , 1939 ) . they are sensitive to skin indentation , respond to sustained mechanical stimuli with slow and intermediate adaptation rates , and form longitudinal lanceolate endings around hair follicles ( abraira and ginty , 2013 , delfini et al . , 2013 , li et al . , 2011 , lou et al . , 2013 , cross comparison of our rna - seq data sets with those of the five nf subsets recently published by usoskin and colleagues ( usoskin et al . , 2015 ) will provide a comprehensive view of the molecular signatures of c - ltmrs and a and a ltmrs . in conclusion , data presented in this study provide a wealth of information about the presence and prevalence of transcripts from known and previously unknown genes in three categories of primary sensory neurons , they highlight the differential expression of a great number of genes in two diametrically opposite classes of cutaneous afferents , the ish screen followed by immunostaining and ib4 binding led to the generation of two open - access libraries that allow monitoring the cellular distribution of 115 genes , they provide the first electrophysiological recording of a low - voltage - gated ca channel exclusively present in c - ltmrs , and they suggest that c - ltmrs share many molecular features with a and a ltmrs . the expression patterns of dn - enriched genes ; the co - expression of c - ltmrs - enriched genes with nf200 neurons ; the supplemental tables enclosing dp- , c - ltmrs- , and dn - enriched genes ; as well as the go analysis are provided in the supplemental information . mice were maintained under standard housing conditions ( 23c , 40% humidity , 12 hr light cycles , and free access to food and water ) . c57bl/6 mice were from charles river laboratories , and heterozygous ginip knockin mice were previously generated in the laboratory ( gaillard et al . , 2014 ) . special effort was made to minimize the number as well as the stress and suffering of mice used in this study . all protocols are in agreement with european union recommendations for animal experimentation . ib4ginip , ib4ginip , ib4ginip , and ib4ginip cells were sorted on facs aria cell sorter ( bd bioscience ) by gating on sytox blue m - cherry for ginip cells and sytox blue m - cherry for ginip cells . drgs cell suspensions from c57bl/6 mouse were used as a negative control of m - cherry fluorescence . cells were sorted directly in ice - cold rlt lysis buffer from rneasy micro kit ( qiagen ) , supplemented with 10% ( v / v ) -mercaptoethanol ( sigma ) , and snap - frozen at 80c before rna extraction . rna - seq libraries were constructed with the truseq rna sample preparation ( low - throughput protocol ) kit from illumina . were performed using the hiseq control software and real - time analysis component provided by illumina . the quality of the data was assessed with fastqc from the babraham institute and the illumina software sav ( sequence analysis viewer ) . demultiplexing was performed using illumina s sequencing analysis software ( casava 1.8.2 ) . the eland_rna module from casava was used to align rna - seq reads to the mus musculus genome ( ucsc mm10 ) with a set of gene - model annotations ( refflat.txt file downloaded from ucsc on october 10 , 2012 ) in order to generate automatically the splice junctions . reads were also aligned to a set of contaminants , including the rrnas , the mitochondrial chromosome , the phix genome ( illumina control ) , and the illumina adapters . then , exon counts were summed by gene and divided by the length of reads ( i.e. , 100 ) to obtain the gene counts . downstream statistical analysis was made on ( 1 ) genes that generated at least ten reads and ( 2 ) genes that generated at least 1,000 reads . differentially expressed genes were identified using the bioconductor ( robinson et al . , 2010 ) package edger ( alexa et al . , 2006 ) version 2.6.2 . data were normalized using the tmm ( robinson and oshlack , 2010 ) normalization factors . genes with adjusted p value less than 1% ( according to the fdr method from benjamini - hochberg ) were declared differentially expressed . genes were then classified using custom rules : ( 1 ) genes declared as differentially expressed in condition a relative to condition b and to condition c and having a fold - change of at least 2 ( absolute value ) in both cases are considered to be specific of condition a. ( 2 ) a gene will be specific of the two conditions a and b simultaneously if it is differentially expressed in a versus c and in b versus c with an absolute fold change > 2 and with an absolute fold change < 2 between a and b. ( 3 ) filtered genes are considered to be specific of none condition . ( 4 ) genes belonging to none of the previous categories are considered to be specific of the three conditions simultaneously . free web access gorilla database was used to perform the go functional annotation of the resulting list of genes as described in eden et al . lumbar drgs were prepared and cultured as described previously ( francois et al . , we incubated cultures with alexa-488-conjugated ib4 ( 1 g / ml ; 5 min at 37c ; invitrogen ) diluted in extracellular solution and washed before recordings . conditions for recordings of voltage - gated calcium currents and of membrane potential were as described in delfini et al . patterns of expression of over 100 genes are enclosed in dp and c - ltmrs ish libraries . for dp , and a. mantilleri performed the in situ analyses , p.m. performed the pcrs for probe synthesis , s.l .

single nanopores attract a great deal of scientific interest as a basis for biosensors and as a system to study the interactions and behavior of molecules in a confined volume . tuning the geometry and surface chemistry of nanopores helps create devices that control transport of ions and molecules in solution . here , we present single conically shaped nanopores whose narrow opening of 8 or 12 nm is modified with single - stranded dna molecules . we find that the dna occludes the narrow opening of nanopores and that the blockade extent decreases with the ionic strength of the background electrolyte . the results are explained by the ionic strength dependence of the persistence length of dna . at low kcl concentrations ( 10 mm ) the molecules assume an extended and rigid conformation , thereby blocking the pore lumen and reducing the flow of ionic current to a greater extent than compacted dna at high salt concentrations . attaching flexible polymers to the pore walls hence creates a system with tunable opening diameters in order to regulate transport of both neutral and charged species . summarycutaneous c - unmyelinated mrgprd+ free nerve endings and c - ltmrs innervating hair follicles convey two opposite aspects of touch sensation : a sensation of pain and a sensation of pleasant touch . the molecular mechanisms underlying these diametrically opposite functions are unknown . here , we used a mouse model that genetically marks c - ltmrs and mrgprd+ neurons in combination with fluorescent cell surface labeling , flow cytometry , and rna deep - sequencing technology ( rna - seq ) . cluster analysis of rna - seq profiles of the purified neuronal subsets revealed 486 and 549 genes differentially expressed in mrgprd - expressing neurons and c - ltmrs , respectively . we validated 48 mrgpd- and 68 c - ltmrs - enriched genes using a triple - staining approach , and the cav3.3 channel , found to be exclusively expressed in c - ltmrs , was validated using electrophysiology . our study greatly expands the molecular characterization of c - ltmrs and suggests that this particular population of neurons shares some molecular features with a and a low - threshold mechanoreceptors .

the endoplasmic reticulum ( er ) is an organelle with crucial biosynthetic and signaling functions in eukaryotic cells . the er is not only the major intracellular calcium ( ca ) storage organelle critically involved in ca homeostasis and ca mediated signaling pathways , but it also provides the environment for the synthesis , folding , and modification of proteins destined to be secreted or embedded in the plasma membrane ( reviewed in [ 1 , 2 ] ) . moreover , the er is the major site for the biosynthesis of steroids , cholesterol , and lipids . proper folding , maturation , and stabilization of the nascent protein in the er require the highly oxidizing and ca - rich er environment , which is essential for the diverse posttranslational and cotranslational modifications , including glycosylation and disulfide bridge formation , to which proteins are subjected after entering the er . these processes are assisted and monitored by several resident chaperones and ca binding proteins , including the glucose - regulated proteins [ such as grp78 or bip ( immunoglobulin heavy - chain binding protein ) ] , calreticulin and calnexin , and several folding enzymes , such as the thioredoxin - like protein disulfide isomerase ( pdi ) . pdi oxidizes cysteine residues in nascent proteins ( i.e. , oxidative folding ) resulting in formation of intra- and intermolecular disulphide bonds , while reduced pdi is in turn oxidized by the thiol oxidoreductase ero1 . ero1 transfers reducing equivalents to molecular oxygen , generating stoichiometric amounts of h2o2 per newly formed disulphide , which is coupled with a depletion of the reduced gluthatione pool . proteins that fail to adopt a correctly folded or native conformation , or a proper oligomeric assembly in case of multisubunit proteins , are retrotranslocated to the cytosol through a process known as er - associated protein degradation ( erad ) , and further degraded by the 26s proteasome . various physiological and pathological conditions , including hypoxia , er - ca depletion , oxidative injury , high - fat diet , hypoglycemia , and viral infections may cause an imbalance between er protein folding load and capacity , leading to the accumulation of unfolded proteins in the er lumen , a condition referred to as er stress . er stress sets in motion an evolutionary conserved and integrated signal transduction pathway known as the unfolded protein response ( upr ) . the upr primarily aims at ameliorating the protein load on the er by coordinating the temporal shut down in protein translation along with a complex program of gene transcription to increase er folding capacity . if this transcriptional program fails to reestablish proper er homeostasis , persistence in er stress induces cell death . severe er stress can cause cell death , usually by activating intrinsic apoptosis . moreover , in order to clear the er from the accumulation of terminally misfolded protein aggregates that can not be degraded by the proteasome , the upr may upregulate the autophagy machinery [ 5 , 6 ] . macroautophagy ( hereafter referred to as autophagy ) is a major lysosomal pathway for the in bulk degradation of cytoplasmic materials , including proteins and damaged organelles , characterized by the sequestration of entire portions of the cytoplasm by a double - membrane bounded vacuole called the autophagosome [ 7 , 8 ] . in spite of its role as a self - digestion mechanism , autophagy is mainly activated to protect against cell death . however , just like in the case of the upr , stimulation of autophagy can under certain circumstances be required to activate the cell death machinery . although both the upr and autophagy can function independently from each other , recent reports show that they may be interlinked and share the functional duality of exerting both a cytoprotective ( under basal or metabolic stress conditions ) and cytocidial activity ( after acute cellular damage ) . tumor cells are bathed in a hostile microenvironment and confronted with chronic metabolic stress conditions that favor the activation of adaptive mechanisms , such as the upr and autophagy [ 10 , 11].moreover , certain promising anticancer regimens have been shown to activate concomitantly er stress and autophagy in cancer cells ( see section 4 ) . the molecular link between the upr and the autophagic response to er stress , and how these stress pathways influence therapeutic outcome , remain largely undefined , making this topic a very important area for future research in cancer therapy . here , we review the molecular mechanisms underlying the emerging connections between the upr and autophagy pathways , and discuss their potential implications in the context of anticancer therapy . the unfolded protein response in mammalian cells is governed by three transmembrane er stress sensors , namely perk ( pkr - like er kinase ) , ire1 ( inositol requiring enzyme 1 ) , and atf6 ( activating transcription factor 6 ) , which are kept in an inactive state by binding to the er chaperone bip , preventing their oligomerization - induced activation . when er homeostasis is perturbed , accumulating misfolded proteins become progressively bound to bip , titrating away bip from interaction with these transmembrane signaling proteins . upon deinhibition and homodimerization these er sensors activate a complex er - to - nucleus signaling pathway that transmits information across the er membrane to an extensive gene - expression program mediated by the activation of downstream transcription factors . the genetic program activated by the upr results in upregulation of the folding machinery along with an expansion of the er lumen and enhanced degradation of terminally misfolded proteins through erad . additional mechanisms include a general translational shutdown as well as the degradation of a select group of secretory mrnas and proteins that are delayed at the translocon , a process also known as pre - emptive quality control . the mechanisms by which upr induction coupled with the failure in reestablishing the er folding capacity leads to cell death and the requirement of upr signaling in autophagy stimulation , are still unsettled questions . furthermore , recent studies have revealed that the er serves as a subcellular platform for the formation of signaling complexes comprising molecular elements of the upr , bcl-2 family members ( both pro- and antiapoptotic ) ( reviewed in ) , and perhaps regulators of autophagy . in the following sections we will discuss current knowledge on the main signaling pathways emanating by each branch of the upr along with their downstream targets ( figure 1 ) . ire is expressed in all cell types and tissues , whereas expression of ire1 is primarily restricted to the epithelial cells of the gastrointestinal tract [ 14 , 15 ] . ire1 is a type i transmembrane protein with an n - terminal luminal sensor domain and a c - terminal cytosolic effector region that contains both kinase and endoribonuclease ( rnase ) domains . in cells undergoing er stress , oligomerization of ire1 results in trans - autophosphorylation and activation of the rnase domains which excise a 26 nt sequence from xbp1u ( unspliced xbp1 ) , producing mature xbp1s mrna ( spliced xbp1 ) . xbp1s encodes an active leucine zipper ( bzip ) transcription factor xbp1s that regulates the transcription of several genes involved in er quality control mechanisms , er / golgi biogenesis , as well as erad components [ 1822 ] and as recently revealed , also genes involved in redox homeostasis and oxidative stress responses . consistent with this , xbp1 deficient cells were found to be more susceptible to exogenous agents causing oxidative stress , such as h2o2 and parthenolide , concomitant with a reduced expression of several antioxidant enzymes including catalase and thioredoxin ( trx1 ) . overexpression of xbp1 restored catalase expression and reduced ros generation after h2o2 , thus implicating a protective role for xbp1 in oxidative stress . intriguingly , this antioxidant effect was mediated by xbp1u ( i.e. , the protein encoded by the unspliced xbp1 mrna ) , whereas xbp1s ( i.e. , the product of ire1 activation ) failed to induce changes in catalase expression in response to ros or following er stress , thus underscoring that ire1 activity is dispensable . although the molecular mechanism underlying the differential function of the unspliced ( xbp1u ) and spliced ( xbp1s ) products of xbp1 is still elusive- and may involve the binding and regulation of selected targets dependent on their relative abundance in different cell types this study highlights a new role for xbp1 in ros signaling , independent of ire1 rnase activity . although ire1 displays intrinsic kinase activity , there are no other substrates known thus far than ire1 itself . however , prolonged activation of ire1 is capable to transmit a map kinase activation cascade . it has been shown that ire1 can serve as a molecular platform for the recruitment of the adaptor protein tnf - receptor associated receptor 2 ( traf2 ) , an e3 ubiquitin ligase , which leads to the activation of apoptosis signal regulating kinase 1 ( ask1 ) , a map3k of the jnk / p38 mapk pathway [ 25 , 26 ] . depending on the cellular context , activation of jnk can either allow the cells to adapt to er stress by initiating autophagy or , as discussed further , promote apoptosis in response to persistent or irrecoverable er stress . like ire1 , perk is a type i transmembrane protein with a luminal sensing domain and a cytosolic kinase domain which becomes activated following dimerization . the resulting transautophosphorylation induces a conformational change that enhances the affinity of perk for eif2 ( eukaryotic initiation factor 2 alpha ) . phosphorylation of eif2 on ser51 by perk results in the rapid shut down of general translation , relieving the protein burden on the stressed er , while the concomitant loss of cyclin d1 arrests the er stressed cells in g1 . in addition , a recent study has shown that eif2 phosphorylation also regulates translation via inhibition of rrna synthesis , coordinately regulating translation and ribosome biogenesis during cellular stress . paradoxically , this translational shutdown leads to the selective translation of the transcription factor atf4 , a member of the bzip family of transcription factors . the perk - eif2-atf4 axis regulates the expression of genes involved in amino acid biosynthesis and transport functions , antioxidant stress responses , and apoptosis . in addition to eif2 , perk also phosphorylates the nuclear factor - e2-related factor 2 ( nrf2 ) . nrf2 is maintained inactive in a cytoplasmic complex with the microtubule associated protein keap1 ( kelch - like ech - associated protein 1 ) . nrf2 phosphorylation promotes its dissociation from keap1 , leading to the nuclear accumulation of nrf2 , binding the antioxidant response element ( are ) in the promotor of genes encoding detoxifying enzymes such as heme oxygenase 1 ( ho-1 ) . in line with these results , it was shown that nrf2 cells are more prone to er stress induced apoptosis . likewise , perk cells , along with an impaired attenuation of protein synthesis , were found to mount a high amount of endogenous peroxides preceding apoptotic induction in response to agents causing perturbation of er functions . interfering with ero1 blocked the increased ros production , thus providing a link between protein oxidation in the er and ros production during er stress . this perk function was linked to the ability of atf4 to regulate the expression of genes involved in glutathione biosynthesis and antioxidant response . these studies suggest that the perk branch of upr bifurcates in two parallel but integrated signaling pathways , perk - eif2-atf4 and perk - nrf2 , with a key role in adaptation to oxidative stress , a metabolic consequence of biosynthesis and posttranslational oxidative processing in the er . both isoforms of atf6 , atf6 and atf6 , are present in all cell lines as type ii transmembrane er proteins . release of bip does not cause atf6 oligomerization , but instead reveals a golgi localization sequence . once translocated to the golgi , atf6 is cleaved at a juxtamembrane site by the site 1 and site 2 proteases ( s1p and s2p ) , which are also involved in the cleavage of the er membrane transcription factor srebp ( sterol response element - binding protein ) [ 35 , 36 ] . processed atf6 moves to the nucleus where it forms active homodimers or dimerizes with other bzip transcription factors like nf - y ( caat binding factor ) as well as xbp1s , to regulate transcription from atf / camp response elements ( cres ) and erses [ 38;39 ] . interestingly , yoshida et al . found that xbp1u interacts directly with the active form of atf6 ( but not atf4 ) , targeting it for proteasomal degradation which may provide a negative feedback loop to decrease xbp1 expression . other transcriptional targets include proteins increasing er chaperone activity and degrading of er client proteins [ 37 , 39 ] . although atf6 is neither essential for basal expression of er chaperones nor for embryonic or postnatal development , it plays an important role in recovery from acute er stress and adapting cells to chronic er stress . additionally , a recent study shows that atf6 also contributes , in an xpb1s - independent manner , to lipid biogenesis and er expansion , an er stress response which was thought to be predominantly mediated by the irei pathway . bcl-2 family proteins , which consist of proapoptotic multidomain proteins ( e.g. , bax , bak ) , antiapoptotic multidomain proteins ( e.g. , bcl-2 ) , and bh3-only proteins ( e.g. , bid , bim , bad ) , are key regulators of mitochondrial apoptosis [ 41 , 42 ] . they function as gatekeepers ( antiapoptotic ; bcl-2 ) or gatecrashers ( proapoptotic ; bax / bak ) of the outer mitochondrial membrane . while the molecular mechanism underlying their mitochondrial action is still a matter of debate , it is becoming clear that bcl-2 family proteins can exert a tight control on apoptosis at different subcellular sites . a constellation of er localized bcl-2 family members , including bax , bak , bik / nbk , and bcl-2 , has been shown to be engaged in the control of er - ca homeostasis [ 4446 ] ( for an extensive review see [ 13 , 47 ] ) . moreover , recent reports have identified bcl-2 members as vital regulators of upr sensor mechanisms and cellular fate following er stress . for instance , atf6 negatively regulates bad proapoptotic activity by upregulating regulator of calcineurin 1 ( rcan1 ) , an endogenous inhibitor of calcineurin ( protein phosphatase b ) . bad dephosphorylation allows its dimerization with antiapoptotic bcl-2 protein family members like bcl - xl , thus inhibiting their activity . this mechanism underscores a prosurvival role for the genetic program activated by atf6 , through the suppression of bad proapoptotic activity . the proapoptotic multidomain proteins bax and bak can form a protein complex with the cytosolic domain of ire1 , and this interaction has been shown to be essential for ire1 signaling . genetic ablation of bax / bak in mice caused abnormal response to tunicamycin - induced er stress in the liver along with extensive tissue damage , decreased expression of xbp1 , and reduced jnk activation . furthermore , the requirement of bax / bak proteins for proper ire1 signaling was confirmed in mefs doubly deficient ( dko ) in these proapoptotic proteins . in a recent report klee et al . showed that reconstituting bak expression at the er membranes in dko cells is sufficient to reestablish ire1-traf2 activation and mitochondrial apoptosis ( as discussed further in section 2.3 ) instigated by reticular forms of the bh3-only proteins bim and puma . interestingly , the ire1 pathway activated by reticular bh3-only effectors was atypical as it did not lead to xbp1 splicing , likely because other arms of the upr required for the upregulation of xbp1 mrna levels , such as atf6 , were not sufficiently activated . however , an alternative and intriguing possibility could involve a differential regulation of the ire1 rnase activity ( required for xbp1 mrna splicing ) and ire1-traf2 complex formation ( required to activate proapoptotic jnk signaling ) by a different subset of proapoptotic proteins at the er membrane . clearly , further studies are required to shed more light into the mechanisms regulating ire1 signal transduction . recently , the er associated bax inhibitor-1 ( bi-1 ) , an evolutionary conserved antiapoptotic protein , has been identified as a new player in the regulation of ire1 by bcl-2 family members and their modulators . bi-1 can block bax - mediated apoptosis following er stress and other intrinsic stress signals by directly interacting with antiapoptotic bcl-2 family members and enhancing their antiapoptotic function . bi-1 at the er was found to be capable to interact through its c - terminus domain with ire1 and to inhibit ire1 signaling , in vitro as well as in mice and flies , conferring increased resistance under conditions of mild er stress . er stressed bi-1 deficient cells displayed ire1 hyperactivation along with an increased xbp1 mrna splicing and expression of xbp1s - dependent genes , thus unraveling a paradoxical role of bi-1 as inhibitor of the cytoprotective ire1 branch of the upr in mildly er stressed cells . interestingly , in another study using human fibrosarcoma cells , overexpression of bi-1 inhibited ros production downstream er stress through the upregulation of ho-1 , an effector of the perk - pathway ( as described previously ) . bi-1 deficiency in mefs did not affect expression of ho-1 , this raises the intriguing possibility that bi-1 may affect ire1 and possibly perk pathway in a cell type specific manner . whereas further mechanistic studies are required to solve these discrepancies , these findings reveal how a subtle cross - talk between molecular sensors of upr and cell death regulators might affect the amplitude and function of the upr . moreover , abundant evidence claims for a critical role of proapoptotic bcl-2 family proteins in the induction of apoptosis following er stress . when the initial cellular responses fail to restore er homeostasis , sustained er stress causes the upr to switch from an adaptive to a cell death pathway . however , the molecular elements of this switch are still elusive . with the exception of few components of the upr for which a dominant prosurvival ( i.e. , bip , ) or proapoptotic ( i.e. , chop , [ 56 , 57 ] ) role has been assigned by genetic studies , each apical upr sensor holds a dualistic role in propagating adaptive as well as a toxic signals . for example , genetic deletion of perk or interference with eif2 phosphorylation impairs cell survival [ 58 , 59 ] and tumor growth under hypoxia , while artificially increasing perk activity increases cell survival . however , lin et al . have shown that sustained perk signaling is lethal , whereas the equivalent duration of ire1 signaling is not , suggesting that transition from protective to proapoptotic upr function involves a switch in ire1 signaling along with enduring perk activity . the main effector of perk - mediated apoptosis is the proapoptotic transcription factor chop ( c / ebp homologous protein ; gadd153 ) which can be induced by atf4 , atf6 , as well as xbp1s . however , the perk - eif2 branch appeared to be essential for chop upregulation as both perk , atf4 and eif2 ser51ala knock - in cells failed to induce chop during er stress [ 32 , 58 , 59 ] . chop activity is also regulated translationally by the limited chop mrna lifetime and posttranslationally by p38mapk phosphorylation , which enhances its proapoptotic activity [ 57 , 64 ] . the latter mechanism may provide a point of convergence between the perk and ire1 signaling pathways since p38mapk is a downstream target of the ire1-traf2-ask1 signaling complex [ 25 , 26 ] . genetic studies have shown that chop loss - of - function results in cytoprotection , whereas chop gain - of - function enhances sensitivity to a variety of stresses perturbing er function [ 56 , 65 ] . chop mediated cell death entails the induction of a variety of genes that may potentiate apoptosis , including gadd34 , ero1 , bim , and trb3 ( tribbles homologue 3 ) . gadd34 is a regulatory subunit of protein phosphatase 1 ( pp1 ) that targets pp1 to dephoshorylate eif2 , which promotes the resumption of protein synthesis . if the protein folding capacity of the er has not been reestablished , a premature deinhibition of translation will increase client protein load in the er and may favor improper disulphide bond formation of unfolded proteins , thus amplifying the damage . in addition , elevated expression of ero1 by chop is thought to instigate hyperoxidizing conditions in the er [ 67 , 68 ] . thus the perk - axis , which is involved in maintaining the redox state during er stress , as discussed before , has also the ability to turn into a prooxidant signal when the transcriptional program of chop is efficiently set in motion . as suggested by a recent study wherein the stability of prosurvival and prodeath mrnas and proteins was studied under conditions of mild or severe er stress , atf4-dependent prosurvival gene expression is likely to be more sustained when perk is activated transiently and to a limited extent . in contrast , as a consequence of the intrinsic instability of the proapoptotic mrnas and proteins , the apoptotic program mediated by the atf4 target chop would be activated only when protective mechanisms fail and require a more sustained perk activation . chop can also regulate the expression of a number of bcl-2 family proteins . by a yet unidentified mechanism , it suppresses the expression of the antiapoptotic bcl-2 while directly promoting the transcription of the proapoptotic bh3-only protein bim . although it is clear that chop fulfills an important role in er stress induced apoptosis , the fact that perk and eif2 ser51ala knock - in cells are unable to induce chop yet are very susceptible to er stress [ 58 , 59 ] unravels the dual role of the perk axis in triggering both adaptive and proapoptotic processes . the increased sensitivity of perk deficient cells could be explained , at least in part , by the impaired activation of the prosurvival pi3k ( phosphatidylinositol 3 kinase)-akt signaling pathway which has been shown to promote the expression of inhibitor of apoptosis proteins ( iaps ) , thus conferring cellular resistance to er stress [ 70 , 71 ] . an interesting molecular switch between the prosurvival and prodeath functions of the perk pathway could involve the human orthologue of the drosophila tribble protein ( trb3 ) , a downstream transcriptional target of chop . . showed that trb3 knock down sensitized the cells to cell death during tunicamycin treatment . remarkably , trb3 could downregulate its own induction by repressing chop / atf4 functions [ 72 , 73 ] . a mechanism was proposed wherein trb3 exerts a negative feedback on chop during mild er stress , allowing the cell to adapt to er stress [ 72 , 73 ] . in contrast , during severe or persistent er stress , induction of trb3 would be more robust , leading to apoptosis through a mechanism involving trb3-mediated inhibition ( dephosphorylation ) of akt [ 74 , 75 ] . this feedback mechanism could facilitate er stress mediated apoptosis in severely er stressed cells that have successfully mounted proapoptotic threshold levels of chop . similar to perk , ire1 signaling has also been implicated in promoting or impairing cell survival . for instance , when unfolded proteins accumulate , artificially extending ire1 's rnase function led to enhanced survival [ 62 , 76 ] and the knock down of xbp1 impaired cell survival , [ 77 , 78 ] pointing to a general protective role for the ire1-xbp1 signaling during er stress . however , in another report , ire1 overexpression in hek293 t cells led to its activation in the absence of er stress and subsequent cell death . as discussed before , ire1 has apparently gained signaling properties independent of xbp1 splicing , which are strongly dependent on interaction with bcl-2 proapoptotics and bcl2 modulators at the er membrane . thus , ire1 can promote cell death by recruiting a traf2-ask1 complex leading to the activation of jnk and p38 mapk cascades [ 25 , 26 ] . jnk , in turn , can exert its proapoptotic effect by activating certain bh3-only proteins , such as bim [ 79 , 80 ] , or by suppressing the antiapoptotic activity of bcl-2 . the apoptotic pathway evoked after upr is still unclear , but mounting observations indicate that the mitochondrial pathway is heavily involved , since cells lacking bax and bak , or apaf-1 are resistant to apoptosis induction by different er stressors [ 44 , 82 , 83 ] . moreover , as mentioned before , several bcl-2 family members localize at the er and regulate both calcium levels as well as signal transduction through the upr . in addition to bim , other bh3-only proteins , such as noxa and puma , are transcriptionally activated , through p53-dependent and independent mechanisms depending on the type of er stressor , thus bridging er stress to bax / bak mediated mitochondrial membrane permeabilization . recently klee and coworkers using bax / bak cells showed that bak targeted at the er membrane is sufficient to engage mitochondrial apoptosis when activated by bh3-only molecules puma and bim at the er , thus bypassing the need to be localized to the mitochondria . reticular bak engaged an atypical ire-traf2 activation pathway , wherein the mobilization of ca facilitated persistent jnk activation . intriguingly , er ca release per se was not able to incite mitochondrial apoptosis unless bak was expressed at the reticulum , whereas it favored nonapoptotic cell death , as shown also in our previous study . whether this pathway has any role in normal cells expressing both mitochondrial and er bax / bak still needs to be proven , however it can already be argued that jnk functions as a master regulator of both apoptosis and perhaps autophagy pathways after er stress . thus all together the emerging consensus is that the amplitude and the temporal activation of specific arms of the upr , along with the repertoire of signaling platforms formed at the er membrane ( upr interactome ) , are crucial elements determining cellular fate following er stress . proteasomal degradation and autophagy are the two main mechanisms that are in charge of protein clearance in the cell . unlike proteasomal degradation ( that digests soluble ubiquitin - conjugated proteins in a specific way ) , autophagy can degrade both soluble and aggregated proteins [ 8 , 86 ] . thus , during the autophagic process , entire cytoplasmic portions including organelles and other cytoplasmic components are engulfed within a double membrane vesicle designated autophagosome . the maturation of these vesicles involves their fusion with lysosomes , which leads in turn to the degradation of the autophagosome components by the lysosomal degradative enzymes [ 8 , 86 ] . as discussed below , a variety of stress signals such as nutrient starvation or treatment with different anticancer agents ( including those that induce er stress ) stimulate the autophagy process which is nowadays considered as an essential cellular process participating in a number of physiological functions within the cell . the molecular mechanisms responsible for the regulation of autophagy have not been completely elucidated yet , although genetic and biochemical analyses performed during the last few years have identified several autophagy genes ( atg ) that participate in the regulation of this cellular process . researchers working in the autophagy field have formally divided the autophagic process in several steps . initiation of autophagy relies on the formation of an isolation membrane ( i m ) at the so - called preautophagosomal site . the autophagy process ends with the fusion of the autophagosome and the lysosome , the digestion of the autophagosome content , and the release of the digested components back to the cytosol [ 8 , 86 ] . in these sections , we will briefly summarize the mechanisms by which the different stages of autophagy are regulated . the normal rate of autophagy in the cell is low and therefore this cellular process only becomes activated in response to certain situations . thus , exposure of the cell to an autophagic stimulus triggers a series of modifications in the autophagic machinery that allow the formation and elongation of the i m . the precise origin of the i m in mammalian cells is still unknown , although it has been proposed that it could be either derived from de novo synthesized lipids or generated by vesicle budding from er , golgi apparatus , or endosomes . the transmembrane proteins atg9 and vmp-1 [ 88 , 89 ] are required for autophagosome formation and it has been suggested that they could play a role in the transport of lipids to the i m as well as in the recruitment of additional proteins involved in the initiation of autophagy . thus , the movement of atg9 from the trans - golgi location to the preautophagosomal site seems to be a crucial event in the initiation of autophagy [ 87 , 90 ] . the relocation of the transmembrane protein atg9 to the autophagosome is thought to require activation of the complex formed by the proteins atg1 , atg13 , and atg17/fip200 .. the activity of the atg1 complex is modulated by the mammalian target of rapamycin complex 1 ( mtorc1 ) . mtorc1 is a protein complex formed by mtor , raptor ( regulatory associated protein of mtor ) , mlst8 , and pras40 ( proline - rich akt substrate 40 kda ) that plays a central role in the control of protein synthesis , cell growth , and cell proliferation through the regulation of several downstream targets . in addition , mtorc1 has been proposed to regulate autophagy by repressing the activity of the atg1-atg13-atg17/fip200 complex [ 9295 ] . regulation of mtorc1 relies on the small g protein rheb ( ras homologue enriched in brain ) which ( through a still not completely elucidated mechanism ) activates mtorc1 . the tuberous sclerosis proteins ( tsc1 and tsc2 ) have gtpase activating protein ( gap ) activity on rheb and therefore promote its inhibition . hence , inactivation of tsc1/2 stimulates rheb and mtorc1 and inhibits autophagy . as a result of its central position in the control of cellular homeostasis one of the most important upstream regulators of mtorc1 is the prosurvival kinase akt , which phosphorylates and inactivates tsc2 as well as pras40 . another important regulator of tsc2 is the amp - activated protein kinase ( ampk ) which phosphorylates tsc2 in a different residue than akt leading to activation of tsc1/2 , inactivation of rheb , and inhibition of mtorc1 . as discussed in the following sections , modulation of mtorc1 activity is one of the mechanisms by which er stress and autophagy become connected . another important step in the initiation of autophagy is the generation of a specific pool of phosphatidylinositol-3-p ( pip3 ) at the autophagosome . in mammals , this event is catalyzed by the class iii phosphatidylinositol 3 kinase ( pi3k ) complex [ which consists of vps34 ( vacuolar protein sorting 34 ) and its regulatory protein p150 ( homolog to the yeast vps15 protein ) ] . accumulation of pip3 seems to be crucial for the recruitment of autophagy proteins such as atg18/wipi-1 to the i m which is important for atg9 trafficking and therefore for the initiation of the autophagic process . in addition , other proteins such as matg2 and dfcp1 ( double fyve domain - containing protein 1 ) may also be regulated by pip3 and play a role in the regulation of the formation and elongation of the autophagosome . underlining the importance of pip3 in the early stage of autophagy , a specific phosphoinositide 3-phosphatase ( jumpy ) importantly , other vps34-interacting proteins are required for autophagy including , vps30/atg6/beclin1 , atg14 and autophagy / beclin-1 regulator 1 ( ambra-1 ) , and uvrag . among the different partners of vps34 beclin-1 has a bh3-only domain that permits the interaction of this protein with the antiapoptotic proteins bcl-2 and bcl - xl . different stimuli , including er stress , modulate the interaction between beclin-1 and bcl-2 family members ( see also the following sections ) which is considered an important mechanism of autophagy regulation . atg14 and uvrag are also interactors of vps34 although their presence in the class iii pi3k complex seems to be mutually exclusive [ 87 , 103 , 104 ] . recent findings support that atg14 plays an important role in the early stages of autophagy activation in response to starvation . in any case , further research is still necessary to understand the complex lipid - protein and protein - protein interactions that regulate the formation of the i m . the elongation of the initial autophagic membrane requires the participation of two ubiquitin - like protein conjugation systems which modify the autophagy proteins atg5 and atg8/lc3 . thus , upon autophagy stimulation , atg5 is conjugated to atg12 . in this process atg12 is activated by the e1 activating enzyme atg7 and transferred to the e2-like protein atg10 . finally atg12 is attached to an internal lysine of atg5 in a process that does not seem to require an e3 ligase protein . is cleaved by the protease atg4 ( which generates a glycine ct residue in atg8/lc3 ) . then , the e1 enzyme atg7 activates atg8 , which is transferred to the e2-like protein atg3 . the last step in atg8/lc3 modification involves the conjugation of this protein to phosphatidylethanolamine ( pe ) , a process that is facilitated by the e3-like activity of the atg12-atg5 conjugate [ 105 , 106 ] . upon autophagy induction , most of atg8/lc3 becomes lipidated and associates with the autophagosome , which is widely used to monitor activation of autophagy by immunofluorescence [ 8 , 86 , 107 ] . the atg16l and atg8/lc3 complexes play a crucial role on the modification of the autophagosomal membrane and therefore in the elongation and closure of the autophagosome . the last step in the autophagic process is the fusion of the autophagosome with lysosomes . the canonical machinery of vacuole membrane fusion seems to participate in the regulation of this process [ 87 , 90 ] . thus , the lysosomal protein lamp2 and the small gtpase rab7 have been implicated in autophagosome - lysosome fusion in mammalian cells . nevertheless , many additional proteins including those belonging to the rab and soluble n - ethylmaleimide sensitive factor attachment protein receptors family ( snare ) are believed to play an important role in the autophagosome - lysosome fusion process [ 8 , 86 ] . the lysosomal degradation of the autophagosomal content relies on several lysosomal hydrolases including cathepsins b , d , and l. the final outcome of the activation of the autophagy program is highly dependent on the cellular context and the strength and duration of the stress - inducing signals . thus , besides its role in cellular homeostasis , autophagy can be a form of programmed cell death or play a cytoprotective role , for example in situations of nutrient starvation [ 108 , 109 ] . accordingly , autophagy plays a dual role in cancer . on one hand , this cellular process may help to overcome the stress evoked by the lack of nutrients and oxygen at the initial steps of tumorigenesis . on the other hand , autophagy has been proposed to play a tumor suppressor function by providing the minimal supply of atp required for dna repair , preventing oxidative stress and reducing intratumoral necrosis and local inflammation [ 110113 ] . moreover , different anticancer treatments activate autophagy in tumor cells , which has been proposed to either enhance cancer cell death or act as a mechanism of resistance to chemotherapy [ 110115 ] . different situations that induce er stress also lead to induction of autophagy . as discussed above , the er stress response is activated to protect the cells from different alterations affecting this organelle . however , when the intensity or duration of the er damage can not be restored by this response , er stress can also lead to cell death . likewise , autophagy can help cells to cope with er stress ( for instance contributing to the elimination of unfolded or aggregated proteins ) or participate in the mechanism of er stress - induced cell death [ 115 , 117119 ] . in this section we will try to delineate some of the proposed mechanisms by which er stress and autophagy become connected under certain cellular situations ( figure 2 ) . as described above , the accumulation of unfolded proteins triggers the upr thus promoting the inhibition of general protein synthesis as well as the increased translation of several transcription factors that enhance the expression of er stress genes ( see the previous sections for further details ) . evidence for a link between upr and autophagy was obtained from ectopic expression of polyglutamine ( polyq ) proteins . in these experiments , a dominant - negative form of perk or genetic substitution of serine 51 of eif2 by ala ( which prevents the phosphorylation of this protein ) prevented polyq protein - induced autophagy , strongly suggesting that perk - dependent eif2 phosphorylation plays an important role in the activation of autophagy in response to the accumulation of unfolded proteins . on the other hand , eif2 phosphorylation seems to be also important for autophagy as induced by other er stress - related or unrelated stimuli [ 75 , 120 , 121 ] . it is important to bear in mind that perk is not the only protein kinase regulating eif2 phosphorylation ( see reference for a review ) as double - stranded rna - activated protein kinase ( pkr ; activated in viral responses ) , general control nonderepressible 2 ( gcn2 ; activated upon aminoacid starvation ) , and heme - regulated inhibitor ( hri ; activated in heme depletion ) also phosphorylate eif2. accordingly , pkr - dependent eif2 phosphorylation modulates autophagy in response to viral infection . likewise , the small heat shock 22 kda protein 8 ( hspb8 ) and its cochaperone bcl-2-associated athanogene 3 ( bag3 ) have been proposed to mediate mutated hungtingtin - induced eif2 phosphorylation and autophagy via gcn2 activation . regarding the signalling pathways by which eif2 phosphorylation can modulate autophagy , kouroku and et al showed that perk - eif2-dependent atg12 upregulation is required for induction of autophagy in response to polyq protein accumulation which suggests that controlling the expression of autophagy - related genes by eif2 downstream targets could be one of the mechanisms connecting both events . on the other hand , we have recently found that treatment of cancer cells with -tetrahydrocannabinol ( thc ) , the active component of marihuana , activates autophagy via er stress and eif2 phosphorylation ( an effect that is not mediated by perk , pkr , or gcn2 , salazar , m. and velasco , g. unpublished observations ) . our data indicate that induction of autophagy in response to thc treatment relies on the eif2 phosphorylation - dependent upregulation of the transcription factors p8 , atf-4 , and chop as well as of the pseudokinase trb3 ( four genes that had been previously identified as essential mediators of thc action in cancer cells [ 122 , 123 ] ) . we also showed that an important step in the induction of autophagy is the inhibition of the akt / mtorc1 axis by the pseudokinase trb3 ( see below for additional details ) ( figure 2 ) . in any case , further research is still necessary to clarify the precise mechanisms by which eif2 phosphorylation regulates autophagy in response to different er stress signals . activation of the ire1 arm of the er stress response has also been shown to regulate autophagy . thus , treatment with tunicamycin or thapsigargin or treatment with proteasome inhibitors induced autophagy on an ire1-dependent manner . the proautophagic actions of ire1 seem to rely on the ability of this protein to interact with the cytosolic adaptor traf-2 and activate jnk ) . of interest , jnk has been proposed to regulate autophagy through bcl-2 phosphorylation , which prevents this protein of interacting ( and inhibiting ) the essential autophagy regulator beclin-1 [ 99 , 124 , 125 ] . in addition , jnk has been shown to control beclin-1 expression to regulate ceramide - induced autophagy . as discussed above , beclin-1 is associated to the vps34 and plays a very important role in the regulation of autophagy ( see below ) ( figure 2 ) . it is therefore conceivable that activation of the ire1/traf2/jnk arm of er stress may regulate autophagy through modulation of beclin-1 function and expression . intriguingly , it has been recently shown that xbp-1 ablation increases autophagy and protects from the toxicity induced by the aggregates of the enzyme superoxide dismutase 1 in a model of amyotrophic lateral sclerosis . these observations suggest that the xbp-1 may play a different role than traf2/jnk on the regulation of autophagy by the ire1 arm of the upr . er stress activation is frequently accompanied by calcium release into the cytosol which leads to the activation of several ca - regulated signalling pathways . different agents ( including er stress inducers ) have been shown to produce an increase in cytosolic calcium concentration and activate autophagy . one of the mechanisms connecting ca release from the er and autophagy is the stimulation of ampk . as explained above , several kinases regulate mtorc1 including ampk , which inhibits mtorc1 by activating tsc2 . ampk is considered an important energy sensor that becomes activated upon atp cellular depletion or phosphorylation by different kinases . three ampk upstream kinases have been identified to date : lkb1 , ca / calmodulin - dependent kinase kinase ( cacmkk ) , and transforming growth factor - beta - activating kinase 1 ( tak1 ) . jttel and coworkers showed that increases in cytosolic ca concentration upon treatment with different er stress inducers stimulate camkk , leading in turn to ampk activation , inhibition of mtorc1 , and autophagy stimulation . the same group has recently shown that trail - induced autophagy is also mediated by ampk , in this case through a mechanism that involves phosphorylation of ampk by tak1 and not by lkb1 or cam - kk . these observations suggest that ampk may play an important role in the regulation of autophagy in response to different ca - dependent and independent stress signals . another ca - activated kinase that regulates autophagy in response to er stress is the death associated protein kinase 1 ( dapk ) . dapk is a ser / thr kinase that plays an important role as tumor suppressor due to its ability to promote apoptosis and autophagy . thus , dapk - deficient mefs are less sensitive to er stress - induced autophagy than their wild - type counterparts . activation of dapk upon er stress relies on the dephosphorylation of an inhibitory autophosphorylation site of the kinase by a pp2a phosphatase , which suggests that additional er stress - activated signals ( apart from ca release ) are required to stimulate the proautophagic activity of the kinase . regarding the mechanisms by which dapk regulates autophagy , it has been recently shown that dapk phosphorylates beclin-1 on the bh3-only domain preventing thus the interaction of this protein with bcl-2 [ 133 , 134 ] . as p53 modulates autophagy through different mechanisms [ 112 , 136138 ] , this could be another way by which dapk could regulate autophagy in response to certain er stress stimuli . the protein kinase c theta ( pkc ) has been also implicated in regulating autophagy in response to er stress in a calcium - dependent manner . thus , knock - down of pkc ( but not inactivation of the upr signalling routes ) prevented autophagy as induced by acute er stress . in this study , inactivation of mtorc1 , under the used concentrations of thapsigargin , thapsigargin were not observed , which suggests that different signalling routes may converge in the regulation of autophagy under er stress situations involving calcium mobilization . another link between ca , er stress , and autophagy relies on the modulation of the inositol 1,4,5-trisphosphate receptor ( ip3r ) . this receptor releases ca from er stores in response to different cellular signals , although it could also play additional functions derived from its ability to interact with different proteins , including members of the bcl-2 family . inhibition of the ip3r with xestospongin b or lithium - induced decrease of myo - inositol-1,4,5-triphosphate ( ip3 ) levels promotes autophagy . intriguingly , these effects seem to be independent of the ca mobilization function of ip3r . thus , it has been recently shown that use of pharmacological inhibitors of the ip3r disrupts the interaction of this protein with beclin-1 which could be an additional way of regulating the pro - autophagic function of this protein . further investigation is nevertheless necessary to clarify whether this mechanism participates in the activation of autophagy in response to er stress . as discussed for the case of er stress , autophagy is currently considered a cell survival mechanism that , under certain cellular settings , can also promote cell death . consequently , depending on whether pharmacological or genetic inhibition of autophagy enhances or prevents cell death , activation of autophagy after er stress has been assigned respectively a cytotoxic [ 75 , 115 , 119 , 132 , 145147 ] or a protective [ 5 , 6 , 115 , 119 , 128 ] role . it is worth noting that depending on the intensity of the stimulus , the cell type ( normal versus cancer cells ) , and the cellular context , ( hypoxia , starvation , treatment with antitumoral agents , or presence of mutations ) the final outcome of autophagy activation could be different . an important problem at the time of predicting whether induction of er stress will activate autophagy in a protective or cytotoxic way is our relative lack of understanding of the molecular mechanisms through which autophagy regulates cell death . thus , autophagy has been proposed to protect from apoptosis , operate as an alternative cell death mechanism ( e.g. , in cells that are defective in apoptosis ) , or act upstream of apoptosis to activate this cellular process , ( reviewed in [ 133 , 148 ] ) . as discussed in the previous section , some of the key regulatory steps in the activation of autophagy upon stimulation of er stress ( such as mtorc1 inhibition or the interaction of beclin-1 with bcl-2 ) can also receive signals derived from different inputs including those not directly related with er stress . moreover , some of the regulatory proteins transmitting these signals such as akt , ampk , dapk , or jnk play also a major role in the modulation of cell survival independently of autophagy . it is therefore essential to consider the cellular context in order to understand how the different er stress signals are integrated to yield a protective or cytotoxic autophagic response . from the above discussion , it is clear that er stress and autophagy can activate both prosurvival mechanisms as well as lethal programs , especially under conditions of enduring er stress and organellar damage . thus activation of the upr and autophagy may either impede or facilitate drug - mediated cell killing , and it is plausible that this will depend on the type of cancer and cytotoxic agents used . while a growing number of reports have started to identify molecular elements of the cross - talk between er stress and autophagy ( see section 3.2 ) , thus unraveling potential druggable targets , knowledge of the functional outcome of the activation of these pathways in cancer cells responding to chemotherapeutics is still very limited . in terms of therapeutic outcome , drugs ( or a combination thereof ) capable of activating the proapoptotic branch of the upr while simultaneously inhibiting its prosurvival function should provide the highest therapeutic benefit . moreover , if autophagy activated following er stress is a survival response restoring er homeostasis ( e.g. , by the removal of protein aggregates ) , its pharmacological blockage could protract upr activation until a critical threshold is reached , which may precipitate its proapoptotic function . on the other hand , autophagy may endorse the proapoptotic functions of certain er stress pathways ( see also section 4.1 ) or become a lethal backup pathway in cancer cells with defect on apoptotic signaling [ 133 , 148 ] . a wide array of conventional and experimental chemotherapeutic agents has been shown to stimulate er stress and activation of upr along with autophagy in cancer cells . for example , tunicamycin , thapsigargin , and brefeldin a activate autophagy in colon and prostate cancer cells thus mitigating er stress and protecting against cell death . however , autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the nontransformed murine embryonic fibroblasts but rather contributes to cell death . the combined administration of vorinostat ( a histone deacetylase inhibitor ) and sorafenib ( a tyrosine kinase inhibitor ) to carcinoma cells promotes cell death although activates at the same time a protective er stress - driven autophagic response . similarly , the resistance to imatinib mesylate ( a bcr / abl tyrosine kinase inhibitor used for the treatment of chronic myeloid leukaemia ) might also rely at least in part on the secondary activation of er stress - induced autophagy . by contrast , cannabinoid treatment activates er stress and autophagy leading to apoptotic cell death of glioma and pancreatic cancer cells but not of nontransformed embryonic fribroblasts or primary astrocytes ( in which neither er stress nor autophagy is activated in response to the treatment with these compounds ) . likewise , other agents such as nelfinavir ( an hiv protease inhibitor with anticancer activity ) [ 151 , 152 ] or melanoma differentiation associated gene-7/interleukin 24 ( mda-7/il-24 ) [ 145 , 153 ] activate an er stress response that promotes autophagy and apoptosis of cancer cells . increased expression of tetraspanins ( a family of proteins that facilitate the spatial organisation and localisation of multiprotein complexes in distinct membranal microdomains ) has also been shown to activate er stress and autophagic cell death . understanding the precise molecular mechanisms that regulate the extent of autophagy activation in response to different triggering signals as well as the ones that control the interplay of this cellular process with apoptosis is therefore crucial to design new antitumoral therapies based on the modulation of the er stress - autophagy response . here we discuss further a selected group of clinically used or promising cytotoxic drugs with a demonstrated ability of inducing both upr and autophagy , as paradigms to discuss the potential of targeting these pathways in cancer therapy . cannabinoids , the active components of marijuana , of which thc is the most important owing to its high abundance and potency , exert a wide variety of biological effects by mimicking endogenous substances , the endocannabinoids , that bind to and activate specific cannabinoid receptors . thus , treatment with these agents has been shown to curb tumor growth in various animal models of cancer [ 157159 ] . the antitumoral action of cannabinoids is based on the ability of these agents to inhibit tumor angiogenesis and activate apoptosis of cancer cells [ 157 , 158 ] . our recent findings have unravelled that cannabinoids induce autophagy in different types of tumor cells , including glioma / astrocytoma and pancreatic cancer cells , whereas they do not activate this cellular process in nontransformed cells ( which are resistant to the cell death - promoting activity of cannabinoids ) . of interest , pharmacological or genetic inhibition of autophagy prevented cannabinoid - induced cell death as well as apoptosis , whereas abrogation of apoptosis prevented cell death but not autophagy as induced by these agents . these observations led us to conclude that induction of autophagy is part of the mechanism by which cannabinoids promote the apoptotic death of cancer cells . the in vivo relevance of these findings was demonstrated by the observation that cannabinoid treatment reduced tumor growth and activated autophagy and apoptosis in subcutaneous tumor xenografts derived from human u87 mg astrocytoma cells and transformed mouse embryonic fibroblasts ( mefs ) . likewise , similar results have been obtained in an orthotopic model of pancreatic cancer , in which we had previously shown a proapoptotic and an antitumoral action of cannabinoids ( [ 122 , 123 ] , salazar , m. and velasco g. , unpublished observations ) . furthermore , autophagy - deficient tumors ( generated by subcutaneous injection of transformed atg5 mefs ) were resistant to thc antitumoral action , strongly supporting that autophagy is essential for the antineoplastic activity of cannabinoids . in addition , analysis of samples obtained from two glioblastoma multiforme patients indicated that thc administration might also trigger autophagy - mediated cell death in human tumors . as discussed in the previous section , the mechanism responsible for the activation of autophagy upon thc administration relies on a cannabinoid receptors - induced early accumulation of de novo - synthesized ceramide [ an event that takes place in the er ] , which leads in turn to er dilation and increased eif2 phosphorylation . activation of this er stress response induces the up - regulation of several genes , including the stress - regulated protein p8 and its downstream targets atf-4 and chop and the pseudokinase trb3 , which are required for the stimulation of autophagy in response to cannabinoid action . trb3 plays a crucial role in the induction of autophagy upon thc administration through its inhibitory interaction with akt , which leads in turn to mtorc1 inhibition . in agreement with these observations , treatment of mice with thc decreased mtorc1 activity , stimulated autophagy and apoptosis , and reduced tumor growth in xenografts generated with p8 cells but not in those generated with p8 cells ( in which trb3 is not up - regulated in response to thc ) , further confirming that the p8/trb3 pathway plays an essential role in the activation of autophagy and cell death by cannabinoids also in vivo . cannabinoids activate therefore a cell death - promoting signalling route that involves the stimulation of er stress , autophagy , and apoptosis in cancer cells . thus , cannabinoids constitute an interesting tool to investigate the differential molecular mechanisms that are responsible for the stimulation of autophagy - mediated cell death . on the other hand , the selectivity of cannabinoids ( which only stimulate the above - described cell death promoting pathway in cancer cells ) together with a low toxicity and good safety profile makes of these agents promising antineoplastic tools . photodynamic therapy ( pdt ) is an anticancer therapy involving the selective photosensitization of malignant cell types , usually involving porphyrins , porphyrin analogs or other agents with suitable photophysical properties . the initial step in the photodynamic process involves localization of the photosensitizing agent at subcellular loci , followed by irradiation with visible light of the appropriate wavelength [ 161 , 162 ] . this results in formation of singlet oxygen and other ros that can cause photodamage at sites where the photosensitizing agent has localized . since singlet oxygen will not migrate more than a fraction of a micron from the site of formation , as a result , photodamage can be quite specific . thus , a distinguished property of pdt is that ros formation is mainly targeted to a particular subcellular site and affects a rather specific subset of molecular targets . pdt with various agents has been shown to induce apoptosis along with autophagy and , in most cases , autophagy is activated as a mean to protect cells from killing . agents found to be clinically useful were reported to show affinity for the er , mitochondria , lysosomes , or combinations of these sites . a well - studied paradigm of er - localizing dye is hypericin , a naturally occurring phototoxin with promising applications in bladder cancer . consistent with its predominant reticular localization in cultured cells , light activation of hypericin is coupled with massive er expansion , preceding ultrastructural features of apoptosis , both in vitro and in bladder cancer bearing rats ( verfaillie , t. and agostinis , p. unpublished observations ) , and stimulation of upr . upr activation is likely the result of immediate ros - damage to the serca pump , depleting er - ca store , which is followed by the concomitant activation of autophagy and mitochondrial apoptosis . this ros paradigm of er stress is linked to a persistent activation of the perk - eif2-chop axis , with proapoptotic function ( verfaillie , t. and agostinis , p. unpublished observations ) . induction of autophagy in er stressed cells unable to mount an apoptotic response ( because of bax / bak deficiency ) results in increased photokilling , suggesting the activation of an conversely , in apoptosis - competent cells , blocking autophagy stimulation following er stress by sirnas that target essential modulators of the autophagic machinery , sensitizes to cell death , thus revealing a cytoprotective role for this pathway ( dewaele , m. and agostinis , p. unpublished results ) . hence , it is tempting to speculate that autophagy inhibition may potentiate the proapoptotic perk pathway resulting in a better therapeutic opportunity , only when the cancer cell 's apoptotic machinery has not been fully disabled . further studies are required to establish whether suppression of the autophagic pathway along with upr stimulation may represent a valuable therapeutic strategy in hypericin - based pdt . targeting proteasomal degradation has proven to be a valuable approach in various cancer treatments , and proteasome inhibitors have emerged as a new class of er stress agents . moreover , recent evidence suggests that when used in combination with certain cytotoxic drugs , such as pdt , proteasomal inhibitors are capable of enhancing their anticancer efficacy , making these agents a very promising class of pharmacological agents in combinatorial therapy . bortezomib ( ps-341 or velcade ) distinguishes itself from other proteasome inhibitors as it specifically inhibits the 26s proteasome by selectively blocking its chymotryptic activity . velcade has been clinically approved for treatment of multiple myeloma and mantle cell lymphoma [ 166 , 167 ] and has been shown to successfully induce apoptosis in various human cancer cell lines including myeloma , prostate , and breast cancers as well as squamous cell carcinoma [ 168170 ] . moreover , preclinical studies indicate that bortezomib displays anticancer activity against pancreatic cancers , one of the most aggressive human diseases . one of the potential mechanisms underlying the apoptotic effects of bortezomib in cancer cells relies on its ability to inhibit the nf-b pathway by blocking the degradation of its cytoplasmic inhibitor ib . however , inhibition of nf-b alone could not fully account for the antitumor effect by bortezomib , suggesting additional pathways being involved . since proteasomal degradation of misfolded proteins retrotranslocated from the er to the cytosol represents the final step in erad , proteasomal inhibition causes an additional burden of unfolded proteins on the er . this explains the high efficacy of bortezomib treatment against types of cancer cells in which the er is already predisposed with a considerable load . for instance , hypoxic cancer cells that otherwise show increased resistance to genotoxic agents as well as myeloma cells producing high amounts of immunoglobulins are hypersensitive to treatment with proteasome inhibitors [ 174 , 175 ] . therefore , therapies that target the er response in combination with bortezomib ought to be more successful . indeed , it was shown that bortezomib sensitized pancreatic cancer cells to er stress mediated apoptosis . additionally , we recently found a significant retardation of tumor growth in vivo in two different murine tumor models when photofrin - based pdt , a pdt approach stimulating the upr , was combined with bortezomib , or other clinically used proteasome inhibitors . this suggests that blocking the proteasome might offer a new therapeutic avenue to potentiate the antitumor effect of pdt . interestingly , schewe and aguirre - ghiso found that myeloma cells surviving bortezomib treatment attenuated eif2 phosphorylation and induction of chop . combined treatment with the gadd34-pp1 complex inhibitor salubrinal restored eif2 phosphorylation and chop induction , maximizing bortezomib induced apoptosis , thus suggesting that strategies capable of sustaining chop expression might be required to successfully eradicate tumors . furthermore , these processes are functionally coupled and proteasomal inhibition has been shown to stimulate autophagy , likely as a compensatory mechanism . surprisingly , whether autophagy enhances apoptosis induced by proteasomal inhibitors or not seems to depend on whether the treated cells are transformed or not . these findings suggest that a combined inhibition of both cellular degradation systems would enhance the antitumoral efficacy . indeed , autophagy was shown to be activated in mcf-7 cells treated with bortezomib , by a mechanism which involved proteasomal stabilization of atf4 and atf4 dependent upregulation of lc3b . this mechanism was suggested to contribute to the resistance of breast cancer cells towards bortezomib . however , a recent study wherein myeloma cells where treated with bortezomib in combination with the autophagy inhibitor 3-methyl adenine ( 3-ma ) resulted in an antagonistic response instead of the expected synergizing effect . research during the last decade has contributed to highlight the important role of er stress and autophagy in the maintenance of the cellular homeostasis . the last few years have also evidenced that both processes are closely related as some of the signalling routes activated during the er stress response are involved in stimulating autophagy . intriguingly , for example , accumulation of unfolded proteins in neurodegenerative diseases may activate a protective autophagy response . by contrast induction of er stress in cancer cells may promote the stimulation of autophagy - mediated cell death or the activation of a protective autophagy that may contribute to the resistance to certain antitumoral therapies . thus , different factors such as the intensity of the er stress signal , the simultaneous activation of additional pathways , the cell type , and so forth , must be integrated to yield a specific autophagic response . considering that escape from drug - mediated cell killing is one of the major obstacles of current cancer therapy , a better understanding of the role played by these processes in cancer cells in response to chemotherapy would help us to devise new and more efficient therapeutic opportunities utilizing inhibitors or activators of these er stress pathways . bevacizumab became standard of care as first - line treatment of metastatic colorectal cancer ( mcrc ) in association with chemotherapy , but not all patients benefit from bevacizumab treatment . cumulative data , gathered from retrospective studies , show that bevacizumab - induced hypertension ( htn ) appears to be associated with a statistically significant improvement in response rates , progression - free survival ( pfs ) and overall survival ( os ) . bevacizumab - related htn may serve as a predictor biomarker of response / survival in patients with mcrc treated with bevacizumb in first - line setting . in our subset of patients , htn g2 - 3 demonstrated to be predictive of response to treatment with bevacizumab and of pfs . a trend towards a favourable os was observed . these results strengthen previous evidence that bevacizumab - related htn may be a potential predictive biomarker for bevacizumab efficacy . in the context of an exponential increase in economic treatment costs , contributed by targeted therapy , the identification of a reliable , non - invasive , feasible and not costly predictive factor that would allow the selection of patients / tumours more likely to benefit from treatment and spare those who would not from additional toxicity is of great value in clinical practice . colorectal cancer ( crc ) incidence and mortality rates vary markedly around the world . globally , crc is the third most commonly diagnosed cancer in males and the second in females.1 approximately 25% of patients present with initial stage iv disease and almost half of the patients with crc will develop metastases with a crc - related 5-year survival rate approaching 60%.2 within the past 10 years , an increase in the number of chemotherapeutic agents available to treat this disease has been seen . novel biologically targeted therapies , which interfere with specific molecular pathways of cancer proliferation and metastasis , are being developed.3 vascular endothelial growth factor ( vegf ) is a key mediator of angiogenesis and plays a pivotal role in the pathogenesis of a wide range of human cancers , being a potential target for new anticancer therapies . bevacizumab , a humanised anti - vegf monoclonal antibody , became standard of care as first - line treatment of mcrc , after showing effectivity in the treatment of mcrc with improvement in response rates and survival outcomes.46 although not all patients benefit from bevacizumab , selection of those who do profit from this therapy is still difficult because no biomarker has yet been identified that allows us to predict the benefit of the association of bevacizumab to chemotherapy in mcrc . different biomarkers , such as b - raf and k - ras mutation , microvessel density , vegf receptor ( vegfr ) expression , have been widely studied , but none has proven to be a reliable predictive factor.7 8 hypertension ( htn ) is a common complication of the treatment with bevacizumab which is easily manageable with antihypertensive agents . the pathogenesis of vegf signal inhibition - induced htn is still unclear with reports suggesting that impaired angiogenesis or endothelial dysfunction associated with bevacizumab treatment may be responsible.5 9 in fact , vegf signal antagonism may lead to inhibition of nitric synthase with a consequent decrease in the nitric oxide level , which in turn leads to vasoconstriction and decrease in sodium renal excretion , ultimately resulting in htn.9 cumulative data from recent studies have suggested that htn may be a biomarker for efficacy of vegf signal inhibition , namely of bevacizumab , due to the underlying mechanism of action . retrospective studies have shown that patients who develop bevacizumab - induced htn showed increased response rates and progression - free survival ( pfs ) in comparison with those who did not . these findings build up the hypothesis that bevacizumab - induced htn may represent a predictive and prognostic biomarker in patients with advanced colorectal cancer receiving first - line bevacizumab.5 10 11 the aim of this study was to retrospectively evaluate if htn grades 2 to 3 were correlated with response to treatment with bevacizumab in first line , as well as with improved pfs and overall survival ( os ) , in a series of patients with mcrc . eligible patients were 18 years of age or older with histologically proven mcrc , with measurable disease , receiving bevacizumab as first - line therapy at a dose of 5 mg / kg every 2 weeks in association with irinotecan , 5-fluorouracil and folinic acid , according to the folfiri regimen , or oxaliplatin , 5-fluorouracil and folinic acid , according to the folfox regimen . tumour response evaluation was performed with the use of ct or mri , depending on which imaging methods were used at baseline . blood pressure measurements were recorded before the infusion of bevacizumab by the nurses and a daily record of the blood pressure values was carried out by the patient . the highest value of arterial blood pressure recorded was taken into account to define the grade of bevacizumab - induced arterial htn according to the grading of the national cancer institute grade 23 arterial htn was the cut - off level for htn definition in accordance with previous reports , indicating this level to be biologically and clinically relevant.3 9 patients were then stratified into two groups , those who did not present htn ( grade 01 ) and those who presented bevacizumab - related htn ( grade 23 ) . tumour response and progression were assessed by radiologists of the home institution according to response evaluation criteria in solid tumors , v.1.1 every 8 weeks . response was not re - evaluated by independent radiologists as this assessment was not performed in a clinical trial setting but in a daily clinical practice context . the best tumour response was taken into account to define responders ( patients showing complete or partial response ) or non - responders ( progressive disease ) . disease control ( dc ) was defined as those patients who achieved complete response , partial response and stable disease . the disease was staged at the time of diagnosis according to the guidelines of the american joint committee on cancer v.7 . the primary end point was objective response rate , defined as the number of patients with a complete or partial response divided by the number of patients evaluated . secondary efficacy end points were duration of pfs , defined as the interval between the start of bevacizumab therapy to clinical progression or death from any cause or last follow - up visit if not progressed , and os , defined as the interval between the start of bevacizumab therapy to death or last follow - up visit . odds ratios ( or ) was used to compare the relative odds of response / disease control , given the development or not of bevacizumab - related htn . data on patients who were lost to follow - up were censored at the time of the last evaluation . a significant level of 0.05 was chosen to assess the statistical significance . to determine if htn was an independent predictive factor for response to treatment with bevacizumab , cox regression model was used and adjusted age , eastern cooperative oncology group ( ecog ) ps ( performance status ) and kras status . eligible patients were 18 years of age or older with histologically proven mcrc , with measurable disease , receiving bevacizumab as first - line therapy at a dose of 5 mg / kg every 2 weeks in association with irinotecan , 5-fluorouracil and folinic acid , according to the folfiri regimen , or oxaliplatin , 5-fluorouracil and folinic acid , according to the folfox regimen . tumour response evaluation was performed with the use of ct or mri , depending on which imaging methods were used at baseline . blood pressure measurements were recorded before the infusion of bevacizumab by the nurses and a daily record of the blood pressure values was carried out by the patient . the highest value of arterial blood pressure recorded was taken into account to define the grade of bevacizumab - induced arterial htn according to the grading of the national cancer institute grade 23 arterial htn was the cut - off level for htn definition in accordance with previous reports , indicating this level to be biologically and clinically relevant.3 9 patients were then stratified into two groups , those who did not present htn ( grade 01 ) and those who presented bevacizumab - related htn ( grade 23 ) . tumour response and progression were assessed by radiologists of the home institution according to response evaluation criteria in solid tumors , v.1.1 every 8 weeks . response was not re - evaluated by independent radiologists as this assessment was not performed in a clinical trial setting but in a daily clinical practice context . the best tumour response was taken into account to define responders ( patients showing complete or partial response ) or non - responders ( progressive disease ) . disease control ( dc ) was defined as those patients who achieved complete response , partial response and stable disease . the disease was staged at the time of diagnosis according to the guidelines of the american joint committee on cancer v.7 . the primary end point was objective response rate , defined as the number of patients with a complete or partial response divided by the number of patients evaluated . secondary efficacy end points were duration of pfs , defined as the interval between the start of bevacizumab therapy to clinical progression or death from any cause or last follow - up visit if not progressed , and os , defined as the interval between the start of bevacizumab therapy to death or last follow - up visit . odds ratios ( or ) was used to compare the relative odds of response / disease control , given the development or not of bevacizumab - related htn . data on patients who were lost to follow - up were censored at the time of the last evaluation . a significant level of 0.05 was chosen to assess the statistical significance . to determine if htn was an independent predictive factor for response to treatment with bevacizumab , cox regression model was used and adjusted age , eastern cooperative oncology group ( ecog ) ps ( performance status ) and kras status . since january 2008 and december 2013 , 79 patients ' records were evaluated with a median follow - up time of 19 months . seventeen patients were excluded because they did not present a consistent record of their blood pressure monitoring . patients ' characteristics according to the development or not of bevacizumab - related hypertension are presented in table 1 below . patients ' characteristics arb , angiotensin receptor blockers ; htn , hypertension ; n / a , not applicable ; os , overall survival . the median age at diagnosis was similar between the two groups ( about 60 years ) with a predominance of the male gender being observed in both arms ( 68.2% vs 65.8% ) . all patients were evaluated in terms of ps presenting an ecog ps 0 - 1 . the majority of patients ( 85.4% ) in the group who developed bevacizumab - related htn had an ecog ps 1 , while in the group who did not , almost 66% presented an ecog ps of 0 . the majority of patients had their primary tumour in the colon ( about 63% in both groups ) . hepatic or lung metastases were more frequently seen in both arms with a similar number of patients presenting both liver and lung metastases . peritoneal lymph node , bone , ovarian and/or penile metastases were observed , although to a lesser extent . in the group of patients who developed bevacizumab - related htn , more patients seem to have been previously submitted to surgery of the primary ( 56.1% vs 36.8% colon ; 19.5% vs 10.5% rectum ) . a similar proportion of patients received chemotherapy for stage ii and iii crc in both groups . the chemotherapy regimen most often used in association with bevacizumab in first - line treatment for mcrc was , undoubtedly , folfiri with more than 90% of the patients in both arms having received this treatment . the majority of patients in both arms were evaluated for kras status , with 14 in the group that developed bevacizumab - related htn , and 15 in the group that did not , presenting a kras mutation . nras status was evaluated in a minority of patients with all of those patients having an nras wild type . about 40% of patients , in both groups , presented a previous diagnosis of htn managed with medical treatment . forty - one patients ( 51.9% ) developed htn g2-g3 during chemotherapy - bevacizumab treatment ( 35 patients ( 85.4% ) grade 2 and 6 patients ( 14.6% ) grade 3 ) . thirty - eight patients ( 92.7% ) , after beginning treatment with adequate antihypertensive therapy , maintained bevacizumab until progression ( 3 suspended : 2 developed thrombotic events and 1 fistula ) . all patients were evaluated in terms of response : 5 patients ( 6.3% ) presented complete response ( cr ) , 32 patients ( 40.5% ) partial response ( pr ) and 28 patients ( 35.4% ) stable disease . in the group of patients who developed bevacizumab - related htn , 30 ( 73.2% ) presented response to treatment ( complete response ( cr)+partial response ( pr ) ) and 40 of them ( 97.6% ) achieved dc ( cr , pr or stable disease ) compared to 7 patients ( 18.4% ) with response and 24 ( 63.2% ) with dc in the group of those who did not develop htn ( or 12.08 ; 95% ci 4.13 to 35.29 ; p<0.001 responders vs non responders ; or 20.8 ; 95% ci 2.56 to 168.91 ; p 0.005 controlled vs non - controlled disease ) ( figures 1 and 2 ) . htn g2 - 3 was an independent predictor of progression - free survival for all adjusted factors but not of os ( see online supplementary data ) . number of patients with response in the groups of grade 23 bevacizumab - induced htn and without bevacizumab - related htn . number of patients with disease control in the groups of grade 23 bevacizumab - induced htn and without bevacizumab - related htn . a significant statistical difference was obtained in pfs between the two groups ( p<0.001 ) ( figure 3 ) . the median os was 28 months ( 22.733.3 ) . in the group that developed bevacizumab - related htn , the median os was 33 months ( 25.740.3 ) and in the group that did not , it was 21 months ( 16.525.5 ) , with no significant statistical difference between the 2 groups ( p 0.114 ) ( figure 4 ) . pfs of patients with colorectal cancer with grade 23 bevacizumab - induced htn and without bevacizumab - related htn ( p<0.001 ) . os of patients with colorectal cancer with grade 23 bevacizumab - induced htn and without bevacizumab - related htn ( p 0.114 ) . target therapies like bevacizumab affect tumour growth by targeting specifically tumour - activated biological pathways . new toxicity profiles with this new agents have emerged , as the acneiform skin rash with the anti - egfr therapy and vascular abnormalities associated with anti - vegfr agents.12 htn is a common adverse event of the treatment with antiangiogenic therapy ( such as bevacizumab ) that has been correlated with the biological inhibition of the vegf - related pathway and may represent a possible clinical marker for treatment efficacy , analogously to what has been demonstrated for skin rash and cetuximab.3 13 cumulative data , gathered from retrospective studies , show that bevacizumab - induced htn appears to be associated with a statistically significant improvement in response rates , pfs and os , raising the hypothesis that bevacizumab - related htn may serve as a predictor biomarker of response and survival in patients with mcrc treated with bevacizumb in a first - line setting . scartozzi et al12 conducted a retrospective study which included 39 patients with mcrc treated with bevacizumab as part of front - line therapy . twenty per cent of the patients developed grades 23 htn with a significant improvement in response rate ( 73.2% vs 18.4% p<0.001 ) and pfs ( 14.5 months vs 3.1 months p<0.001 ) being observed in the group of patients who developed bevacizumab - related htn with no statistically significant difference in the median os . in the sterlund et al14 study , 56% of the patients presented htn with results showing that htn predicted bevacizumab treatment efficacy regardless of the analysed end point ( os , pfs or rr ) . early htn , defined as htn within 3 months of treatment initiation , was shown to be predictive for os in this study . similar data in metastatic renal cell carcinoma , breast cancer and lung cancer have also been published.1517 dahlberg et al16 showed that htn within 1 month in bevacizumab therapy for lung cancer was predictive for survival . in our study , 51.9% patients developed htn g2-g3 during chemotherapy - bevacizumab treatment , which is superior to that reported in other retrospective trials.6 12 16 according to a recently published large meta - analysis , the incidence of htn in bevacizumab - treated patients with cancer was 23.6%.18 a possible explanation may be that our study population presents a high predominance of the male gender being more frequently affected by primary htn , suggesting that bevacizumab - related htn may present the same biological pathway , or that this increase may be possibly due to selection bias since it is a retrospective trial . our results seem to be in line with published data , with patients who developed bevacizumab - related htn showing an improvement in clinical outcomes , namely response rates ( 73.2% vs 18.4% p<0.001 responders vs non responders ) , disease control ( 97.6% vs 63.2% p 0.005 controlled disease vs non - controlled ) and pfs ( p<0.001 ) . in our group , no statistical difference in the median os was observed ( 33 months vs 21 months p 0.114 ) , although a trend towards a favourable outcome was seen in those patients with bevacizumab - induced htn . htn g2 - 3 was an independent predictor of progression - free survival for all adjusted factors ( age , ecog ps and kras status ) but not of os . this apparently conflicting result might be explained by the fact that htn is an important risk factor for cardiovascular disease being associated with an increase in cardiovascular mortality and so possibly counteracting , in the long term , the potential beneficial impact on os . the fact that more patients were submitted to surgery of the primary tumour in the group of patients who developed bevacizumab - related htn may reflect a population with less advanced disease at diagnosis . only a minority of the patients were evaluated for nras status , which is explained by the fact that this mutation analysis only became available for clinical use in our institution recently . the fact that it is a retrospective analysis , as the majority of the previous studies on this subject , presents some limitations as the lack of randomisation ( presenting possible selection bias ) and accuracy of data ( as it is carried out in a retrospective fashion ) . the small sample size may also be a limitation , although this sample is larger than that of some of the previous ones reported ( scartozzi or sterlund ) . it would be interesting to perform analysis about the potential effect of antihypertensive medication on treatment efficacy in bevacizumab - treated mcrc . owing to the sample size , the authors did not have adequate statistical power to evaluate this hypothesis . it is known that inflammation might affect blood pressure ; however , owing to the fact that the c reactive protein level at baseline was not systematically evaluated for all patients , since it is normally requested in an infectious setting , this effect could not be assessed . the accumulating evidence seems to imply that the identification of a reliable clinical factor such as bevacizumab - induced grades 23 arterial htn may constitute an early indicator efficacy , whereas lack of this side effect could represent a warning of lack of activity and maybe justify an early change in the treatment strategy , for example , egfr inhibitors.9 10 in the context of an exponential increase in economic treatment costs , contributed by targeted therapy , the identification of a reliable , non - invasive , feasible and not costly predictive factor that would allow the selection of patients / tumours more likely to benefit from treatment and spare those who would not from additional toxicity is urgent.3 19 in conclusion , in this subset of patients , htn g2 - 3 demonstrated to be predictive of response to treatment with bevacizumab and of pfs . bevacizumab treatment - related htn is a very interesting subject at the moment and prospective studies , to disclose whether bevacizumab - associated htn predicts outcome and the exact mechanism for the development of anti - vegf related htn , are needed .

different physiological and pathological conditions can perturb protein folding in the endoplasmic reticulum , leading to a condition known as er stress . er stress activates a complex intracellular signal transduction pathway , called unfolded protein response ( upr ) . the upr is tailored essentially to reestablish er homeostasis also through adaptive mechanisms involving the stimulation of autophagy . however , when persistent , er stress can switch the cytoprotective functions of upr and autophagy into cell death promoting mechanisms . recently , a variety of anticancer therapies have been linked to the induction of er stress in cancer cells , suggesting that strategies devised to stimulate its prodeath function or block its prosurvival function , could be envisaged to improve their tumoricidial action . a better understanding of the molecular mechanisms that determine the final outcome of upr and autophagy activation by chemotherapeutic agents , will offer new opportunities to improve existing cancer therapies as well as unravel novel targets for cancer treatment . backgroundbevacizumab has become standard of care as first - line treatment of metastatic colorectal cancer ( mcrc ) , after proving increased response rates and improvement in survival outcomes . hypertension ( htn ) is a common complication of the treatment with bevacizumab and , owing to its close relation with antiangiogenic mechanism , may represent a clinical biomarker to predict the efficacy of the treatment . the aim of this study was to retrospectively evaluate if htn grades 2 to 3 were correlated with response to treatment with bevacizumab in first line , as well as with improved progression - free survival ( pfs ) and overall survival ( os ) , in a series of patients with mcrc.methodsretrospective evaluation of clinical records of patients with histologically proven mcrc , treated with bevacizumab as first - line treatment , between january 2008 and december 2013.results79 patients were evaluated . 51.9% of patients developed htn g2-g3 during chemotherapy - bevacizumab treatment . in the group of patients who developed bevacizumab - related htn , 73.2% showed response to treatment ( complete response ( cr)+ partial response ( pr ) ) and 97.6% achieved disease control ( cr , pr or stable disease ) compared to 18.4% of patients with response and 63.2% with disease control in the group that did not ( or 12.08 ; 95% ci 4.13 to 35.29 ; p<0.001 responders vs non - responders ; or 20.8 ; 95% ci 2.56 to 168.91 ; p 0.005 controlled vs non controlled disease ) . the median os was 28 months ( 22.733.3 ) . significant statistical difference was obtained in pfs between the two groups ( p<0.001 ) . in the group that developed bevacizumab - related htn , the median os was 33 months ( 25.740.3 ) , and in the group that did not , it was 21 months ( 16.525.5 ) with no significant statistical difference between the two groups ( p 0.114).conclusionsin this subset of patients , htn g2 - 3 was predictive of response to treatment with bevacizumab and of pfs but not of os .

the need to advance saliva research is strongly recognized by the strategic plan of the national institute of dental and craniofacial research . the ability to monitor health status , disease onset , progression , recurrence and treatment outcome through non - invasive means is highly important to advancing health care management . saliva ( oral fluid ) is a perfect medium to be explored , offering the potential for a non - invasive , easy to obtain means for detecting and monitoring disease . the adoption of saliva testing would allow a patient to collect their own specimens at home , yielding savings in health costs , convenience for the patient and facilitating multiple sampling . specimen collection is less objectionable to patients than in the case of other bodily fluids and easier in children and older individuals . the analysis of saliva can thus provide a cost - effective approach for the screening of large populations . due to these significant advantages , developing biomarkers in saliva for the detection of serious illnesses such as oral and systemic cancers has been on the national healthcare agenda for several years ( government performance & results act 2008 ) . one mandate formulated in the government performance & results act report is that by the year 2013 proof of principle will be obtained for the ability of saliva to monitor health and diagnose for one systemic disease . a vast amount of saliva omics data has been generated by recent studies using high throughput technologies . however , there are still barriers which researchers must overcome before such data can be exploited , such as lack of computationally accessible salivary data and information , and inability to cross - reference the salivaomics data that could potentially be made available through different proteomics , transcriptomics , genomics and metabolomics studies . for these reasons , there is an urgent need to create the salivaomics knowledge base ( skb ) , a data management system and web resource constructed to support saliva diagnostics research , and we present below the informatics advances brought about through the skb and through the associated tools and resources . ontologies are controlled structured vocabularies designed to provide consensus - based means to ensure consistent description of data by scientists working in disparate domains . as applied in the biomedical domain , ontology plays a key role in providing consensus - based controlled vocabularies serving the consistent annotation of biological and medical data and information , most conspicuously within the framework of the gene ontology and now of its sister ontologies within the open biomedical ontologies foundry ( http://obofoundry.org ) . the basic formal ontology ( bfo ) is a formal ontological framework developed by barry smith , pierre grenon and others , which serves as the starting point for some 100 ontology projects primarily in the biomedical domain ( http://www.ifomis.uni-saarland.de/bfo/ ) . the bfo framework can be readily extended to the treatment of families of ontologies of other types , above all to the treatment of relations between ontologies of different levels of granularity , from genes to species and from a single patient to epidemics at a geographical scale ( combining applications of bfo to the medical and to the geographical domain ) . the framework may also be used as a tool for dealing with the relations between distinct perspectives on the biomedical domain , including culturally generated perspectives of the sort which are studied by linguists and anthropologists . two bfo - based ontologies of special significance for our work here are the ontology for biomedical investigations ( obi ) and the ontology for general medical sciences . the obi is an ontology designed to serve the coordinated representation of designs , protocols , instrumentation , materials , processes , data and types of analysis in all areas of biological and biomedical investigation . ontology for general medical science is an ontology of the entities involved in the clinical encounter . thus , it includes very general terms that are used across medical disciplines , including : disease ' , disorder ' , disease course ' , diagnosis ' , patient ' and healthcare provider ' . to advance the consistency of data in the dental research community , smith et al . propose an approach to building a consensus - based ontology to support dental research ( odr ) . in analogy to efforts in other fields , a consortium of research groups specializing in different areas of study would undertake such an effort , each building different components of ontology to support dental research . initial efforts in this direction , by scientists in dental research and biomedical ontology at university at buffalo and university of california , include work on the ontology of oral pathology , oral maxillofacial anatomy , dental disease and dental procedures , and as we discuss below , the saliva ontology . integral to his work is a plan to allow a seamless connection between the use of ontology to support dental research in the dental domain and the use of existing ontology resources developed in other areas of biology and medicine , by reusing elements and strategies from them . the work on dental diseases is carried out in conjunction with the development of ontology for general medical science . the saliva ontology ( salo ) ( figure 1 ) is a detailed ontology of this bodily fluid that is optimized to meet the needs of both the clinical diagnostic community and the cross - disciplinary community of omics researchers . the salo is created through cross - disciplinary interaction with saliva experts , protein experts , diagnosticians and ontologists . to aid development and testing of salo , we develop a corpus of saliva - relevant literature in skb to assist in characterizing core terms and synonyms within the ontology and to provide links between salo content and relevant items in pubmed . skb will also incorporate the results of experiments in data and text mining using the ontology . salo will incorporate links to existing ontologies and terminology resources involving treatment of saliva - relevant phenomena . we will also identify and represent within salo relationships to saliva - relevant types represented in ontologies such as the gene ontology , the protein ontology and the chemical entities of biological interest ontology , and also provide links to corresponding snomed ct terms where available . each term in the ontology has its own url which points to a webpage providing definitions , pubmed sources , references to annotations to skb and to external databases . it is cross - platform and supports many popular relational database managements systems , including mysql , oracle , postgresql , sql server and db2 . the software is data - agnostic , and can therefore be easily adapted to existing data sets . it is expandable and customizable through a plug - in system , and is open - source so the community can participate in deeper development . furthermore , biomart can seamlessly connect geographically disparate databases , facilitating collaboration between different groups . these features have catalyzed the creation of biomart central portal , a first of its kind community - supported effort to create a single access point integrating many different , independently administered biological databases . anybody can contribute an independently maintained resource to the central portal , allowing it to be exposed to and shared with the research community , and linking it with the other resources in the portal . users can take advantage of the common interface to quickly utilize different sources without learning a new system for each . the system also simplifies cross - database searches that might otherwise require several complicated steps . several integrated tools streamline common tasks , such as converting between i d formats and retrieving sequences . the combination of a wide variety of databases , an easy - to - use interface , robust programmatic access and the array of tools make central portal a one - stop shop for biological data querying . sdxmart is a biomart data portal that hosts salivary proteomic , transcriptomic , metabolomic and microrna data and offers access to the data by using the biomart interface and querying environment . the sdxmart is designed to provide a variety of queries to facilitate saliva biomarker discovery including complex queries that integrate genomic , clinical and functional information . the sdxmart holds data from projects of oral diseases and systemic diseases including oral cancer , sjgren 's syndrome , pancreatic cancer and breast cancer . the types of datasets are : ( i ) proteomics ; ( ii ) transcriptomics ; ( iii ) microrna ; and ( iv ) metabolomics . in addition , the sdxmart is imported with several public databases including ensembl genome database ( ensembl release 37 ) , and the number of resources is continuously growing . it is being built in tandem with the salo and sdxmart which will allow the skb to interoperate with other omics databases as part of a general strategy to facilitate integration of heterogeneous and disparate data sources that enable system biology approaches . either salo or sdxmart is a first and only resource of its kind in the field of dentistry . emergence of new concepts in the management of oncologic malignancies and the fast - changing trends of modern medicine necessitate the development of strategies for customized treatment suited to the patient 's variable requirements . response to chemotherapy can not be predicted with certainty in an individual presently . moreover , the histopathological changes after chemotherapy vary in patients . studies have systematically described various histopathological changes seen after neoadjuvant chemotherapy in a variety of tumors including breast carcinoma , rectal carcinoma , ovarian carcinoma , head and neck carcinomas , esophageal carcinoma , wilms tumor , and non - small - cell lung carcinoma . however , the emphasis of these studies varied and no study described the pathologic changes following chemotherapy comprehensively . this study describes the various histopathologic changes seen after neoadjuvant chemotherapy in breast malignancies , squamous cell carcinomas , adenocarcinomas , and wilms tumor . the present prospective study was carried out on 60 patients including 40 patients with carcinoma breast and 20 patients with other malignancies who received neoadjuvant chemotherapy . during a study period of one - and - a half years , a total of 355 patients were treated for breast carcinoma with neoadjuvant chemotherapy . of these , 40 patients fulfilling the inclusion criteria as per the protocol of the thesis project were included in the study . inclusion criteria was patients with prechemotherapy clinicoimaging assessment of tumor size , with an established histological diagnosis of carcinoma on biopsy , and who had received at least two cycles of neoadjuvant chemotherapy . the study group was divided into three subgroups ; group a ( n = 30 ) and group b ( n = 10 ) included breast carcinoma patients with initial pathologic material submitted as trucut or wedge biopsy in group a and lumpectomy specimens in group b. group c ( n = 20 ) included cases of other malignancies besides breast carcinoma who received neoadjuvant chemotherapy . after initial tissue diagnosis , the patients were clinically examined in detail and investigated including radiological , imaging , and other laboratory tests to decide the stage of the cancer . the initial biopsies were subjected to routine formalin fixation and paraffin processing with microscopic analysis on hematoxylin- and eosin - stained sections supplemented by special stains including immunohistochemistry to decide histological type and grade of the tumor according to the world helath organization ( who ) classification . detailed histopathological examination was carried out especially looking for chemotherapy - induced histopathologic changes like necrosis , fibrosis , inflammatory reactions , and other retrogressive changes . for carcinoma breast , neoadjuvant chemotherapy regimens either included cyclophosphamide 50 to 60 mg / m2 iv ( iv : intravenous ) , doxorubicin 40 to 50 mg / m2 iv , and 5-fluorouracil 500 to 800 mg / m2 iv ( caf ) , or cyclophosphamide , epirubicin , and 5-fluorouracil ( cef ) , 21 days apart . number of chemotherapy cycles varied from two to six depending on the initial size of the tumor to make them operable . the drugs and doses of neoadjuvant chemotherapy given to the patient were recorded in the pro forma . the tumor cells were evaluated for dissociation , dyscohesion , and loss of organization of the tumor cells and necrobiotic changes such as necrosis , vacuolation of nucleus and cytoplasm , karyorrhexis , pyknosis , and karyolysis . any change in pattern or type of carcinoma was noted . the stroma was examined for host response including fibrosis , elastosis , and collagenization , and infiltration by lymphocytes , plasma cells , fibroblasts , histiocytes , and giant cell formation was observed . epithelial - to - stromal ratio was calculated as the mean of readings in all sections and viable - to - nonviable tumor cell ratios were calculated in both pretreatment biopsies and postchemotherapy specimens , viability being defined as distinct nuclear chromatin with intact nuclear and cytoplasmic membrane in the absence of the criteria of necrosis ( karyorrhexis , karyolysis , pyknosis ) . a note was made on the tumor type and any change after chemotherapy ; tumor grading was done based on the elston and ellis modification of bloom - richardson ( mrb ) grading system and nottingham prognostic index ( npi ) was calculated . lymphocytic response was graded as : grade 1 , scattered lymphocytes between tumor cells ; grade 2 , formation of microaggregates of lymphocytes ; grade 3 , dense infiltration of lymphocytes destroying tumor cells or forming masses . the presence of lymphovascular embolization and in situ disease / cancerization of ducts were separately noted . twenty cases of other malignancies ( group c ) besides breast included 12 cases of squamous cell carcinoma ( seven from head and neck , four of cervix , and one of esophagus ) , five cases of adenocarcinoma , and three of wilms tumor . neoadjuvant chemotherapy for squamous cell carcinoma of head and neck included chemotherapy based on docetaxel , cisplatin , and 5-fluorouracil ( tpf regimen ) . patients with carcinoma cervix received paclitaxel and carboplatin regimen , and for esophageal carcinoma , cisplatin and 5-fluorouracil was given . chemotherapy given for ovarian carcinoma was paclitaxel and carboplatin , and patients with anorectal carcinoma received a 5-fluorouracil- and oxaliplatin - based regimen ( folfox ) . children with wilms tumor received vincristine and dactinomycin cycles varying from three to four cycles , as was considered appropriate for reducing the bulk of the tumor . histologic sections from all the malignancies were observed for any change in differentiation , changes in architecture , necrobiotic changes , and host tissue response . pearson 's coefficient of correlation was applied to correlate the three types of response patterns with all the parameters observed . breast tumors which showed complete response after neoadjuvant chemotherapy included five of ductal carcinoma - not otherwise specified ( nos ) type , and one each of schirrhous , apocrine , and tubular types . apocrine change and tubular and papillary pattern disappeared in postchemotherapy specimens ; mucinous change and metaplastic change appeared in two each of ductal nos types . ductal nos with metaplastic change differentiated to completely metaplastic in mastectomy specimens [ table 1 ] . carcinoma breast : histological types of the tumor before and after chemotherapy number of cases with metastasis was 22 ; number of lymph nodes with metastatic deposits was 88 . the most common pathologic changes noted were necrosis , elastosis / collagenization , and lymphocytic response . there was no relation of lymphocytic response with the clinical or pathologic response irrespective of their presence in the pre or postchemotherapy specimens . giant cell response was significantly correlated to all the types of response grades ( p < 0.05 ) . collagenization was significantly correlated to pathologic and tumor regression grade ( p < 0.05 ) [ tables 2 and 3 ] . carcinoma breast : inflammatory and stromal response seen after neoadjuvant chemotherapy carcinoma breast : inflammatory and stromal response seen after neoadjuvant chemotherapy in axillary lymph nodes in the present study , of the total 20 cases of group c , 12 cases were of squamous cell carcinoma ( seven from head and neck , four of cervix , and one of esophagus ) , five cases were of adenocarcinoma , and three cases were of wilms tumor . of the 12 cases of squamous cell carcinoma in the present study , there was improvement in histological differentiation from moderate to well - differentiated carcinoma in two cases of head and neck cancers , whereas there was complete pathologic disappearance of tumor in another two cases comprising one case each of carcinoma tongue and cervical carcinoma . other histological changes observed were increase in keratinization with formation of keratin pearls , acellular keratin with islands of nonviable tumor cells , histiocytic giant cells , and increase in lymphocytes surrounding residual tumor cells . of the five cases of adenocarcinoma , a change in histological differentiation was seen in a case of anorectal carcinoma which revealed poor differentiation with solid sheets , a small amount of mucin in the initial biopsy , and moderately differentiated mucin - secreting adenocarcinoma with large mucin pools after chemotherapy . in serous papillary adenocarcinoma of the ovary , degenerative changes were observed with karyorrhexis , pyknosis , smudging , dyscohesion , and loss of papillary architecture in some areas . in a single case of esophageal adenocarcinoma , an increase in signet ring cells adenocarcinoma : histological differentiation before and after chemotherapy of the three wilms tumors , one was biphasic and two were triphasic . in the two cases revealing triphasic wilms tumor in the pretreatment biopsy , blastemal component decreased with marked necrobiotic changes and totally disappeared in the one with biphasic wilms tumor . mesenchymal component revealed rhabdoid differentiation in one and chondroid differentiation in the other with the presence of smooth and skeletal muscles . in the present prospective study , a spectrum of histopathologic changes was observed with the use of chemotherapy . the effects have been divided into two , as pathologic changes in tumor cells and changes in the stroma . in the tumor cells , nuclear enlargement , nuclear shrinkage , necrosis , vacuolation of nucleus , cytoplasm , pyknotic nuclei , and degenerative changes have been described in the literature.[159 ] in the present study , loss of architecture , dyscohesion , and shrinkage of tumor cells with retrogressive changes like karyorrhexis , karyolysis , and pyknosis were observed in addition to the above - mentioned changes . in view of the presence of a large variety of retrogressive changes , the emphasis was more on trying to recognize viable tumor cells , morphologically identified as cells with distinct nuclear chromatin with intact nuclear and cytoplasmic membrane in the absence of criteria of necrosis ( karyorrhexis , karyolysis , pyknosis ) . necrosis was the most common event observed . in the stroma , fibrosis , elastosis , collagenization , hyalinization , microcalcification , and neovascularization have been observed and described.[56813 ] in the present study , in addition , some prominent findings were elastosis/ collagenization of the stroma , hyalinization of the walls of the blood vessels , and atrophy of the adjacent breast parenchyma and cancerization of ducts even in atrophic lobules . collagenization was found to be significantly correlated to pathologic response and tumor regression grade ( p < 0.05 ) . lymphocytic reaction and the presence of plasma cells and macrophages with the formation of histiocytic giant cells observed in many studies may be indicative of host tissue response to necrobiotic tumor.[5101214 ] the most common inflammatory host response observed in the present study was lymphocytic ; others included mixed inflammation , plasmacytic , prominent histiocytic , and giant cell types . giant cell reaction was significantly correlated to all types of tumor responses ( p < 0.05 ) . pathologically similar changes were observed in response to chemotherapy in draining lymph nodes but not as pronounced as in the primary site . though these changes have been observed by many workers , positive correlation of the presence of these changes with the effect on chemotherapy has not been significant in many studies . according to them , the presence of fibrosis , elastosis , collagenization , hyalinization , necrosis , inflammatory infiltrate , lymphocytic response , giant cell reaction , plasma cells , foamy macrophages , microcalcification , and neovascularization were not significantly related to the response to chemotherapy . honkoop et al . concluded that none of the pretreatment pathologic or biologic characteristics were predictive of a good pathologic response . in the present study , the most common type was ductal carcinoma nos ; mucinous change appeared in three patients after chemotherapy who were diagnosed with ductal carcinoma nos type in the initial biopsy , and a transformation from ductal to metaplastic carcinoma and from papillary pattern to ductal nos was seen in one case each . a change to either a higher or lower grade was noted by rasbridge et al . complete response was seen in one case of apocrine carcinoma and disappearance of apocrine component in mixed ductal with apocrine differentiation in another . the response in the case of ductal carcinoma nos was not significantly different from the response in invasive lobular carcinoma . in the present study , the clinical and pathologic response was not found to have a significant correlation with the prechemotherapy grade . however , significant correlation was observed between the postchemotherapy grade and the pathologic and tumor regression grades but not with clinical response . so , a better response to chemotherapy was observed in the poorly differentiated malignancies with higher mrb and npi grades and a poorer response with well - differentiated ones ( p < 0.05 ) [ figure 1 ] . in contrast , sanchez et al . observed a poor response in poorly differentiated carcinomas . carcinoma breast : occasional nests of viable tumor cells left post chemotherapy ( h and e ; 100 ) post neoadjuvant chemotherapy , hyalinization of the blood vessel wall was a common finding . the adjoining breast showed atrophy of parenchyma ; even then , terminal duct lobular units ( tdlus ) revealed ductal carcinoma in situ ( dcis)/ cancerization of ducts by surviving tumor cells in some cases . no correlation of size with the response grades was observed , that is , size reduction after chemotherapy was seen in both small and large tumors and was not restricted to any one group . in squamous cell carcinoma , changes observed were improvement in histologic differentiation in some , complete pathologic disappearance of the tumor in a few , an increase in keratinization with formation of keratin pearls , acellular keratin with islands of nonviable tumor cells , histiocytic giant cells , and increase in lymphocytes surrounding residual tumor cells in most cases [ figure 2 ] . in adenocarcinoma , an improvement in histologic differentiation was seen with large mucin pools after chemotherapy , degenerative changes were observed in serous papillary adenocarcinoma of the ovary , and an increase in signet ring cells was noticed in an esophageal carcinoma [ figure 3 ] . in wilms tumor , there was an almost complete disappearance of blastemal component and retrogressive changes in the epithelial component . squamous cell carcinoma : after chemotherapy , only necrotic masses of keratin with areas of calcification are left ( h and e ; 100 ) adenocarcinoma : abundant pools of mucin with occasional scattered tumor cells after chemotherapy ( h and e ; 40 ) wilms tumor : only few bizarre tumor cells persisting along with the mesenchymal component with complete disappearance of the blastemal component which was a triphasic tumor before chemotherapy ( h and e ; 200 ) the results of this study again reveal that the response to chemotherapy may be markedly variable in patients . the desired response in some may be achieved with a fewer number of cycles , whereas in other patients , the tumor may resist even with the maximum number of neoadjuvant chemotherapy cycles presently employed , thus defeating the very purpose even at the potential risk of toxicity . it was also concluded that the tumor grade decreases and differentiation improves , in addition to the retrogressive changes and increase in stromal component as a result of chemotherapy in carcinoma breast as well as in other malignancies .

there is a need recognized by the national institute of dental & craniofacial research and the national cancer institute to advance basic , translational and clinical saliva research . the goal of the salivaomics knowledge base ( skb ) is to create a data management system and web resource constructed to support human salivaomics research . to maximize the utility of the skb for retrieval , integration and analysis of data , we have developed the saliva ontology and sdxmart . this article reviews the informatics advances in saliva diagnostics made possible by the saliva ontology and sdxmart . background : various histopathological changes have been observed following neoadjuvant chemotherapy in individual tumors in the literature.aims and objectives : to observe histopathologic changes seen after neoadjuvant chemotherapy in breast malignancies , squamous cell carcinomas , adenocarcinomas , and wilms tumor using breast cancer predominantly as the model.materials and methods : the present prospective study was carried out on 60 patients including 40 patients with carcinoma breast and 20 patients with other malignancies who received neoadjuvant chemotherapy.results:post neoadjuvant chemotherapy , mastectomy specimens revealed nuclear enlargement , nuclear shrinkage , necrosis , vacuolation of nucleus , vacuolation of cytoplasm , dyscohesion , and shrinkage of tumor cells with nuclear changes of nonviability like karyorrhexis , karyolysis , and pyknosis . stromal reactions manifested as fibrosis , elastosis , collagenization , hyalinization , microcalcification , and neovascularization . areas of necrosis included both vascular and avascular pattern . the stroma also revealed fibrinoid necrosis and mucinous change . hyalinization of the blood vessel wall was a common finding . the most common inflammatory host response observed in the present study was lymphocytic ; others included mixed inflammation , plasmacytic , prominent histiocytic , and giant cell types . giant cell reaction was significantly correlated to all types of tumor responses ( p < 0.05 ) . similar changes were also observed in other malignancies . a detailed review of the literature has also been done and presented.conclusion:the tumor grade decreases and differentiation improves , in addition to the retrogressive changes and increase in stromal component , as a result of chemotherapy in carcinoma breast as well as in other malignancies .

acute myeloid leukemia ( aml ) is a clinically , morphologically and genetically heterogeneous disease characterized by clonal expansion of myeloid blasts in peripheral bone marrow ( bm ) , blood or other tissues . the incidence of aml increases with age , particularly after 65 years old , and the median age at diagnosis is around 70 years . age in itself is one of the most powerful prognostic factors for survival in aml , , . elderly patients not only present more comorbidities and worse performance status but also have more adverse molecular features compared to younger patients , , . due to the presence of comorbidities and poor performance status , intensive chemotherapy ( ic ) or allogeneic stem cell transplant is often unsuitable for a large proportion of elderly patients with aml , resulting in early mortality and prolonged hospitalization , . however , when feasible , treatment is associated with better survival when compared to best supportive care only but the median overall survival achieved with ic in the elderly is only 513 months , . therefore , there is an increasing need for new treatment options for elderly patients with aml . the aza-001 phase iii trial has demonstrated effectiveness of azacitidine ( aza ) in a dose of 75 mg / m2 daily for 7 consecutive days in patients with high - risk myelodysplastic syndromes . a subset analysis of the aza-001 trial restricted to patients with 2030% bm blasts , demonstrated superior efficacy of aza when compared with conventional care regimens , leading to aza international approval for the treatment of low bm blast count ( 2030% ) who - defined aml . these results raise the question of the potential use of aza in aml irrespectively of the bm blast count . this is supported by the identification of somatic mutations of genes involved in epigenetic regulation in approximately 30% of aml patients . consequently several groups have assessed and reported the efficacy of aza in patients with aml , , , . more recently a randomized phase iii trial comparing first - line aza to conventional therapies , including intensive chemotherapy , in elderly patients with aml revealed that aza achieved clinically meaningful survival benefit compared to conventional treatments . we present herein a multi - centre retrospective analysis of 77 aml patients who were treated with aza in first line or due to relapsed / refractory disease . the main aim was the characterization of the efficacy and safety of aza treatment in aml patients in portugal . we also conducted an exploratory comparison of overall survival between patients receiving 1st line treatment with aza and elderly aml patients treated with ic alone . patients with aml who received at least 1 cycle of aza were selected for inclusion . the comparator group comprised 50 matched patients from a historical cohort of aml patients from a single institute . patient data was collected retrospectively from patient files from 4 portuguese institutions following written informed consent . the study was approved by the ethical committee of instituto portugus de oncologia de lisboa , francisco gentil , epe and all data treated according to the declaration of helsinki . overall survival ( os ) was defined as time from start of treatment with aza ( either 1st or 2nd line ) or ict ( 1st line ) to death from any cause or last follow - up . progression - free survival ( pfs ) was defined as time from start of treatment until disease relapse / progression or death from any cause . survival curves of the different treatment groups were compared using the log - rank test ( univariate analysis ) . all tests were two - tailed and p - values less than 0.05 were considered to be statistically significant . seventy - seven patients treated with aza and 50 elderly aml patients treated with ict were included . 1line n=51ict n ( % ) n=50p - value aza1st line vs ictage at diagnosis69 years [ 2789]73 years ( 4689)74.5 years ( 6686)0.015median [ min - max]33 ( 65% ) > 70yrs41 ( 82% ) > 70yrsgendermale54 ( 70%)36 ( 72%)28 ( 56%)0.13female23 ( 30%)15 ( 29%)22 ( 44%)percentage blasts at start of treatmentmedian [ min - max]32 [ 093]35 [ 2092]50 [ 20100]0.06mean ( standard deviation)37 ( 23)40 ( 20)52 ( 30)haemoglobin10 g / dl58 ( 75%)38 ( 74.5%)38 ( 76%)0.39wbc ( x109/l)0.72 [ 0.01 30.60]0.71 [ 0.03 17.05]0.81 [ 0.01 23.91]0.56neutrophils0.5 x10/l26 ( 34%)20 ( 39%)13 ( 26%)0.05platelets50 x10/l34 ( 44%)23 ( 45%)24 ( 48%)0.59aml subtypeprimary44 ( 57%)33 ( 64%)30 ( 60%)0.21secondary33 ( 43%)18 ( 36%)20 ( 40%)cytogenetics risk groupnormal36 ( 47%)17 ( 33%)16 ( 32%)intermediate*22 ( 29%)23 ( 45%)17 ( 34%)0.45poor**17 ( 22%)11 ( 21%)17 ( 34%)unknown2 ( 3%)*all others.**chromosome 7 ; > 3 abnormalities . other than higher age in the ict control group , there was no significant difference between patients treated with aza or ict in first line . table 2treatment details.table 2aza n ( % ) n=77aza 1line n ( % ) n=51ict n ( % ) n=50p - valueaza1st line vs icttime from diagnosis to start of aza , months1.4 [ 089.3]0.6 [ 048.3]1.3 [ 09.2]0.195median [ min - max]first line treatmentaza , n ( % ) 51 ( 100%)ict ( 3 + 7)24 ( 31%)n / adauno 90 + arac 10010 ( 20%)dauno 60 + arac 10021 ( 42%)ida 12 + arac 1009 ( 18%)total no of cyclesmedian [ min - max]6 [ 128]1.7 ( 13)n / aaza = azacitidine ; ict = intensive chemotherapy ; dauno 90+arac 100=daunorubicin 90 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; dauno 60+arac 100=daunorubicin 60 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; ida 12 + arac 100=idarubicin 12 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 . aza = azacitidine ; ict = intensive chemotherapy ; dauno 90+arac 100=daunorubicin 90 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; dauno 60+arac 100=daunorubicin 60 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 ; ida 12 + arac 100=idarubicin 12 mg / m d1 - 3 , cytarabine 100 mg / m d1 - 7 . between start of aza and table 3response to therapy.table 3n ( % ) aza 1line n=51ict n ( % ) n=50p - value aza1st line vs ictresponse ratesoverall response ( pr+cr)34 ( 44%)26 ( 51%)38 ( 76%)partial response10 ( 31%)7 ( 14%)11 ( 22%)complete response24 ( 13%)19 ( 37%)27 ( 54%)0.02stable disease22 ( 29%)13 ( 25%)progression / refractory21 ( 27%)12 ( 24%)12 ( 24%)transfusion independenceno44 ( 57%)27 ( 53%)yes30 ( 39%)24 ( 47%)n / an / aunknown3 ( 4% ) four ( 3.3% ) patients treated with aza were not evaluable for survival . median follow - up time in patients was 12.4 months in patients receiving aza as first line treatment and 6.9 months in patients treated in second or subsequent lines . during this period 1 . survival estimates for patients treated with aza according to line of treatment . fig . median age at diagnosis of the comparator group was 74.5 years - old ( range : 6686 ) and 44% ( 22 patients ) were female ( table 1 ) . the median time from diagnosis to start of ic in this comparator group was 0.13 months ( range : 09.2 months ) ( table 2 ) . all patients treated with oct alone received only supportive care prior to induction chemotherapy . fig . 4survival of aml patients treated with aza first line compared to those treated with ict alone.fig . 4 . survival of aml patients treated with aza first line compared to those treated with ict alone . table 3response to therapy.table 3n ( % ) aza 1line n=51ict n ( % ) n=50p - value aza1st line vs ictresponse ratesoverall response ( pr+cr)34 ( 44%)26 ( 51%)38 ( 76%)partial response10 ( 31%)7 ( 14%)11 ( 22%)complete response24 ( 13%)19 ( 37%)27 ( 54%)0.02stable disease22 ( 29%)13 ( 25%)progression / refractory21 ( 27%)12 ( 24%)12 ( 24%)transfusion independenceno44 ( 57%)27 ( 53%)yes30 ( 39%)24 ( 47%)n / an / aunknown3 ( 4% ) four ( 3.3% ) patients treated with aza were not evaluable for survival . median follow - up time in patients was 12.4 months in patients receiving aza as first line treatment and 6.9 months in patients treated in second or subsequent lines . during this period 1 . survival estimates for patients treated with aza according to line of treatment . fig . median age at diagnosis of the comparator group was 74.5 years - old ( range : 6686 ) and 44% ( 22 patients ) were female ( table 1 ) . the median time from diagnosis to start of ic in this comparator group was 0.13 months ( range : 09.2 months ) ( table 2 ) . 4survival of aml patients treated with aza first line compared to those treated with ict alone.fig . 4 . survival of aml patients treated with aza first line compared to those treated with ict alone . their frailty imposes limitations upon the aggressiveness of the treatment which can be administered , . in addition , the poor prognostic disease features often seen in this population mean that refractoriness and relapse are more frequent . treatments for this population require an optimal balance of tolerability and efficacy . the main aim in most cases is to prolong survival and reduce the impact of cytopenias without undue toxicity . aza has demonstrated efficacy in high risk mds , reducing transfusion requirements and prolonging survival . in this setting it had proven to be well tolerated including by those more elderly . based on this experience , several groups have tested the efficacy and safety of aza in elderly patients with aml with promising results , , , . our results are in line with previous reports that aza is efficacious in the treatment of aml regardless of the blast count . the response rates and the overall survival observed in our study are in consonance to those reported by other groups , , , . it is noteworthy that the rates of cr obtained with aza are equivalent to those obtained in some published series of elderly patients treated with ic . improved survival in our cohort was significantly associated with any quality of response , including maintenance of stable disease . this suggests that aza is capable of delaying progression , and thus prolong survival , even if no hematological or marrow improvement are obtained . this supports its use in this population until frank progression is detected or until the patient receives another more definitive treatment , such as allogeneic bone marrow transplant . another significant finding of our analysis is the efficacy of aza in those who are refractory or relapsed following ic . the fact that survival from start of aza is similar in first and second line patients indicates that relapsed / refractory patients benefit equally . the comparison of the cohort treated with aza first line with the comparator group treated only with ic highlights the advantages brought by the former treatment modality . it is important to note that this finding has not been confirmed in a randomized trial . however , 41% of the trial patients were treated with aza following failure with ict whereas our comparator group never received aza . as aza has demonstrated efficacy as second line therapy , this could explain the difference in our findings . the significantly lower survival of the ic cohort indicates that this population benefits from less intensive treatment options which target other pathophysiological processes . the retrospective nature and the limited numbers of patients in the collaborative nature of the study helps reduce bias which may be introduced by single centre data . however our study adds new evidence to the use of aza in elderly patients with aml and indicates that it is at least not inferior to conventional ic . the response rates and survival data here presented support its use in first line in those who are not candidates for ic and in second line in those who are refractory or relapsed following ic . this and future collaborative studies will add to trial data to better identify those patients who benefit most from new treatment modalities , thus improving the management of elderly patients with aml . one of the most important organ systems is cardiovascular that it 's disorders specially ischemic heart diseases can cause disability or death . some people specially workers who work in special industries are more in the risk of same disorders . there were many studies work on workers health but there were some of them , study on cardiovascular system because these disorders have more than one etiology and one of the modifiable risk factors or etiology is work exposure . according to these realities ; cardiovascular disorders risk factors were assessed and measured such as triglyceride , total cholesterol , low density lipoprotein ( ldl ) , high density lipoprotein ( hdl ) , blood pressure , obesity , smoking , genetic . also shift work , stress , carboxi hemoglobin ( cohb ) , solvents , noise , vibration , temperature of environment , air pollution can find the high risk personnel with non occupational and occupational risk factors of cardiovascular disorders . exposure to fumes , solvents , additives and drugs , carbon monoxide , carbon disulfide , pesticides , heavy metals , cold or warm environment , air pollution , vibration and noise can cause occupational cardiovascular disorders had demonstrated work as a farmer , have a heavy and manual work , exposure to noise and some physical or chemical were cardiovascular disorders risk factors . ghiasvand et al . in their study had shown that work in rail road industry was a risk factor and can cause hyper low density lipoprotein , specially in workers who had shift work . in study of national heart , lung , and blood institute had been shown that the work in tunnel can cause exposure to carbon monoxide and other study on foundry workers can cause the same exposure . kim et al . in other study had demonstrated that work in refrigerated industry was not a risk factor but workers who work in cold place and environment had diastolic blood pressure in more amount , it was in comparison between exposure to cold and non exposure to it . had shown that work as a engine driver had cardiovascular risk factors , because they had more hypertension and hyperlipidemia than control group . kang et al . in their study had found that work in companies and related job mental stress specially in the background of decision making it was related to amount of cholesterol and triglyceride , in the background of demands of work these were related to systolic blood pressure . chin - ming lin et al . had demonstrated that medical technicians had more hypertensive disorders than other healthcare workers . also kim had shown the relationship between cold exposure and increasing of blood pressure or hypertension . the objective of this study was comparison of cardiovascular risk factors between workers in different industries of iran . this study was performed in khorasan razavi and tehran industries in 2007 - 2011 , workers who divided to three groups ; automobile industry who work in production saloon , food industry and light works such as office worker , administrative , healthcare , sanitary workers . in a cross - sectional study with simple randomized sampling method workers had been selected . with = 0.05 , = 0.80 , p = 25% population were at least 150 person in each group from 10 factories of automobile , food industries and light work . this study has been done by completing checklist from medical issues of workers . for validity and reliability of research tool ; checklist have been written and improved in educational department with professors opinions , it had pilot study with correlation coefficient of 85% and it have been used . data were gathered from medical issues because all workers had periodic physical and paraclinic examinations such as blood pressure blood sampling for triglyceride , total cholesterol , low density lipoprotein , high density lipoprotein , fasting blood sugar , age , body mass index , work duration , smoking , shift work . inclusion criteria was at least three year work duration in the same industries and exclusion criteria were previous cardiovascular disorders or dyslipidemias . three groups had been observed for age , work duration , body mass index , shift work . data were gathered in spss 11.5 and analyzed for calculation of frequency , mean , anova for quantitative variables , chi-2 for qualitative variables and multi nominal logistic regression with p < 0.05 . in regression cardiovascular risk factors such as hypertension , hyper triglyceridemia , hyper cholesterolemia , hyper low density lipoproteinemia ( ldl ) , lower high density lipoproteinemia ( hdl ) , diabetes mellitus , smoking has been analyzed . this study has been approved by university board , for research ethics ; with oral satisfaction and i told that cumulative data have been used result will be without name of industries . this study was performed in khorasan razavi and tehran industries in 2007 - 2011 , workers who divided to three groups ; automobile industry who work in production saloon , food industry and light works such as office worker , administrative , healthcare , sanitary workers . in a cross - sectional study with simple randomized sampling method workers had been selected . with = 0.05 , = 0.80 , p = 25% population were at least 150 person in each group from 10 factories of automobile , food industries and light work . this study has been done by completing checklist from medical issues of workers . for validity and reliability of research tool ; checklist have been written and improved in educational department with professors opinions , it had pilot study with correlation coefficient of 85% and it have been used . data were gathered from medical issues because all workers had periodic physical and paraclinic examinations such as blood pressure blood sampling for triglyceride , total cholesterol , low density lipoprotein , high density lipoprotein , fasting blood sugar , age , body mass index , work duration , smoking , shift work . inclusion criteria was at least three year work duration in the same industries and exclusion criteria were previous cardiovascular disorders or dyslipidemias . three groups had been observed for age , work duration , body mass index , shift work . data were gathered in spss 11.5 and analyzed for calculation of frequency , mean , anova for quantitative variables , chi-2 for qualitative variables and multi nominal logistic regression with p < 0.05 . in regression cardiovascular risk factors such as hypertension , hyper triglyceridemia , hyper cholesterolemia , hyper low density lipoproteinemia ( ldl ) , lower high density lipoproteinemia ( hdl ) , diabetes mellitus , smoking has been analyzed . this study has been approved by university board , for research ethics ; with oral satisfaction and i told that cumulative data have been used result will be without name of industries . results are in three sections , general information , specific information and cardiovascular risk factors from 875 workers of industries . 403 ( 46.0% ) from automobile industry , 166 ( 19% ) from food industry and 304 ( 35% ) from light works had been participated in this study . in general mean of age , work duration , body mass index , smoking had been calculated . the mean of age in automobile industry was 34.87 6.19 years old , in food industry was 34.13 8.64 years old and in light works was 31.40 5.70 years old with f = 25.146 and p < 0.001 . the mean of work duration in automobile industry was 10.37 4.86 years , in food industry was 5.94 2.46 years and in light works was 5.16 1.98 years with f = 117.286 and p < 0.001 . 76 ( 18.8% ) of automobile industry workers were smoker , none of the food industry worker and 11 ( 3.6% ) of light works were smoker . in specific information mean of systolic blood pressure , diastolic blood pressure , triglyceride , total cholesterol , low density lipoprotein ( ldl ) , high density lipoprotein ( hdl ) , fasting blood sugar had been measured . in table 1 these information had been demonstrated and there were comparison of related variables to cardiovascular disorders between industries workers . comparison of risk factors to cardiovascular disorders between industries workers then number and percent of cardiovascular risk factors such as obesity , hypertension , hyper triglyceridemia , hyper cholestrolemia , hyper low density lipoprotein , lower high density lipoprotein , diabetes mellitus , were determined and compared in table 2 . although the person - time in automobile industry , food industry and light works were 4179.11 , 986.04 and 1568.64 . according to the results workers of food industry more healthy than other industries for cardiovascular disorders , mean of high density lipoprotein ( hdl ) was more than 63 mg / dl and it was preventive factor . also according to results automobile industry workers had more obesity , hypertension , hyper triglyceridemia , hyper low density lipoprotein , lower the high density lipoprotein , diabetes mellitus . after the regression analysis , hypertension , hyper triglyceridemia , lower high density lipoprotein had significant difference and more in automobile industries . there were some same results in previous studies , but total cholesterol , systolic and diastolic blood pressure were more in food industry workers but in the normal range . sancini et al . had demonstrated that exposure to noise and some physical or chemical were cardiovascular disorders risk factors . had demonstrated that work as a engine driver had cardiovascular risk factors , because they had more hypertension and hyperlipidemia than control group . ghiasvand et al . in their study had shown that work in railroad industry was a risk factor for cardiovascular diseases and can cause hyper low density lipoprotein and total cholesterol in blood , specially in workers who had shift work . cardiovascular disorders such as hypertension can cause stroke , heart diseases , and had relation to long time disability and out of work . forbidden the smoking , control of temperature were popular advices for managers of industries to have healthy workers . there were fumes , solvents , stress , heavy metals , in automobile industry that affect the cardiovascular system , although were controlled . in study of national heart , lung , and blood institute had been shown that the work in tunnel can cause exposure to carbon monoxide and other study on foundry workers can cause the same exposure . but about food industry temperature of environment is more important risk factor , that was controlled in food industries of iran . the workers in automobile industries had more work duration , older and body mass index . also light works such as office worker , administrative , healthcare , sanitary workers had more risk factors than food industry workers , that may be had better health with follow up of screening results . screening of cardiovascular disorders risk factors in all works and industries can helpful and benefit . according to other studies in some industries controls of shift work , stress , noise , vibration and chemical exposures are helpful . in this study , after multi nominal logistic regression ; hypertension , hyper triglyceridemia , lower the high density lipoprotein were significant and the most in workers of automobile industry . automobile industry have a lot of cardiovascular risk factors , these were fumes and other chemicals , physicals , control of them can reduce the risk of occupational cardiovascular disorders , one of the ways to control was engineering method , then administrative and at the end personal protective devices . medical surveillance with periodic examination for screening of non occupational and occupational disorders , specially risk factors of cardiovascular disorders can be helpful . screening of cardiovascular disorders risk factors were important and helpful in industries specially automobile industry , that might be preventive method for these disorders in the future .

retrospective data was collected from 77 elderly acute myeloid leukemia ( aml ) patients treated with azacitidine ( aza ) and 50 elderly aml patients treated with intensive chemotherapy ( ic ) from 4 portuguese hospitals.median os was 10.6 months in those receiving aza as 1st line . response ( overall response rate 44% ) had a significant impact on overall survival ( p=<0.0001 ) . median os of the comparator ic cohort was significantly inferior to that observed in the cohort treated with aza in first line ( p=0.0104).these results support the efficacy of aza in aml in the elderly in any line of treatment . background : disorders of cardiovascular system can cause disability or death , screening is necessary specially in workers who maybe had risk factors . hypertension , hyperlipidemia , obesity , smoking , genetic , exposure to chemicals , fumes , solvents , coldness are non occupational and occupational risk factors . objective was comparison of cardiovascular disorders risk factors between workers in different industries of iran.methods:in a cross - sectional study , workers of automobile , food industries and light works had been selected and cardiovascular disorders risk factors had been gathered then data analyzed in spss with one - way anova , chi-2 and multi nominal logistic regression with p < 0.05.results:875 workers had been participated in the study , all of the cardiovascular disorders risk factors were in the normal range . mean of high density lipoprotein ( hdl ) in food industry workers was 63.83 17.42 mg / dl and it was protective , but in workers who work in automobile industry was 38.97 11.08 mg / dl and the lowest , also hypertension and hypertriglyceridemia were more prominent in this industry and after regression with p < 0.05 , the differences were significant.conclusions:screening of cardiovascular disorders risk factors were important and helpful in industries specially automobile industry , that might be preventive method for these disorders in the future .

vesicoureteral reflux ( vur ) occurs commonly in children , resulting in potentially serious complications . this could result in renal scarring , hypertension , and even end - stage renal failure . treatment options for children with vur include continuous antibiotic prophylaxis , endoscopic injections , and laparoscopic or open ureteroneocystostomy . since hutch introduced open ureteral reimplantation to correct vur in 1952 , many such techniques have been developed . in 1984 , endoscopic injections of tissueaugmenting material for the correction of vur were introduced ; since then , many urologists prefer this procedure . endoscopic injection is frequently used now as a firstline treatment because it involves less pain and a shorter hospitalization period and leaves no scar , in contrast to open reimplantation . however , when an injection treatment fails , open ureteral reimplantation should be considered for treating persistent vur . we aimed to test the usefulness of open reimplantations , which were performed primarily and after failed endoscopic injection procedures . we analyzed the records of 81 children who underwent open reimplantation surgery over about 10 years . among them , 64 underwent open reimplantation as a primary procedure ( group a ) , and 17 underwent it after failed endoscopic injection ( group b ) . we retrospectively analyzed the clinical data of both groups , including age , sex , preoperative reflux grade , operation time , laterality of surgery , postoperative complications , and hospitalization period . all patients were classed in terms of preoperative reflux grade using voiding cystourethrography ( vcug ) . reflux grade was classed as grades i - v according to the international classification system of the international reflux study committee . we classified patients with reflux grades i - iii as a low - grade reflux group , and patients with grades iv and v as a high - grade reflux group . success of surgery was defined as resolution of the vur as determined by vcug 6 months after surgery . in addition , we conducted a telephone - based survey of the parents of group b about their preference for endoscopic injection or open reimplantation , and the reasons for their preference ; we asked the parents " which surgery do you choose primarily between endoscopic injection again and ureteral reimplantation if your child have to undergo surgery for vesicoureteral reflux again ? and what is the reason ? " . statistical analysis was performed using ibm spss statistics ver . 20.0 ( ibm co. , armonk student t - tests and chi - square tests were applied to compare the parameters of each group ; p - values < 0.05 were considered statistically significant . the mean ages of groups a and b were 49.637.1 months and 56.522.5 months , respectively . the male - to - female ratios of groups a and b were 43:21 and 12:5 , respectively . there were no statistically significant differences in age or sex distribution between the 2 groups ( p=0.236 and p=0.312 , respectively ) . the low - grade reflux rates in groups a and b were 12 ( 18.8% , grade ii were 2 [ 3.2% ] and grade iii were 10 [ 15.6% ] ) and 8 ( 47.1% , grade ii were 3 [ 17.7% ] and grade iii were 5 [ 29.4% ] ) , respectively , and the high - grade reflux rates were 52 ( 81.2% , grade iv were 34 [ 53.1% ] and grade v were 18 [ 28.1% ] ) and 9 ( 52.9% , grade iv were 7 [ 41.3% ] and grade v were 2 [ 22.2% ] ) , respectively . patients with high - grade reflux were significantly more common in group a than b ( p=0.022 ) . at 6 months after endoscopic surgery in group b , the numbers of patients with low- and high - grade reflux were 10 ( 58.8% , grade ii were 5 [ 29.4% ] and grade iii were 5 [ 29.4% ] ) and seven ( 41.2% , grade iv were 6 [ 35.3% ] and grade v were 1 [ 5.9% ] ) , respectively . there was no significant change in the numbers before and after primary endoscopic surgery in group b ( p=0.738 ) . however , group a still had more patients with high - grade reflux after endoscopic surgery than did group b ( p=0.020 ) . the operation times for unilateral cases in groups a and b were 16736 minutes and 15518 minutes , respectively . those for bilateral cases in groups a and b were 21533 minutes and 21623 minutes , respectively . the mean hospitalization periods for groups a and b were 10.45 days and 8.82 days , respectively . there were no serious postoperative complications except that bleeding occurred in both groups . in groups a and b , 7 ( 11.0% ) and 2 patients ( 11.9% ) , respectively , needed postoperative blood transfusions because of anemia . however , this difference was not statistically significant ( p=0.107 ) . at 6 months after surgery , resolution of vur was found in all patients who underwent vcug . in the survey of the parents of children in group b , eleven parents ( 64.7% ) answered that they would choose endoscopic injection first in the same situation . the reasons included the convenience of endoscopic treatment , that there was no need for hospitalization , that it involved less pain than open surgery , and that it avoided the discomfort from urethral catheterization after open surgery . the main reason was that they did not want their children to have multiple procedures and anesthesia . among the eleven parents who chose primary endoscopic surgery , 7 of their children had low - grade reflux , and 4 had high - grade reflux . among the 6 parents who chose primary open surgery , one of them had a child with low - grade reflux , and 5 had a child with high - grade reflux . parents of children with low - grade reflux preferred endoscopic surgery ; however , there was no statistically significant difference ( p=0.064 ) ( table 2 ) . selecting the treatment option for different grades of vur reflux depends on the clinical presentation and renal function . however , minimally invasive treatments such as endoscopic injection have advantages over open surgery , including less postoperative pain and fewer bladder spasms and infections , and the absence of surgical scarring . furthermore , this procedure can be performed in a short operation time , in an outpatient setting , and with minimal use of postoperative analgesics . therefore , endoscopic treatments have been preferred as the first - line treatment for children with vur . in a meta - analysis , elder et al . showed that after one injection the reflux resolution rate was 78.5% for grades i and ii reflux , 73% for grade iii , 63% for grade iv , and 51% for grade v . the american urological association ( aua ) vesicoureteral reflux guideline update committee analyzed data from 17,972 patients , and reported that the overall success rate of a single endoscopic treatment was 83.0% . as seen above , endoscopic treatment of children with vur had many advantages and a high success rate , but there were also many failures , especially in the children with more severe reflux . when the first endoscopic treatment fails , endoscopic treatment needs to be repeated or reimplantation surgery is indicated . in this study , we analyzed the characteristics of our patients who underwent open reimplantation surgery . patients who underwent reimplantation as a primary treatment did not differ from those who underwent it after endoscopic injection in terms of age , sex , operation time , hospitalization period , or postoperative complications . it is generally assumed that a secondary operation after failed a primary procedure is more difficult and has more complications . in open reimplantation for vur after failed endoscopic surgery , we also supposed that it may show less success rate , more operation time , more complications , and more hospitalization periods because of ureteral adhesion or inflammation due to primary injection or because each patient had more severe vur . however , as in our findings , several studies have reported that previous endoscopic treatment does not have an adverse effect on the success of subsequent open reimplantation . in many studies , open reimplantation for vur showed much higher success rates than endoscopic treatment for all grades of reflux . in the aua guidelines , the success rate for open surgical procedures was 98.1% . chertin et al . reported the success rate of open reimplantation following failed deflux injections to be 100% . a similar study by moreira - pinto et al sencan et al . showed that previous endoscopic injections could cause difficulties in dissection of the ureter because of fibrosis ; however , they did not alter the success rate or complications following open surgery . parental preference has a major role in the selection of endoscopic treatment compared with long - term antibiotic treatment . in a survey , 80% of parents preferred endoscopic treatment rather than antibiotic prophylaxis or open surgery when given the option of any of the three treatment modalities . this is due to the failed cases of endoscopic injection is very small , because of high success rate of endoscopic injection . these patients were received endoscopic injection again or ureteral reimplantation to treat vur after failed endoscopic injection according to patients conditions and parents demand . therefore , the number of patients underwent ureteral reimplantation was very little , and this study was not analyzed by patient matched - anlaysis . in addition , the follow - up duration after surgery was not long , and several patients did not undergo a follow - up vcug . accordingly , further studies with more patients and a longer follow - up duration are needed . however , it is meaningful that this study showed open reimplantation can be applied effectively for failed endoscopic surgery . in addition , even a parent whose child experienced failed endoscopic injection prefer endoscopic injection as primary treatment for vur . patients with high - grade vur tended to have open ureteral reimplantation as their primary surgery rather than endoscopic injections . however , there were no significant differences in operation time , postoperative complications , hospitalization period , or surgical success rates between open ureteral reimplantation surgery after failed endoscopic injection for vur and primary open reimplantation . we conclude that open reimplantation can be conducted safely and effectively after failed endoscopic treatment . in addition , parents prefer endoscopic injection as the first - line treatment option for their children with vur because of its convenience and reduced postoperative discomfort . in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs . health and social services today face groups of patients who have composite problems and are often unstable . they include , very obviously , elderly persons with multiple problems , chronically ill children , and persons suffering mental ill - health . they have continuing need of care and in search of care they move between primary care , hospital care and municipality care , such as that provided for elderly persons . their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected . individuals do not see themselves as multi - ill , but as needing support for their needs as they know them . it must be said that from the 1970s onwards a number of integrative approaches have been tried out , not least in education . although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs . so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives . much of the evidence indicates that the problem we face is a result of the prevailing mindset . how can we increase our understanding of health and social services that are located in different organizations ? in all well developed societies there tend to be barriers between different organizations and different professions , even when those professions want to co - operate to help individuals to satisfy their needs . health and social services today face groups of patients who have composite problems and are often unstable . they include , very obviously , elderly persons with multiple problems , chronically ill children , and persons suffering mental ill - health . they have continuing need of care and in search of care they move between primary care , hospital care and municipality care , such as that provided for elderly persons . their situation demands some form of integration between health and social services [ 35 ] , the benefits of which have been identified as including reduced hospital use , a strong focus on prevention and keeping patients healthy , and the provision of care closer to home . from the perspective of the person seeking care , medical and social needs are connected . individuals do not see themselves as multi - ill , but as needing support for their needs as they know them . it must be said that from the 1970s onwards a number of integrative approaches have been tried out , not least in education . although there are exceptions to learn from , generally speaking european health and social services are fragmented and poorly equipped to take care of patients with composite needs . so far , and to a great extent , the task of integrating different delivery systems , of managing the transitions from one provider to another , has fallen on the shoulders of patients themselves or their relatives . much of the evidence indicates that the problem we face is a result of the prevailing mindset . how can we increase our understanding of health and social services that are located in different organizations ? lindberg observed examples of meaningful co - operation at the local level , with colleagues from different organizations meeting and pooling their knowledge of local conditions with the patient or user as the focal point . the phenomenon has been variously called the chain of care , integrated care , seamless care or shared care . this co - operation aims at creating a continuing relationship with the patient / user regardless of who at a given moment is the responsible provider . edgren and stenberg found these practical attributes of co - operation in cass : a common task is shared among the co - working agents.collaboration is in people s minds , it is instinctive behaviour.each actor s capabilities are known and respected.a combination of monetary and non - monetary incentives exists in order to create lasting mutually acceptable solutions . collaboration is in people s minds , it is instinctive behaviour . each actor s capabilities are known and respected . a combination of monetary and non - monetary incentives exists in order to create lasting mutually acceptable solutions . according to brommels in his presentation to the ehma - conference 2006 , the cas approach means , among other things : identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts.avoiding strict definitions of roles and concentrating on agreed actions.giving agents their freedom to organize current activities . identifying and supporting constructive relations between agents within the system and understanding tensions and conflicts . avoiding strict definitions of roles and concentrating on agreed actions . giving agents their freedom to organize current activities . if we are to improve our understanding of how a health or social service provider functions as an integrated part of a locally driven health and social service system , we need an innovative , in terms of changing practice , model to guide our thinking . traditional models view systems as machines . it helps us understand what happens in dynamic living systems , where many agents are interconnected . in order to make clear its significance let us first recapitulate the machine model . for a long time effective organizations the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate . hospitals and other health services organizations are usually designed to function according to principles of scientific management [ 15 , 16 ] . rationality , objectivity , stability and predictability are the terms we associate with this approach . rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system . change is seen as a linier and predictable process , controlled by top managers and carried out by works managers . plans are made and are to be followed , and the intended change , takes place as a direct consequence . if problems do arise during the implementation of change , then either there is wrong expectations or there is an inability or refusal to take prescribed action . the change takes time and energy and the outcome is difficult to take in . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened . if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . instead of theories assuming cause and effect linkages between separate details , we need theories to deal with patterns and principles . a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown . davis coined the expression any time , any place when he describes how time and space restrictions this has given us enormous possibilities to communicate , to become connected , to network . as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . indeed , most of our assumptions about organizations are at least fifty years old . so why turn to complexity science and to complex adaptive systems ( cas ) ? according to zimmerman et al . they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . in such a way complexity science can serve as a sense making tool . it also enables us to develop locally adapted solutions in order to manage complex tasks , such as we find in advanced home health care . complexity can be expressed as the amount of information needed to describe or understand something . and the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . a cas consists of several subsystems called agents , which act in dependence of one another . . they may either compete or co - operate according to their sense of their interests and what will bring them an advantage . self - organization is about creating order or increasing the regularity of the system without help from the outside . good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere . researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically . prominent figures in the field include nobel laureates ilya prigogine in chemistry [ 26 , 27 ] and murray gell - mann in theoretical physics . within medicine we find stuart kauffman [ 30 , 31 ] and physics russ marion and mary uhl - bien . according to augustinsson , one way to explain the phenomenon of complexity is by reference to the possibility to apply routines to carry out a particular task . the more a task is characterized by regularities the more we can think in terms of applying routines . when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity . although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services . the agents see the point , because they create order out of many local interactions . complexity science offers new ways to understand how complementary knowledge organizes for co - operation . we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system . emergency treatment is a clear example of independent agents interacting locally with other independent agents . another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants . the objective was to identify how local politicians and managers understand problems and goals regarding the structures and processes involved in the care of the elderly . a common vision , easy to understand and to communicate , there is no external constructor , no superior centrally located source to govern the design of the system . when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration . complexity science emphasizes the inherent power of development and self - organizing nature of the system . attempts from above to reduce the complexity of the system in order to gain control , are often counterproductive . in practice we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient . such top down attempts are usually made in order to make it simpler for the provider not the person seeking care . when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them . one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation . later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other . so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace . positive feedback loops enhance or amplify changes and tend to move a system away from equilibrium stage . for example in general terms , there is a positive feedback loop between income and consumption in an economy . the bigger the income of the individuals , negative feedbacks tend to dampen or buffer changes and hold a system to some equilibrium state , like a thermostat in a fridge . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science positive and negative feedback there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . instead holden talks about direction without directives . it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours . the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . with their local knowledge of needs , they are so much better placed to act than any centrally located management ever could be . they sense that they have control over their own work situation , perhaps the most important change needed to create the good workplace . in this way we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole . general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas . others may argue that this is only one of several possible approaches to promote integrated care . communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted . a certain level of system inertia may also develop over time . to learn to use a different approach to organizing may mean insecurity and risk similar to being expected to accept new technology . seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management . the cas approach raises some ethical concerns that refer to decision making that can neither be supported by science nor by objective criteria . first this is due to the nature of the system which is determined by the sum of choices made in it . secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! we know that all our choices to some extent represent a step in the dark , and therefore , we can not but be responsible for them . but the cas approach lays out the considerations to be taken into account more clearly than the alternatives . for a long time effective organizations have been looked upon like well - oiled machines . the machine has a constructor , the top manager , who describes the integral parts and how they are supposed to co - operate . hospitals and other health services organizations are usually designed to function according to principles of scientific management [ 15 , 16 ] . rationality , objectivity , stability and predictability are the terms we associate with this approach . rationality , for example , requires that all integral parts act on perfect information , have the same background and similar values and work towards the same goals , and that there is a system designer , the top manager , who is from outside the system . change is seen as a linier and predictable process , controlled by top managers and carried out by works managers . plans are made and are to be followed , and the intended change , takes place as a direct consequence . if problems do arise during the implementation of change , then either there is wrong expectations or there is an inability or refusal to take prescribed action . the change takes time and energy and the outcome is difficult to take in . when political decisions setting precise goals are centralized and detailed rules are laid down and lines of action specified , and when there is constant top - down monitoring and assessment , there is a very real risk of destroying capacity and motivation at the so - called and then , when the unforeseen happens , the whole system breaks down , because there is no readiness or capability to adapt all solutions have been pre - programmed beforehand . from this we learn that when there is no motivation to renew the system , when there is an inability to innovate , the survival of the system is threatened . if health and social service providers are to meet changing demands and expectations from patients or users , they must be able to move quickly to find mutually acceptable , locally developed forms of integration at their points of intersection , that is , where their separate services should be coming together . instead of theories assuming cause and effect linkages between separate details , we need theories to deal with patterns and principles . a few years ago concepts such as the knowledge society and learning organizations , and the associated mechanisms and technologies the internet , e - mail , mobile phones and digital imaging were all unknown . davis coined the expression any time , any place when he describes how time and space restrictions this has given us enormous possibilities to communicate , to become connected , to network . as we advance in the knowledge society , the basic assumptions behind much of what is taught and practiced in the name of management are now hopelessly out of date . indeed , most of our assumptions about organizations are at least fifty years old . so why turn to complexity science and to complex adaptive systems ( cas ) ? according to zimmerman et al . they provide an alternative to traditional management principles , that is they offer patterns and principles whereby we can better act in an increasingly complex world , as when we attempt to harness health and social care and other services to meet the particular needs of the individual . in such a way complexity science can serve as a sense making tool . it also enables us to develop locally adapted solutions in order to manage complex tasks , such as we find in advanced home health care . complexity can be expressed as the amount of information needed to describe or understand something . and an important part of complexity science is the complex adaptive system ( cas ) . the term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . a cas consists of several subsystems called agents , which act in dependence of one another . . they may either compete or co - operate according to their sense of their interests and what will bring them an advantage . self - organization is about creating order or increasing the regularity of the system without help from the outside . good examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . important work has been carried out at the new england complex system institute and the santa fe institute and elsewhere . researchers in chemistry , physics , biology , medicine , anthropology economics , and sociology have been asking fundamental questions about living systems , living systems that are not fixed , but change , grow , heal up , adjust , renew and develop organically . prominent figures in the field include nobel laureates ilya prigogine in chemistry [ 26 , 27 ] and murray gell - mann in theoretical physics . within medicine we find stuart kauffman [ 30 , 31 ] and physics russ marion and mary uhl - bien . according to augustinsson , one way to explain the phenomenon of complexity is by reference to the possibility to apply routines to carry out a particular task . the more a task is characterized by regularities the more we can think in terms of applying routines . when everyday work is characterized by both regularities and irregularities , by a mix of the predictable and the unpredictable , then we have the highest degree of complexity . although more research is needed to achieve greater understanding of complex adaptive systems and to strengthen the knowledge base for action , we do have a growing number of examples that show the cas concept gaining ground within the health and social services . the agents see the point , because they create order out of many local interactions . complexity science offers new ways to understand how complementary knowledge organizes for co - operation . we can regard integrated care partners as partners in a common system and we can regard them logically as agents in this system . emergency treatment is a clear example of independent agents interacting locally with other independent agents . another example comes from elderly care where the cas concept has been applied to secure agreement between politicians and civil servants . the objective was to identify how local politicians and managers understand problems and goals regarding the structures and processes involved in the care of the elderly . a common vision , easy to understand and to communicate , was created to connect the two groups . there is no external constructor , no superior centrally located source to govern the design of the system . when we study a cas , the focus is on the interaction and communication between agents . rewriting the old clich , that the whole is greater than the sum of the parts , the whole is the relations between the parts . whether between two persons or between human being and machine or between machine and machine , it is the intensity of relations that determines the complexity of the system , the constant change , adaptation and development of the system , which will be in an unforeseeable non - linear way . today digital imaging as a diagnostic technology brings together the specialist radiologist and the primary practitioner treating the patient in a way that was virtually impossible previously . and thereby generate new possibilities of professional interaction and patient involvement as well as new forms of inter - organizational collaboration . complexity science emphasizes the inherent power of development and self - organizing nature of the system . attempts from above to reduce the complexity of the system in order to gain control , are often counterproductive . in practice we assume that any provider s top down attempt at specifying tasks will risk more complexity for the patient . such top down attempts are usually made in order to make it simpler for the provider not the person seeking care . working in isolation the burden of coordination passes to the patient . when we address a given community s health and social problems , the process is local : both in intelligence gathering , using multiple local sources to build up the knowledge base for action , and in connecting all those locally based agencies / institutions and individuals with competence to do something about them . one important aspect of complexity science is that it has shown how complementary knowledge organizes for co - operation . later studies of cass have emphasized how the interplay between the environment and the system results in a sort of co - development , whereby each influences the other . so , for example , a hospital inpatient can at the same time be a part both of the hospital system and another system at his workplace . positive feedback loops enhance or amplify changes and tend to move a system away from equilibrium stage . for example in general terms , there is a positive feedback loop between income and consumption in an economy . the bigger the income of the individuals , the more the whole population consume , which further increasing their income as individuals . negative feedbacks tend to dampen or buffer changes and hold a system to some equilibrium state , like a thermostat in a fridge . these loops are carriers of information , material and energy between the agents of the system , and facilitate the adaptability of the entire system . in complexity science there is not much sense in agents spending time separately on detailed planning since the functioning of the system is a result of their interactions . instead holden talks about direction without directives it has been found that for purposes of fostering connectivity among diverse agents , effective coupling of structures , ideas and innovations , and ensuring that they are neither too loose nor too tightly interdependent , complex systems are better led by indirect than by direct leadership behaviours . the cas approach helps agents to see themselves as co - workers , part of an innovative team with great potential . with their local knowledge of needs , they are so much better placed to act than any centrally located management ever could be . they sense that they have control over their own work situation , perhaps the most important change needed to create the good workplace . in this way we see that the cas approach satisfies the fundamental human need to participate , to have a feeling of solidarity , to be part of a greater whole . general criticisms of the cas approach coming from practitioners concern a lack of recommendations as to how they should behave being part of a cas . some theorists would claim that cas is nothing but the emperor s new clothes . others may argue that this is only one of several possible approaches to promote integrated care . communication- and co - ordination conflicts among participating agents , or rather free riding agents , have been noted . a certain level of system inertia may also develop over time . to learn to use a different approach to organizing may mean insecurity and risk similar to being expected to accept new technology . seen from a staff perspective the cas approach may mean increased insecurity , greater responsibility , more decision - making and more elements of risk management . the cas approach raises some ethical concerns that refer to decision making that can neither be supported by science nor by objective criteria . first this is due to the nature of the system which is determined by the sum of choices made in it . secondly , when there appears to be no simple final objective or calculable ground for our decisions , we can not shift the responsibility for the decision onto something or somebody else do nt blame me ; the genetic algorithm said we should sell ! we know that all our choices to some extent represent a step in the dark , and therefore , we can not but be responsible for them . but the cas approach lays out the considerations to be taken into account more clearly than the alternatives . when the competence necessary for carrying out a given task does not lie within one individual provider organization , co - operation between agents within the system comes into play to discharge that task . we are no longer talking about the individual organization / agent but shaping overall workable solutions taking a patient / user perspective . this applies to matching care with the needs of different patient groups and of individual patients . the cas approach helps the management to understand why the traditional top down way of managing may meet with problems in organizations with complex tasks . an important discussion is about how the top management in fact executes its steering function . leaders may consider accepting complexity instead of trying to reduce it , formulate few simple and concrete goals , communicate and give feedback and measure performance . when we perceive health and social service organizations as cass we should gain more insight into the processes that go on within and between organizations . are we willing to face the interdependence between health and social services , the dependence on collaboration to deliver appropriate integrated care ? if we do , complexity science could be an important step towards fresh thinking in order to fulfil our patients and users presently unfulfilled needs . edgar borgenhammar , phd ( economics ) , mba ( berkeley ) , dsi ( lund ) , professor emeritus , from the nordic school of public health in gteborg , former hospital chief executive , sweden peter carswell , dr . , centre for health services research and policy , faculty of medical and health sciences , university of auckland , auckland , new zealand alene hokenstad , project director , united hospital fund , new york , usa

purposeas endoscopic treatment for vesicoureteral reflux ( vur ) has increased , secondary ureteral reimplantation ( ur ) after failure of endoscopic treatment has increased . we studied the clinical feature and efficacy of secondary ur after failure of endoscopic treatment compared with primary ur.materials and methodseighty - one children who had ur for vur were enrolled . charts were reviewed retrospectively for age , sex , grade of vur before surgery , operative time , hospitalization period , postoperative complication , and success rate . primary ur ( group a , n=64 ) was compared with secondary ur after failed endoscopic treatment ( group b , n=17 ) . in group b , telephone survey for the satisfaction of endoscopic treatment and surgery was done.resultsmean age of each group was 49.637.1 and 56.622.5 months ( p=0.236 ) . there was no significant difference between each group in sex , mean operative time , postoperative transfusion , complication rate , and success rate . as telephone survey in group b , eleven responders preferred endoscopic treatment as primary treatment of vur because it was a simple method and no hospitalization.conclusionssecondary ur after failure of endoscopic treatment was similar to primary ur . parents preferred endoscopic treatment as first line treatment for vur in spite of the need for secondary ur after failure of endoscopic treatment . introductionorganizations can be regarded as systems . the traditional model of systems views them as machines . this seems to be insufficient when it comes to understanding and organizing complex tasks . to better understand integrated care we should approach organizations as constantly changing living organisms , where many agents are interconnected in so - called complex adaptive systems ( cas).theory and discussionthe term complex emphasizes that the necessary competence to perform a task is not owned by any one part , but comes as a result of co - operation within the system . adaptive means that system change occurs through successive adaptations . a cas consists of several subsystems called agents , which act in dependence of one another . examples would be the ant - hill , the human immune defence , the financial market and the surgical operating theatre team . studying a cas , the focus is on the interaction and communication between agents . although these thoughts are not new , the cas - approach has not yet been widely applied to the management of integrated care . this helps the management to understand why the traditional top down way of managing , following the machine model thinking , may meet with problems in interdependent organizations with complex tasks.conclusionwhen we perceive health and social services as cass we should gain more insight into the processes that go on within and between organizations and how top management , for example within a hospital , in fact executes its steering function .

we will introduce recent advances on the study of post - translational processing , modification , and targeting of cathepsins and cystatins . almost all the intracellular proteins are passed through principally similar processes from the synthesis to their degradation in general . therefore , i would like to introduce the general fate of intracellular proteins , from the post - translational processing , modification , and targeting to the ordered particles . as figure 1 shows , the intracellular proteins are synthesized as pre - promature complex in polysomes and prepart is removed cotranslationally , and then the promature parts are translocated into golgi - apparatus , and then glycosylated by mannose - rich sugar . the glycosylated mature part is translocated into target organelles and the degradation was started by the splitting from the ordered nicked bonds to make hydrophobic peptides . these hydrophobic peptides are secreted to cytoplasm and are incorporated into the phagosomes or proteosomes after ubiquitination . biological merit of post - translational processing and modifications of proteins are possible considerations . the capability to take variable forms on the way of biosynthesis is important to keep adaptability to the changing of biological requirements and intracellular translocation during the maturation must be regulated . pro - parts or bound sugar are the targeting signals in some cases . in the cases of carbamoyl phosphate synthetase ( cps ) and ornithine transcarbamylase ( otc ) , their pro - parts play the role of signals to be recognized by their receptors located on target organella membrane , such as lysosomes , as shown in figure 4 . the glycosylated cathepsins are targeted into lysosomes mediated by mannose-6 phosphate receptors which are located on the lysosomal . as figures 4(a ) and 4(b ) show , the lysosomes in which cathepsin h or b is located are attached to the cell - membrane . on the contrary , cathepsin l is located in the lysosomes which are distributed diffusely in liver cells . as figure 4(a ) shows , using immunodouble gold - particle staining , cathepsin b ( small gold particle ) and cathepsin h ( large gold particle ) are located clearly in different lysosomes . cathepsin l is translated as 17 amino acids of prepart , 96 amino acids of pro - part and 221 amino acids of mature - part [ 9 , 10 ] . the prepart is removed cotranslationally and formed procathepsin is translocated into golgi - apparatus , and then the 108-asn and the 155-asn in mature parts are glycosylated by high - mannose - type sugar , as shown in figure 2(a ) . the initiation of the degradation is started from the nicking bond ( 178 bond ) cleavage by some cysteine protease . cathepsin b is translated as 17 amino acids of prepart , 62 amino acids of pro - part , and 252 amino acids of mature part [ 12 , 13 ] . the prepart is also removed cotranslationally and the formed procathepsins are translocated into golgi - apparatus and then the 38th - asn in pro part and the 111th - asn in the mature - part are glycosylated by high - mannose - type sugar . then the mannose-6-phosphate - moities play a role as the targeting marker to the lysosomes . cathepsin h is translated as 21 amino acids of prepart , 114 amino acids of pro - part , and 217 amino acids of mature part . the pro - part has two carbohydrate chains at the 70th - asn and the 90th - asn and the mature part consisted of 217 amino acids , and one carbohydrate chain is bound at the 99th - asn [ 8 , 15 ] . the initiation of degradation is started from the 177th nicking bond by cysteine protease . intracellular protein degradation (= autophagosome formation ) is regulated by nutritional and hormonal conditions . fasting or insulin enrichment caused stimulation of the phagosome formation and cathepsin l amount are increased ; on the contrary , refeeding and glucagon enrichment resulted in the suppression of phagosome formation and decreasing of cathepsin l amount . as you see in figure 2 , cathepsins b , l , and h have individual ordered nicking bond as the initiation of their degradations ; these bonds are cleaved by cysteine protease , therefore as table 1 shows , the half - lives ( t1/2 ) of cathepsins and the contents in lysosomes clearly increased by treatment by e-64 ( inhibitor for all cysteine proteases ) . the initiations of degradation of cathepsins are started from the ordered limited proteolysis by cysteine protease in the lysosomes , as figure 2 shows . the formed hydrophobic products were translocated into autophagosome or proteasome ( after ubiquitinated ) to degrade to amino acids . procathepsins or mature cathepsins are secreted from the various cells and play individual physiological , or pathological roles . as figure 5 shows , bone metabolism is consisted of functional balances between osteoblastic cell function and osteoclastic cell function . bone collagen is degraded mainly by cathepsin l and k , therefore they are called collagenolytic cathepsins . the secretion of these cathepsins from osteoclasts is stimulated by pht ( parathyroid hormone ) and suppressed by vitamin d3 , e-64 , ca-148 eta . therefore , bone metastasis of cancers is inhibited by cathepsin inhibitors , such as by e-64 or clik-148 . as pit formation test in table 4 shows , bone - matrix degradation is catalyzed by cathepsin l , but not by cathepsin b. endogenous low - molecular weight cathepsin inhibitors are consisted of two big groups , one is only located in skin and the other is ubiquitously located in all organs . cystatin ( a ) is only located in epidermis , cystatin ( b ) is located ubiquitously in all cells ( organs ) . the secretary formed cystatins are cystatin c in eggwhite and cystatin s in the saliva . another secretary group having high - molecular weight ( repeated low molecular cystatins ) is kininogen family in serum . each cystatin shows different inhibitory specificity for individual cathepsins , for example , cystatin does not inhibit cathepsin b activity but strongly inhibit cathepsin l activity and cathepsin h activities [ 7 , 19 ] . the amino - acid sequences of low - molecular weight cystatin family have strong homology , however , their post - translational processing are quite different . because their localizations and biological functions is quite different . when skin was stained by immunohistochemistry using anticystatin antibody , only the cornified - envelope of skin was stained . the sphingosine treatment to new born skin resulted in the strong suppression of the targeting of cystatin into the cornified envelope . the sphingosine is a powerful inhibitor for protein kinase c. as shown in figures 7(a ) and 7(b ) , threonine residue in near c - terminus ( the 92th threonine in figure 9 shows ) of cystatin is phosphorylated by protein kinase c , and the phosphorglated cystatin is incorporated into cornified envelope . the participation of protein kinase c in the phosphorylation of cystatin was also confirmed using specific inhibitor of protein kinase c , ( hidaka h-7 ) inhibited the incorporation of p into cystatin . the phosphorylated cystatin was incorporated into skin cornified envelop . in the envelop , the phosphorylated cystatin was conjugated with filaggrin - linker sediment peptide which is rich in glutamine residues , mediated by epidermal transglutaminase in the presence of calcium , and yield a high - molecular weight protein ( skin fiber ) , as shown in figure 10 . the phosphorylated cystatin showed strong inhibition to cathepsin l , but not to the cathepsin b and h , it also showed strong inhibition to the bacterial cysteine proteases and virus cathepsins . therefore , cystatin and phosphorylated cystatin showed strong protection from infections of bacteria or viruses in skin . post - translational covalent modification of cystatin and the changing of their inhibitory activities . the third position of the n - termius - cysteine residue of cystatin reacts with glutathione to form a mixed disulfate complex and also is able to make dimmer by the disulfate bond . as figure 8 shows , n - terminus of cystatins was inserted into the substrate binding pocket of cathepsins , the glutathionated or the dimmer form were unable to insert into the binding pocket of cathepsins . the coefficient of oxidized or reduced forms of glutathiones regulates the inhibitory activity of cystatin . therefore , the activities of cathepsins are regulated by the intracellular redox potentials in spite of the changes of the inhibitory activities of cystatin . as figure 7(b ) shows , using -cell of pancreas , the cystatin is located in insulin secretion particles , therefore , cystatin is secreted with insulin in the pancreas . endocrine disorders may complicate , cause or mimic otolaryngologic disorders , some of which may be anatomical , due to an enlargement of the thyroid gland , while others are physiological , resulting from increased or decreased glandular activity.1 hypothyroidism is characterized by the slowing of mental and motor activity , depression , constipation , cold intolerance menorrhagia , stiff muscles , carpal tunnel syndrome , sleep apnea , dry hair and skin , weight gain , snoring and a hoarse voice.1 less common symptoms involve the heart , muscles , joints , and blood.2 dysarthria as the presenting symptom of hypothyroidism has only been reported once before.3 here , we present an unusual case of hypo - thyroidism presenting with dysarthria . a 39-year - old female presented to the clinic with dysarthria of six months duration . , it was revealed that there had been episodes of dysphonea , snoring , sleep apnea , dysphagia and choking during eating or drinking . her past medical history was normal apart from one occasion of delayed recovery from anaesthesia during surgery for a fractured femur the year before . clinical examination , including the central nervous system , was normal apart from a slightly puffy face . there was no abnormality in the movement of the tongue or pharygeal / palate muscles . pmol / l ( normal 9.2 - 23.9 pmol / l ) , a thyroid stimulating hormone of 82.7 miu / l ( normal 0.32 - 5.00 miu / l ) , antithyroglobulin antibodies were 1:320 u / ml ( normal 1:40 u / ml ) , antiperoxidase autoantibodies of 1:1600 u / ml ( normal 1:40 u / ml ) . a complete blood count film was consistent with iron deficiency , a haemoglobin of 7grams ( normal 11 - 16 grams ) , low serum iron of 2 umol / l ( normal 10 - 28 umol / l ) , increased red cell distribution width ( rdw ) of 17.2 ( normal 11.6 - 13.7% ) . other biochemical abnormalities were high serum cholesterol of 6.9 mmol / l ( normal 3.6 - 6.8 , hypo - thyroidism was diagnosed . in the light of the patient 's diagnosis , a second history was taken which showed that she suffered from other symptoms of hypothyroidism such as , dry skin , generalised weakness , excessive sleeping , hoarse voice , and menorrhagia . two months after the initiation of therapy , the patient had no more dysarthria or other associated symptoms . dysarthria is a disturbance of articulation that may be caused by a neuromuscular lesion , or an abnormality of the vocal cords . the first may result from damage to the central or peripheral nervous system such as head trauma , brain stem infarction , bulbar palsy , motor neuron disease , peripheral neuropathy , huntington 's chorea , parkinson 's disease , multiple sclerosis , myasthenia gravis , or muscle disease.4 the second may be attributable to congenital , traumatic , inflammatory , tumors , or post - operative lesions of the vocal cords . these causes were unlikely in this patient , because she showed no associated clinical features of these diseases besides the normal neurological examination and investigations . other causes such as congenital or aquired storage disorders such as amyloidosis , and such endocrine disorders as acromegaly or hypo - thyroidism,3 as in the presented case , may lead to an enlargement of one or more of the components of the vocal cords.56 the most likely cause for the dysarthria in this patient was hypothyroidism . this was supported by the abnormal thyroid functions and the response of the dysarthria to thyroxin . dysarthria due to hypothyroidism had been reported only once previous to this case.3 the pathophysiology of dysarthria in hypothyroidism can be explained by edematous swelling of laryngeal and hypopharyngeal structures in combination with macroglossia.3 it has been shown that macroglossia in hypothyroidism is caused by a thickening of the epithelial tissue.6 these changes can also explain the choking and the dysphagia which this patient experienced . there have been a few reports of hypothyroidism responsible for secondary dysphagia.1012 her sleep apnea may also be a manifestation of hypothyroidism , most likely caused by edema and myopathy.7 sleep apnea attributable to hypothyroidism is reversible with thyroxin replacement therapy.8 the episodic hoarseness of voice can also be explained by hypothyroidism,1 as well as the delayed recovery from anaethesia the year before , most probably the result of undiagnosed hypothyroidism.91314 unfortunately , thyroid function tests had not been performed on our patient at that time . iron deficiency anemia in this patient was due to menorrhagia , which is one of the characteristic features of the disease.1 hyperlipidaemia may also be due to hypothyroidism , a known association.1 clinical implication of the presented patient was that dysarthria may be the presenting symptom of hypothyroidism , even if other symptoms had been present for a long time . hypothyroidism as the cause of dysarthria was confirmed with the discovery of additional symptoms in the patient 's history . otolaryngeal symptoms should therefore be considered possible symptoms of hypothyroidism.15 prompt recovery of dysarthria is expected after hormone replacement therapy .

cathepsins are an essential protease family in all living cells . the cathepsins play an essential roles such as protein catabolism and protein synthesis . to targeting to various organella and to regulate their activity , the post translational - processing and modification play an important role cathepsins are translated in polysome as the pre - pro - mature forms . the pre - peptide is removed cotranslationally and then translocated to golgi - apparatus and the pro - part is removed and the mature - part is glycosylated , and the mature - part is targeted into the lysosome mediated by mannose-6-phosphate signal and the mature - part is bound with their coenzymes . the degradation of the mature - part is started by the limited proteolysis of the ordered nicked bonds to make hydrophobic peptides . the peptides are incorporated into phagosome or proteasome after ubiquitinated and are degrade into amino - acids . cystatins are endogenous inhibitors of cathepsins . cystatin which is only located in skin is phosphorylated at the near c - terminus by protein kinase - c , and the phosphorylate - cystatin is incorporated into cornified envelope and conjugated with filaggrin - fiber by transglutaminase to form the linker - fiber of skin . the cystatin is modified by glutathione or make their dimmer , and they are inactive . those modifications are regulated by the redox - potential by the glutathione . hypothyroidism is a common endocrine disorder with characteristic clinical symptoms and signs . typical symptoms of hypothyroidism are lethargy , cold intolerance , slowing of intellectual and motor activity , decreased appetite , weight gain , and dry skin . a 39-year - old female presented to the clinic with dysarthria as the chief symptom . subsequent questions revealed that other symptoms were confined to the otolaryngeal region , which were episodes of mild dysphonia , dysphagia , sleep apnea , and snoring . laboratory data revealed marked hypothyroidism and positive tests for antithyroglobulin and antimicrosomal antibodies . after administration of thyroxin , the dysarthria and the other symptoms rapidly disappeared . dysarthria may be the presenting symptom of hypothyroidism and can be resolved after hormone replacement therapy .

bicipital groove ( bg ) is an indentation on the anterior aspect of proximal part of humerus . this groove allows tendon of long head of biceps brachi muscle enveloped in synovial sheath and ascending branch of anterior circumflex humeral artery to pass through it . it is bounded medially by lesser tubercle , laterally by greater tubercle , and superiorly by bridging of transverse humeral ligament /muscle fibers of subscapularis , supraspinatus and pectoralis major muscles [ 2 , 3 ] . this groove with transverse humeral ligament / muscle fibers bridging it provides stability and smooth functioning of tendon of long head of biceps brachi muscle and prevents its subluxation during multidirectional biomechanical movements of arms . apart from this , the greater function of biceps brachi muscle whose tendon is enshrined in bicipital groove is suppination , flexion , and screwing biomechanical movements . on motion of humerus , the proximal humerus moves in relation to fixed biceps tendon which is firmly held in place at the level of intertubercular sulcus by tuberosities and humeral transverse ligament . with elevation of arm , humerus moves about 3.8 cm on the fixed tendon . in the dynamics of external rotation to internal rotation , the tendon is forced medially against lesser tubercle and superiorly against transverse humeral ligament . coracohumeral ligament directly overlies the transverse humeral ligament / muscle fibres and is continuous with rotator cuff . morphometry of bg may influence the functions of surrounding structures leading to various pathological conditions [ 8 , 9 ] . supratubercular ridge originally described by meyer in 1928 and later by hitchcock and bechtol in 1948 consists of bony protuberance and is continuous with superior aspect of lesser tuberosity . it allows the tendon a more gradual change in direction as it enters the bicipital groove by elevating and forcing it laterally . therefore , incidence of spurs and supratubercular ridge in indian population through this study has also been observed . apart from this , the bicipital groove is important landmark for replacement of prosthesis of shoulder . thus knowledge of bg is highly useful in prosthetic sizing , positioning , and designing . bicipital groove also acts as an important landmark for placement of lateral fin of prosthesis in shoulder arthroplasty and humeral head replacement in fractures of upper end of humerus . in a series of classic reports by various authors , the papers in [ 4 , 1215 ] have discussed primary versus secondary biceps tendinitis and different treatment regimens for each of these entities [ 16 , 17 ] . the association of shoulder pain with pathology of the lhb is currently attributed to inflammation ( synovitis ) , impingement , prerupture , or instability of the tendon at the entry into the bicipital groove ( subluxation or dislocation ) [ 16 , 1824 ] . lesions affecting the tendon of lhb brachii have been postulated to be among the most frequent causes of pain and disability in the shoulder . this pain may be caused by rotator cuff , supraspinatus , and biceps tendon diseases . pathologies of the biceps tendon can be broadly divided into two classes , namely , as follows . primary tendonitis : berlemann and bayley reported the long term results of 14 patients ( 15 shoulders ) following keyhole biceps tenodesis . fifty - three percent of patients had previously undergone a subacromial decompression but symptoms persisted until the biceps tenodesis was carried out . this would suggest that biceps tendinitis is a primary event.secondary tendonitis [ 14 , 15 , 17 , 27 ] : this may further be subdivided into three main types , inflammatory , instability , and traumatic . clearly , there is a huge overlap between these categories and in fact biceps pathology is very rarely a single entity . apart from this , the most recent biomechanical data come from youm et al . who found that loading of the long head of biceps tendon significantly affects the glenohumeral joint , range of movement , translations , and kinematics . therefore , varied anatomical knowledge of the bg is important as abnormalities of the bicipital tendon and its synovial sheath have been implicated in a variety of causes of shoulder pain and disability [ 6 , 7 ] . a radiological study recommended that the entire length of the bg be examined to determine the osseous anatomy of the groove . few authors have studied the morphology of the upper end of the humerus in geographically diversified regions [ 10 , 3133 ] . primary tendonitis : berlemann and bayley reported the long term results of 14 patients ( 15 shoulders ) following keyhole biceps tenodesis . fifty - three percent of patients had previously undergone a subacromial decompression but symptoms persisted until the biceps tenodesis was carried out . secondary tendonitis [ 14 , 15 , 17 , 27 ] : this may further be subdivided into three main types , inflammatory , instability , and traumatic . clearly , there is a huge overlap between these categories and in fact biceps pathology is very rarely a single entity . apart from this , the most recent biomechanical data come from youm et al . who found that loading of the long head of biceps tendon significantly affects the glenohumeral joint , range of movement , translations , and kinematics . therefore , varied anatomical knowledge of the bg is important as abnormalities of the bicipital tendon and its synovial sheath have been implicated in a variety of causes of shoulder pain and disability [ 6 , 7 ] . a radiological study recommended that the entire length of the bg be examined to determine the osseous anatomy of the groove . few authors have studied the morphology of the upper end of the humerus in geographically diversified regions [ 10 , 3133 ] . hence an attempt has been made to examine the length , width , depth , and opening angle of bicipital groove statistically to correlate with clinical implications in north indian population along with a review of the literature . the study was carried out using hundred and one humeri of assorted sex and pair . the subjects consisted of 45 left and 56 right humeri obtained from osteology laboratory of kg medical university , lucknow , up , india . the lengths of medial , lateral walls , depth , lengths , and width of bicipital groove ( figure 1 ) were measured by digital vernier callipers . the statistical analyses consisting of not only incidences of supratuberecular ridge of meyer and presence of bony spurs but also mean , standard deviation , range , median and mode of length of medial and lateral walls , width , depth , and opening / medial wall angles of bg were carried out . the opening / medial wall angles of bg ( figure 2 ) have been computed . the narrowness and shallowness of bg have been redefined objectively in relation to dimensions of both bicipital groove and biceps tendon for adequate prediction of pathologies of biceps tendon . though the precise definition of narrowness / shallowness of bg is difficult yet systematic , relative and constrained definition depending on the dimensions of bicep tendon and its natural abode , that is , bicipital groove , is formulated as follows.if the width of bg is less than the width of biceps tendon , it is a narrow bg which may produce attritional changes causing impingement , inflammation , and degeneration . similarly , if the depth of the groove is less than height of the bicep tendon , it is shallow causing subluxation or dislocation which in the long run may cause degenerative changes and rupture . if the width of bg is less than the width of biceps tendon , it is a narrow bg which may produce attritional changes causing impingement , inflammation , and degeneration . similarly , if the depth of the groove is less than height of the bicep tendon , it is shallow causing subluxation or dislocation which in the long run may cause degenerative changes and rupture . as the new definition of narrowness or shallowness requires the width and height of biceps tendon , so the width and height of 4 biceps tendons from 2 cadavers have also been measured to provide more realistic definition of narrow / shallow of bicipital groove . the means standard deviation of lengths of medial and lateral walls , width , depth and opening / medial wall angles of bg have been computed as shown in table 1 . mean length of medial wall of bg on right side was 22 4 and that on left side was 23 5 mm ( table 1 ) . mean length of lateral wall of bg on right side was 31 6 mm and that on left side was 31 5 mm . mean width on the superior part of bg on right side was 8 2 mm and that on left side was 8 2 mm . the depth of bg on right side was 5 1 mm and that on left side was 6 1 mm . the incidence of supratubercular ridge was 37% total , 17% on right side , and 20% on left side . the average length of bg is 26.7% of total length of the humerus and average width of bg is 52% of average width of humerus . the width and height of biceps tendon ( figure 3 ) have been displayed in table 2 . the morphometric study carried out by various authors [ 25 , 3437 ] has been compared with present study as depicted in table 3 . lengths of medial wall and lateral walls of bg have not been reported so far except in the present study , so there is no data for comparison . length of bg in present study is comparable with that of murlimanju but slightly higher than that observed by wafae et al . the width of bg under present study is close to that of cone et al . but slightly higher than that of murlimanju and lower than that of wafae et al . median of width of bg on right and left sides are 8 and 10 mm , respectively , and mode is 8 mm on each side . it indicates that width of bg in most of the north indian population is 8 mm . in the present study depth of bg more than 3 mm and depth ranging 46 mm are 98% and 96% of humeri , respectively , as against 90% and 86% in the study of cone et al . median and mode of depth in right / left side are 5/6 mm each . medial wall angle of bg is slightly higher than that of joseph and lower than cone et al . medial wall angle ( mwa ) in my study is lower than cone and higher than joseph . the median and mode of these morphometric parameters of bg are very useful for prosthetic sizing , positioning , and designing . the supratubercular ridge in present study is found in 17% in right and 20% in left totalling to 37% in all humerii . as per hitchcock and bechtol cone et al . from their radiographic interpretations observed this ridge in 50% of cases and reported that it was not pathologically significant . observed this ridge in 88% on right side and 57% on left side and emphasised that it was more important on right side than left to prevent medial displacement of long head of biceps from the bg . in present study mean width and height of biceps tendon ( figure 3 ) are 10.5 and 1.6 mm , respectively , which is higher than that observed by lam and mok . similarly these dimensions of tendon at the exit from bg are 7 and 1.8 mm , respectively . the width of tendon at the exit is more and height is less than that observed by lam and mok . if the tendon is not encased by median and lateral walls of bg due to its shallow depth it may be dislocated , either partially or fully by biomechanical movements of arms . this dislocation of biceps tendon associated with impingement may cause degeneration leading to partial or full rupture with the passage of time . apart from this , if the movements of tendon are not free in narrow or in presence of bony spurs in bg during biomechanical movements of shoulder joint , its constant fraying might give rise to pathologies of biceps tendon . reported that wide grooves ( i.e. , > 17 mm ) were often shallow . but in a groove 3 mm deep or less , it should be viewed with suspicion in managing pathologic conditions of the shoulder on patient radiographs as per cone 's view . pfahler et al . found a flat groove angle associated with radiologic depth less than 2 mm . they found a significant accumulation of pathologically changed biceps tendons when a flat groove angle was present . according to several authors subluxation and dislocation of biceps tendon were more common in presence of shallow bicipital groove [ 1 , 4 , 5 , 7 , 39 ] . it is also reported that with shallow bicipital groove , the tendon is susceptible to chronic trauma due to impingement by the overlying acromion , rotator cuff , and coracoacromial arch during shoulder movement . a shallow intertubercular groove is vulnerable to impingement damage and subluxation [ 37 , 40 ] . rupture of the biceps tendon most commonly occurs proximally near the glenoid labrum and distally in the bicipital groove . smith designated bicipital groove types as narrow , normal and shallow depending on mean opening angle less than 66 , 94 , and 118. as seen from above description , the objective and realistic definition has not been given by any author . therefore , present author has attempted reliable , realistic , and objective definition of shallowness of bg in relation to biceps tendon as given in section 2 of this paper . this definition of shallowness can be realised in situ in live patients only but is expected to elucidate the pathologies associated with shallowness of the groove . as per morphometric data ( table 3 ) of bg and biceps tendon as observed in two cadavers in the present study , the groove is not shallow according to new definition of shallowness . figure 3 shows the biceps tendon and bg in a dissected cadaver wherein the biceps tendon is positioned and perfectly well protected in bg . the bg in this case is not shallow as seen in the above - mentioned figure , and the height of biceps tendon is less than depth of bg . continual mechanical stress at anatomically narrow sites ( i.e. , distal bicipital groove , beneath the acromion or the coracoacromial ligament ) and impingement of the biceps tendon in the coracoacromial arch during flexion may cause these well - known degenerative changes [ 19 , 41 ] . in the present study the data of bg and biceps tendon observed from two cadavers do not indicate narrow bg as per new definition of narrowness and shallowness given in section 2 . did not find any conspicuous anatomic findings of the bicipital groove in the shoulders effected by rotator cuff diseases on mri such as a narrow groove , flat groove , or small medial groove that were predictive of biceps pathology at the time of arthroscopy [ 37 , 38 ] . this may be because of the following limitations of josheph 's study , subjective / qualitative definition of shallowness and narrowness . limitations of josheph 's study , subjective / qualitative definition of shallowness and narrowness . the limitations to josheph 's study were as follows : clearly a selection bias to surgical patients suffering from primary rotator cuff disease , only patients having mri done , classification of biceps tendon pathology was arbitrary and based purely on visual inspection not on histopathologic changes , mri is often considered to be less accurate than x - ray or computed tomography scan in evaluating bony dimensions . clearly a selection bias to surgical patients suffering from primary rotator cuff disease , only patients having mri done , to surgical patients suffering from primary rotator cuff disease , only patients having mri done , classification of biceps tendon pathology was arbitrary and based purely on visual inspection not on histopathologic changes , mri is often considered to be less accurate than x - ray or computed tomography scan in evaluating bony dimensions . lesions due to pathology of biceps tendon have been postulated to be among the most frequent causes of pain and disability in the shoulder . biceps tendon pathology has been visualized in three main categories , namely , instability , inflammatory , and traumatic . abboud et al . divided the biceps tendon pathology in normal , inflamed , partially torn , or ruptured tendon . acute inflammatory and chronic degenerative alterations causing partial / complete rupture and subluxation / dislocation can be found in the long head of the biceps tendon . instability of biceps tendon besides other factors may be attributed to length of medial / lateral walls , opening / medial wall angles depending on width / depth constituting shallowness of bg , and presence of supratubercular ridge [ 37 , 40 , 41 ] . the implication of longer walls is expected to ensure greater stability to biceps tendon lying in the bicipital groove than the shorter walls during multidirectional biomechanical movements . but the rider to this fact is that it may also cause attritional friction in a longer length of biceps tendon surrounded by longer walls creating inflammation under narrow conditions of bg . as the lengths of medial and lateral walls decrease , the instability increases and the tendon is likely to be damaged . the inference drawn is based on reconstruction of anatomical model of this part of the human body advancing the knowledge and experience of anatomy and clinical studies recorded in the literature supported by logical force as the study is on dry bones . range provides an idea of length of these walls in north indian population , whereas the mean sd reveals the average size of bg . the median may be very useful in planning surgical procedures in this part of the body . mode is representative of most frequent incidence of lengths of these walls in the subject population . if the instability of biceps tendon is studied in relation to lengths of bg most frequent value of length of walls may play a vital role in diagnostics of tendon instability or attritional damage . felt that a groove 3 mm deep or less and more than 17 mm wide may predispose to tendon subluxation or dislocation on patient radiographs . the flat groove of pfahler et al . was found to depict significant accumulation of pathologic changes in biceps tendon in 62% of cases on sonography . the supratubercular ridge of meyer and a prematurely shallow bicipital or intertubercular sulcus have been postulated to result in a variety of lesions after repetitive use or acute trauma [ 37 , 40 , 41 ] . these include acute or chronic peritendonitis , varying degrees of attrition or damage to the tendon , and subluxation or complete dislocation . as the biceps tendon is enshrined in bg , width may influence the pathology occurring in this tendon . in wider groove the tendon the morphometry of the bicipital groove in terms of length of media wall , lateral wall , length of bg , width , depth medial wall angle , and opening angle has been elucidated with reference to north indian population.the data on morphometry of bg will be of utmost use for anatomist , radiologists , orthopaedic surgeons , and physicians.the new definition of narrow / shallow bg has been given . the morphometry of the bicipital groove in terms of length of media wall , lateral wall , length of bg , width , depth medial wall angle , and opening angle has been elucidated with reference to north indian population . the data on morphometry of bg will be of utmost use for anatomist , radiologists , orthopaedic surgeons , and physicians . the new definition of narrow / shallow bg has been given . five of the world s most fatal work safety accidents in the past decade ( 20012010 ) occurred in china ( 1 ) . work safety accidents has become the first cause of deaths of chinese citizens below 44 yr old ( 2 ) . work safety accidents pose a great threat to people 's life , especially major accidents , defined as ones that result in at least 10 deaths . the chinese government has established a surveillance system for monitoring work safety accident since 2001 under state of saws ( 3 ) , which is the only authority system for work safety accidents inquiry in china . injuries related to transportation , mining , fire , explosion had the majority of occurrence ( 46 ) , and the most accidents occurred in construction on certain geographic regions of china ( 7 , 8) . previous researches had focused on the characteristics of work safety accidents , while there is very few research analyzing major accidents with mass casualties . there has no clear answer yet that whether accidents with mass casualties are a simple enlargement of general accidents , or there is inherent mechanism of accident cause and special risk factors or not . the in - depth research focused on cause mechanism of work accidents with at least 10 fatalities has important theoretical value and practical meaning to improve relevance and effectiveness of accident prevention . to examine the national profile of the extent and type of major accidents in work safety , which causes more than 10 fatalities per accident , we analyzed data from accident inquiry system on saws website for the period 2003 to 2012 and provide scientific basis for prevention measures and strategies to reduce major work safety accidents and deaths in china . data from the state saws corresponding to the period 20032012 were used in this analysis . saws is the competent agency under the state council in charge of the comprehensive supervision and administration of work safety and coalmines of the whole country . china has made mandatory provisions focusing on the implementation of safety standards , risk forecasting , accident reporting , accident accountability and other aspects through the development of relevant laws and regulations , departmental regulations and safety standards . the saws is law - based administration to strengthen the comprehensive supervision and administration of work safety and coal mines of the whole country . the saws collects information from provinces , cities and local data sources , including details regarding fatal accidents with more than three deaths and non - fatal injuries , and associated economic losses . the information of work safety accidents and coalmine incidents are required to report from enterprise involved to the regional agency and provincial department , and then finally submitted to the saws . after conducting an investigation , the saws announces the findings and determines about penalty . in this study , the data covered most industries except the military forces and private enterprises . additionally , we checked statistical year - book of road traffic accidents and public literature to supplement information and details . the analysis was conducted using the registered data with more than 10 deaths per incident . major accidents in transportation involved victim who was the operator , passenger , or a pedestrian stuck in or on the side of the road . after extraction , details including types of the event , year , region , number of deaths and injuries , causes of accidents were transformed into spss v13.0 ( chicago , il , usa ) . we performed analysis on the frequency of accidents and deaths , the trend , geographic distribution and type of major accidents . we analyzed the geographic distribution using hierarchical cluster analysis , focused on the frequency of accidents and deaths . incidence of major accident shows the occurrence probability of any person , and major accident mortality shows the dying probability . the incidence and mortality of major accident are defined as follows : incidence = number of major accident/ ( total national population106)mortality = number of death/ ( total national population 106 ) data from the state saws corresponding to the period 20032012 were used in this analysis . saws is the competent agency under the state council in charge of the comprehensive supervision and administration of work safety and coalmines of the whole country . china has made mandatory provisions focusing on the implementation of safety standards , risk forecasting , accident reporting , accident accountability and other aspects through the development of relevant laws and regulations , departmental regulations and safety standards . the saws is law - based administration to strengthen the comprehensive supervision and administration of work safety and coal mines of the whole country . the saws collects information from provinces , cities and local data sources , including details regarding fatal accidents with more than three deaths and non - fatal injuries , and associated economic losses . the information of work safety accidents and coalmine incidents are required to report from enterprise involved to the regional agency and provincial department , and then finally submitted to the saws . after conducting an investigation , the saws announces the findings and determines about penalty . in this study , the data covered most industries except the military forces and private enterprises . additionally , we checked statistical year - book of road traffic accidents and public literature to supplement information and details . the analysis was conducted using the registered data with more than 10 deaths per incident . major accidents in transportation involved victim who was the operator , passenger , or a pedestrian stuck in or on the side of the road . after extraction , details including types of the event , year , region , number of deaths and injuries , causes of accidents were transformed into spss v13.0 ( chicago , il , usa ) . we performed analysis on the frequency of accidents and deaths , the trend , geographic distribution and type of major accidents . we analyzed the geographic distribution using hierarchical cluster analysis , focused on the frequency of accidents and deaths . incidence of major accident shows the occurrence probability of any person , and major accident mortality shows the dying probability . the incidence and mortality of major accident are defined as follows : incidence = number of major accident/ ( total national population106)mortality = number of death/ ( total national population 106 ) we found a dramatic decline in both numbers of accidents and deaths since 2006 . the number of accidents and deaths dropped by 52.0% and 66.6% from the peak year ( 2005 ) to 2012 , respectively . in the 10-yr period from 2003 to 2012 , there were 866 major accidents in work safety in china , resulting in 16795 deaths and 9183 injuries . the incidence of major accident and the mean mortality rate were 1.93 and 1.34 per one million populations annual . in recent years , both the major accident mortality rate and incidence of major accident have declined ( fig . 1 ) . mortality rate and incidence of major accidents in work safety in china , by year , 20032012 fig . the deeper the color in the region , the higher number of accidents and deaths occurred . using hierarchical cluster analysis , we found that the regions reporting the highest number of injuries and deaths were hunan ( 62/936 ) , guizhou ( 69/1101 ) , henan ( 54/1302 ) , shanxi ( 79/2045 ) , with a total of 264 incidents and 5384 deaths , both accounting for 30% nation - wide . geographic distribution of major work safety accidents in china , 20032012 ( number of incidents / number of deaths ) transportation - related accident was responsible for the highest frequency of deaths from 2003 to 2012 ( n=437 ) . table 1 shows that transportation and mining caused more injuries and deaths during the dates evaluated . the number of injuries declined in the last years , as did the number of injuries caused by all types of industries . however , we have not noticed such a tendency in manufacturing , construction and service . furthermore , mining declined in percentage of injuries , while percentage of transportation - related accident increased . type of industry - related accident in china by year , 20032012 mining contributed to the highest number of deaths ( n=7646 ) . from 2003 to 2012 , there were 309 mining - related accidents , each of which caused 24.7 fatalities . as indicated in table 2 , more than 80% of major work safety accident - related deaths occurred in mining and transportation , and both of them declined in recent years . number of deaths by industry in china by year , 20032012 table 3 displays the number of deaths per accident by industry by year . the average number of death per accident in service and mining shows a decline in recent years , while we have not found the similar trend in manufacturing , construction and transportation . coal mine - related and road traffic - related accidents were dominant and nearly accounted for four - fifths of all types of major accidents . the evaluation of all types of accidents has revealed that tailing dam related accident was the most severe in terms of number of fatalities per incident , causing 105 deaths per incident . number of deaths per accident by industry in china by year , 20032012 aviation - related and railway - related accidents caused 49.5 and 43.3 deaths per incident , respectively ( table 4 ) . the most lethal type of major work safety accident , with the highest death injury ratio was tailing dam accident . machinery , collapse , electric shock accident and coalmine accident were specifically higher in death injury ratio , while aviation accident and railway accident were specifically higher in the number of deaths per incident ( fig . 3 ) . number of deaths and frequency by type of accidents in china , 20032012 death per incident and death injury ratio in different types of accident we found a dramatic decline in both numbers of accidents and deaths since 2006 . the number of accidents and deaths dropped by 52.0% and 66.6% from the peak year ( 2005 ) to 2012 , respectively . in the 10-yr period from 2003 to 2012 , there were 866 major accidents in work safety in china , resulting in 16795 deaths and 9183 injuries . the incidence of major accident and the mean mortality rate were 1.93 and 1.34 per one million populations annual . in recent years , both the major accident mortality rate and incidence of major accident have declined ( fig . 1 ) . mortality rate and incidence of major accidents in work safety in china , by year , 20032012 fig . 2 shows major work safety accidents and deaths in relation to their geographic distribution . the deeper the color in the region , the higher number of accidents and deaths occurred . using hierarchical cluster analysis , we found that the regions reporting the highest number of injuries and deaths were hunan ( 62/936 ) , guizhou ( 69/1101 ) , henan ( 54/1302 ) , shanxi ( 79/2045 ) , with a total of 264 incidents and 5384 deaths , both accounting for 30% nation - wide . geographic distribution of major work safety accidents in china , 20032012 ( number of incidents / number of deaths ) transportation - related accident was responsible for the highest frequency of deaths from 2003 to 2012 ( n=437 ) . table 1 shows that transportation and mining caused more injuries and deaths during the dates evaluated . the number of injuries declined in the last years , as did the number of injuries caused by all types of industries . however , we have not noticed such a tendency in manufacturing , construction and service . furthermore , mining declined in percentage of injuries , while percentage of transportation - related accident increased . type of industry - related accident in china by year , 20032012 mining contributed to the highest number of deaths ( n=7646 ) . from 2003 to 2012 , there were 309 mining - related accidents , each of which caused 24.7 fatalities . as indicated in table 2 , more than 80% of major work safety accident - related deaths occurred in mining and transportation , and both of them declined in recent years . the average number of death per accident in service and mining shows a decline in recent years , while we have not found the similar trend in manufacturing , construction and transportation . coal mine - related and road traffic - related accidents were dominant and nearly accounted for four - fifths of all types of major accidents . the evaluation of all types of accidents has revealed that tailing dam related accident was the most severe in terms of number of fatalities per incident , causing 105 deaths per incident . number of deaths per accident by industry in china by year , 20032012 aviation - related and railway - related accidents caused 49.5 and 43.3 deaths per incident , respectively ( table 4 ) . the most lethal type of major work safety accident , with the highest death injury ratio was tailing dam accident . machinery , collapse , electric shock accident and coalmine accident were specifically higher in death injury ratio , while aviation accident and railway accident were specifically higher in the number of deaths per incident ( fig . 3 ) . number of deaths and frequency by type of accidents in china , 20032012 death per incident and death injury ratio in different types of accident economic development is related to the incidence of accident . in developing countries such as china , increases in the growth rate of economic may have caused work safety accident rate to rise ( 9 ) . in china , both major work safety accidents and deaths appear to have decreased since 2006 , while the growth rate of gdp shows an increase steadily ( varying in the range of 7.8% to 14.2% ) ( 10 ) . efforts in regulations and surveillance , safety initiatives and innovations in safety controls by chinese government can attribute to the decline . to improve the whole country s work safety , chinese government has issued a series of supervision regulations especially for fatal serious work safety accidents , such as fire regulation , transportation regulation , coal mine and non - coal mine regulations , and the 11 five - year plan for national economic and social development of the people s republic of china ( 11 ) . as part of this move , chinese government has also strengthened the penalties for fatal major work safety . as the formulation and implementation of the regulations , provinces have made great improvements in preventing serious and major accidents . the number of accidents and deaths show a sharp drop in ten years , which are broadly consistent with other published studies ( 1217 ) . while there has been a decline in the frequency of accidents and deaths in recent years , the mortality rate was still relatively high . according to zhang et al . ( 12 ) , the risk of death was 6.1 per million populations in work safety in whole country , which was higher than other countries ( 18 ) . using hierarchical cluster analysis , we recognized that provinces of hunan , guizhou , henan , shanxi reported the highest number of major accidents and deaths , while provinces of shanghai , qinghai , hainan , ningxia , beijing , tianjin reported the least , which is consistent with the regional distribution of work safety accidents in whole country ( 12 ) . after detailed analyzing , we found that hunan , guizhou , henan and shanxi had more than 55 major accidents , resulting in more than 900 deaths . among these accidents , mining and transportation contributed to the majority , accounting for more than 89% of all . these statistics may relate to the differences in natural environment , working conditions , the level of economic development and economic patterns . this percentage is comparable with the other published studies of work safety accidents in china ( 19 , 20 ) . based on counts and proportions of accidents , transportation and mining that mostly produce casualties were still high - risk industrial safety accident types in china . in transportation section data analysis revealed that one major road traffic accident occurred every 10 days on average in china , while the average number of deaths in aviation was 49.5 . this may be an important reason why fewer aviation accidents caused more widespread concern in the international community . road transportation regulations of people 's republic of china was issued at the end of 2004 ( 21 ) , and efforts were made to better disseminate the regulation , safety procedures , and tips to operators to increase drivers awareness of risk and to improve emergency response capabilities . to reduce traffic accidents , some prevention strategies including driver training , working conditions , weather conditions , hours - of - service regulations , and safety culture should be better considered ( 2225 ) . in mining section , coalmine accidents contributed to the majority . literature reported that coal - mine accidents in china occurred ten times more frequently than that in india ( 1 ) , and the death rate per million tons was 6.66 during the past 24 yr , which was ninety - four times more larger than in america ( 0.07 ) ( 26 ) . the distribution and exploitation of mineral resources are an important factor for the significant difference between china and america . fortunately , with the provisions on the prevention of coal mine accidents at the end of 2005 ( 27 ) and the closure of half of the operating mines in 2006 , the death rate per million tons showed a rapid decline since 2006 ( 26 ) , and the number of mining accidents and deaths including major mining accidents declined . however , several safety concerns still persist in some mines , such as ignoring safety professionals , lack of emergency rescue measures and employee professional safety training . some effective interventions are needed to mitigate the worst health and safety performance in china . this study used death per incident and death injury ratio as a surrogate measure to describe the severity of major accidents and the urgency of emergency rescue . the declines in the number of death per service and mining accident indicated improvement on the special intervention . however , obstacles exist in transportation , construction and manufacturing intervention . as show in fig . 4 , the occurrence of tailing dam accident associated with major casualties and economic losses , and it will cause high case - fatality once occurred . like tailing dam incident , collapse , therefore , full risk assessment and prevention measures are the primary intervention to prevent these accidents . aviation and railway accidents were recognized to result in relatively higher case - fatality and higher number of occupational injuries . we discussed the major work safety accidents nationwide and the severity and emergency rescue urgency of accident using a scatter plot by death per accident and death injury ratio , the improvement compared with the similar studies ( 7 , 12 ) . additionally , we searched the published literature and statistical yearbook to supplement the detailed information . the information registered in the saws system were not comprehensive , such as the cause of per incident , thus we could not do further analyze . ten years work safety data indicate that the frequency of incidents and number of deaths of major work safety accidents were declined . 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 .

the variant morphometry of bicipital groove is reported to be associated with pathologies of biceps tendon and is useful in surgical procedures in this region . the pathologies of biceps tendon are frequent causes of shoulder pain . therefore , under the condition of paucity of data pertaining to north indians , not only morphometric analysis of bicipital groove and a new definition of narrow / shallow groove to provide logical explanation for dependence of pathologies of biceps tendon on groove morphology is done but also a review of the literature has been carried out . various dimensions such as lengths of medial and lateral walls , width , depth , medial wall , and opening angles including incidence of supratubercular ridge of bicipital groove from 101 humerii are 23 5 , 32 5 , 8 2 , 6 1 , 48.91 10.31 , 82.20 22.62 , and 37% , respectively . the average height along with average width of biceps tendon and average width along with average depth of bicipital groove from two cadavers are 1.8 , 10.5 , 11.3 , and 5.5 mm , respectively . the knowledge of bicipital groove will be of paramount importance to anatomists for new data , for orthopaedic surgeons in carrying out surgical procedures in this region , and for physicians in the management of anterior shoulder pain in north indian population . background : this study provides a national profile of major work safety accidents in china , which cause more than 10 fatalities per accident , intended to provide scientific basis for prevention measures and strategies to reduce major work safety accidents and deaths.methods:data from 20032012 census of major work safety accidents were collected from state administration of work safety system ( saws ) . published literature and statistical yearbook were also included to implement information . we analyzed the frequency of accidents and deaths , trend , geographic distribution and injury types . additionally , we discussed the severity and urgency of emergency rescue by types of accidents.results:a total of 877 major work safety accidents were reported , resulting in 16,795 deaths and 9,183 injuries . the numbers of accidents and deaths , mortality rate and incidence of major accidents have declined in recent years . the mortality rate and incidence was 0.71 and 1.20 per 106 populations in 2012 , respectively . transportation and mining contributed to the highest number of major accidents and deaths . major aviation and railway accidents caused more casualties per incident , while collapse , machinery , electrical shock accidents and tailing dam accidents were the most severe situation that resulted in bigger proportion of death.conclusion:ten years major work safety accident data indicate that the frequency of accidents and number of eaths was declined and several safety concerns persist in some segments .

previously described growth conditions including those for inducing development were used for a. nidulans [ 17 , 18 ] . expression of anea via niia promoter is induced by 0.6% sodium nitrate and repressed by 0.2% ammonium tartrate . an anea disruption cassette ( dc ) was constructed according to methods described by yu et al . . using genomic dna prepared from a. nidulans fgsc a4 , dna fragments for the dc were amplified by pcr by using appropriate primer sets ( anea - a1/-a2 , anea - b1/-b2 , and argb - for/-rev ) . the complete dc was amplified by a nested - pcr primer set ( anea - c1/-c2 ) , purified , and then used to transform the tj1 strain . nucleic acid preparation and northern blot analysis were performed as described previously . for southern blotting , genomic dna was treated with appropriate restriction enzymes and then separated on 1% agarose gels . the gels were washed with distilled water ( dw ) and soaked twice in a depurination solution ( 250 mm hcl ) for 15 min . after the reaction , the gel was washed with dw again and then soaked in denaturation solution ( 1.0 m nacl and 0.4 m naoh ) for 1 hr . the genomic dna contained in the processed gel was transferred onto a hybond - n membrane ( amersham biosciences , pittsburg , pa , usa ) . gene - specific probes were prepared from the pcr - generated fragments and were labeled using the ecl direct nucleic acid labeling system ( amersham biosciences ) . previously described growth conditions including those for inducing development were used for a. nidulans [ 17 , 18 ] . expression of anea via niia promoter is induced by 0.6% sodium nitrate and repressed by 0.2% ammonium tartrate . an anea disruption cassette ( dc ) was constructed according to methods described by yu et al . . using genomic dna prepared from a. nidulans fgsc a4 , dna fragments for the dc were amplified by pcr by using appropriate primer sets ( anea - a1/-a2 , anea - b1/-b2 , and argb - for/-rev ) . the complete dc was amplified by a nested - pcr primer set ( anea - c1/-c2 ) , purified , and then used to transform the tj1 strain . nucleic acid preparation and northern blot analysis were performed as described previously . for southern blotting , genomic dna was treated with appropriate restriction enzymes and then separated on 1% agarose gels . the gels were washed with distilled water ( dw ) and soaked twice in a depurination solution ( 250 mm hcl ) for 15 min . after the reaction , the gel was washed with dw again and then soaked in denaturation solution ( 1.0 m nacl and 0.4 m naoh ) for 1 hr . the genomic dna contained in the processed gel was transferred onto a hybond - n membrane ( amersham biosciences , pittsburg , pa , usa ) . gene - specific probes were prepared from the pcr - generated fragments and were labeled using the ecl direct nucleic acid labeling system ( amersham biosciences ) . the dc for deletion of the anea gene was constructed and used to transform the recipient strain . southern blot analysis of pst i - digested genomic dna from candidate strains with a pcr - amplified probe specific for the 5'-flanking region of anea confirmed the deletion of the chromosomal anea gene by revealing an expected band of 5.7 kb ( fig . 1 ) . to investigate the function of anea in development of a. nidulans , the pattern of hyphal growth including radial growth , septation , and asexual sporulation were examined in one of the deletion mutant strains . however , we observed no differences between the wild - type and deletion mutant ( data not shown ) . these results indicated that -cop is not essential for the viability of a. nidulans ; these results are similar to those obtained in yeasts . recently , copi proteins were reported to play roles in responses to er stress and thermal stress in yeast [ 11 , 13 ] . here , we tested the effect of -cop deletion on sensitivity to environmental stresses caused by treatment of the fungi with several drugs that are known to exert adverse effects on cellular processes related to the stress response . no detectable changes were observed in the -cop - defective strain by treatment with drugs including inhibitors of cell - wall biosynthesis ( calcofluor white , congo red , and caspofungin ) , n - glycosylation ( tunicamycin ) , ergosterol biosynthesis ( terbinafine ) , and glycerol biosynthesis ( fludioxonil ) , an inducer of apoptosis ( farnesol ) , and a ca chelator ( egta ) . thermal stress , which makes the -cop - defective yeast strain unviable , showed no effect on viability and vegetative growth in the -cop deletion strains ( data not shown ) . taken together , these results show that -cop in a. nidulans is neither essential for viability nor involved in stress responses unlike in yeast . in a. nidulans , a mutation in the sodc gene copi depletion also results in the failure of cytokinesis and a reduction in the number of overlapping central spindle microtubules during meiotic divisions for spermatogenesis in drosophila . in the -cop - depleted ( anea ) a. nidulans strain , we observed no defects in asexual development ( data not shown ) , but significant defects in sexual development ( fig . even after culturing in conditions favorable for cleistothecium development , with hypoxic treatment with 1% glucose in the dark , the anea strain showed more than 50% decrease of fruiting body formation ( table 3 ) . in addition , in 1% glucose without hypoxic treatment , fruiting body formation was almost completely abolished by -cop depletion . in 2% lactose without hypoxic treatment , which predominantly induces the formation of fruiting bodies , -cop depletion showed no deleterious effect on fruiting body formation ( table 3 ) . because the effect of c - sources and hypoxic treatment was tested in the presence of 0.1% sodium nitrate as a nitrogen source , we tested 0.2% yeast extract , which preferentially induces sexual development , and found that the defect in fruiting body formation on 1% glucose without hypoxic treatment was recovered in anea deletion strain ( data not shown ) . although further experiments are required to determine the underlying mechanisms , our results suggested that -cop plays a role in sexual development in a. nidulans under certain environmental conditions , possibly by affecting cytokinesis and/or construction of er - based spindle envelopes , as observed in fruit fly spermatogenesis . our previous study showed that the c - terminal domains of both -cop and -cop are essential for their interaction , and that the n - terminal wd40 motif of -cop and the tpr region of -cop are involved in controlling the interaction between these two cops in a. nidulans . we also reported that a. nidulans -cop can substitute for s. cerevisiae -cop functions in vivo . in yeast , -cop is known to stabilize the thermo - sensitive -cop mutation . thus , over - expression of -cop confers viability to the -cop mutant at elevated temperatures . therefore , we investigated whether the phenotype of the sodc1 mutant was suppressed by over - expression of anea in a. nidulans . when a prg3-ama1 plasmid containing the anea gene encoding -cop was introduced into the sodc1 mutant , over - expression of anea however , over - expression of -cop could not rescue the thermo - sensitive phenotype of the sodc1 mutant at a non - permissive temperature ( 42 ) ( fig . these results indicate that -cop is able to interact with -cop , but does not stabilize -cop at elevated temperatures unlike in yeast . in summary , our results indicate that -cop in a. nidulans is not essential but related to formation of the sexual reproductive organ in response to particular environmental factor(s ) . it is worth highlighting that recent reports indicate that the function of the copi complex ( and its subunits ) is not confined to intracellular vesicular trafficking . the copi complex is also involved in other cellular events such as chromosome disjunction in a. nidulans , male development in chicken embryos , er stress responses in yeast , association with calcineurin during heat stress , meiotic divisions for spermatogenesis in drosophila , induction of productive autophagy and cellular survival , intercompartmental trafficking of specific rnas in neuronal cells , and neurite outgrowth . although further studies are necessary to confirm the actual role of -cop ( and/or copi ) in sexual development of fungi , our results open up interesting avenues for further studies on the function of the copi complex . the dc for deletion of the anea gene was constructed and used to transform the recipient strain . southern blot analysis of pst i - digested genomic dna from candidate strains with a pcr - amplified probe specific for the 5'-flanking region of anea confirmed the deletion of the chromosomal anea gene by revealing an expected band of 5.7 kb ( fig . 1 ) . to investigate the function of anea in development of a. nidulans , the pattern of hyphal growth including radial growth , septation , and asexual sporulation were examined in one of the deletion mutant strains . however , we observed no differences between the wild - type and deletion mutant ( data not shown ) . these results indicated that -cop is not essential for the viability of a. nidulans ; these results are similar to those obtained in yeasts . recently , copi proteins were reported to play roles in responses to er stress and thermal stress in yeast [ 11 , 13 ] . here , we tested the effect of -cop deletion on sensitivity to environmental stresses caused by treatment of the fungi with several drugs that are known to exert adverse effects on cellular processes related to the stress response . no detectable changes were observed in the -cop - defective strain by treatment with drugs including inhibitors of cell - wall biosynthesis ( calcofluor white , congo red , and caspofungin ) , n - glycosylation ( tunicamycin ) , ergosterol biosynthesis ( terbinafine ) , and glycerol biosynthesis ( fludioxonil ) , an inducer of apoptosis ( farnesol ) , and a ca chelator ( egta ) . thermal stress , which makes the -cop - defective yeast strain unviable , showed no effect on viability and vegetative growth in the -cop deletion strains ( data not shown ) . taken together , these results show that -cop in a. nidulans is neither essential for viability nor involved in stress responses unlike in yeast . in a. nidulans , a mutation in the sodc gene encoding -cop is responsible for non - disjunction of the chromosome during cell division . copi depletion also results in the failure of cytokinesis and a reduction in the number of overlapping central spindle microtubules during meiotic divisions for spermatogenesis in drosophila . in the -cop - depleted ( anea ) a. nidulans strain , we observed no defects in asexual development ( data not shown ) , but significant defects in sexual development ( fig . even after culturing in conditions favorable for cleistothecium development , with hypoxic treatment with 1% glucose in the dark , the anea strain showed more than 50% decrease of fruiting body formation ( table 3 ) . in addition , in 1% glucose without hypoxic treatment , fruiting body formation was almost completely abolished by -cop depletion . in 2% lactose without hypoxic treatment , which predominantly induces the formation of fruiting bodies , -cop depletion showed no deleterious effect on fruiting body formation ( table 3 ) . because the effect of c - sources and hypoxic treatment was tested in the presence of 0.1% sodium nitrate as a nitrogen source , we tested 0.2% yeast extract , which preferentially induces sexual development , and found that the defect in fruiting body formation on 1% glucose without hypoxic treatment was recovered in anea deletion strain ( data not shown ) . although further experiments are required to determine the underlying mechanisms , our results suggested that -cop plays a role in sexual development in a. nidulans under certain environmental conditions , possibly by affecting cytokinesis and/or construction of er - based spindle envelopes , as observed in fruit fly spermatogenesis . our previous study showed that the c - terminal domains of both -cop and -cop are essential for their interaction , and that the n - terminal wd40 motif of -cop and the tpr region of -cop are involved in controlling the interaction between these two cops in a. nidulans . we also reported that a. nidulans -cop can substitute for s. cerevisiae -cop functions in vivo . in yeast , -cop is known to stabilize the thermo - sensitive -cop mutation . thus , over - expression of -cop confers viability to the -cop mutant at elevated temperatures . therefore , we investigated whether the phenotype of the sodc1 mutant was suppressed by over - expression of anea in a. nidulans . when a prg3-ama1 plasmid containing the anea gene encoding -cop was introduced into the sodc1 mutant , over - expression of anea however , over - expression of -cop could not rescue the thermo - sensitive phenotype of the sodc1 mutant at a non - permissive temperature ( 42 ) ( fig . these results indicate that -cop is able to interact with -cop , but does not stabilize -cop at elevated temperatures unlike in yeast . in summary , our results indicate that -cop in a. nidulans is not essential but related to formation of the sexual reproductive organ in response to particular environmental factor(s ) . it is worth highlighting that recent reports indicate that the function of the copi complex ( and its subunits ) is not confined to intracellular vesicular trafficking . the copi complex is also involved in other cellular events such as chromosome disjunction in a. nidulans , male development in chicken embryos , er stress responses in yeast , association with calcineurin during heat stress , meiotic divisions for spermatogenesis in drosophila , induction of productive autophagy and cellular survival , intercompartmental trafficking of specific rnas in neuronal cells , and neurite outgrowth . although further studies are necessary to confirm the actual role of -cop ( and/or copi ) in sexual development of fungi , our results open up interesting avenues for further studies on the function of the copi complex . the diagnosis and extent of resection in the management of hd depend on the sensitive and specific identification of ganglion cells.[13 ] however , documenting aganglionosis is often difficult and tedious on routine hematoxylin - eosin ( h and e ) stained sections . acetylcholinesterase ( ache ) has evolved as the gold standard in diagnosing hd ; however , this histochemical analysis is technically challenging , and to date , has not gained worldwide utilization and applicability . the aim of this study was to evaluate the efficacy of calretinin immunostaining in the ganglionic and aganglionic hd colon biopsy specimens and correlate with the h and e , thereby exploring its utility in suspicious cases of hd . our standard protocol of care for a child presenting with hd is barium enema study at presentation followed by a laparotomy where multiple biopsies are taken from the spastic aganglionic segment , transition zone , and the normal colon . a colostomy is sited at the level of the junction between the normal colon and the transition zone . the definitive surgery of choice is duhamel 's pull - through after 6 months of age . thirty - six were full - thickness rectal biopsies ( for suspected hd ) and 24 were bowel segments resected during the definitive pull - through surgery . calretinin ( monoclonal mouse antihuman antibody ( dako ) , ( clone : dak calret 1 , code : ir627 ) immunohistochemistry ( ihc ) staining was done on all paraffin - embedded blocks after routine h and e examination . the age of patients ranged from 1 day to 14 years ( mean 8.2 months ) , and there were 46 boys and 14 girls ( m : f = 3.2 - 3.3:1 ; normal ratio 3:1 - 4:1 ) . twenty - three patients ( 63.8% ) presented at less than 1 month of age . five ( 8.3% ) had long segment disease , 6 ( 10% ) had total colonic aganglionosis . during the analysis of 36 initial full - thickness colon biopsy specimens , h and e staining revealed absence of ganglion cells ( negative ) in 19 cases ( 52.7% ) , presence of ganglion cells ( positive ) in 2 cases ( 0.05% ) , and suspected presence of ganglion cells in 15 cases ( 41.6% ) . of the 19 cases reported negative through h and e staining , 17 ( 47.22% ) were reported negative , and 2 ( 0.05% ) positive for calretinin histochemistry for the ganglion cells and nerve fibers [ table 1 ] . hematoxylin and eosin staining versus calretinin in rectal biopsies in 15 patients , the h and e sections were suspicious of a presence of ganglion cell . calretinin ihc showed immunopositivity in 3 slides , whereas 5 slides showed immunopositivity in nerve fibers . among the 5 slides positive for nerve fibers , in 2 of the cases ( 2/60 ) , calretinin gave a slight positive staining of nerve fibers , but with no staining of other areas . the slight calretinin positivity in the 2 specimens was observed in some large bundles with no staining in other areas , and thus indicating the beginning of transition zone . in the 24 patients where resected specimens from the definitive surgery were sent , serial biopsies were taken from the aganglionic segment , transition zone , and the ganglionic segment [ table 2 ] . calretinin was not expressed in the ganglion cells and nerve fibers of submucosal and myenteric plexus of 24 aganglionic ( spastic ) segments . in the transition zone , calretinin staining was positive in the ganglion cells in 20 cases ( 83.3% ) and was focally positive in the nerve fibers of 22 cases ( 91.6% ) , both in the submucosal and myenteric plexus . in ganglionic bowel segments of hd , calretinin showed immunopositivity in > 90% of ganglion cells and nerve fibers of submucosal and myenteric plexus . the histological diagnosis of hd is challenging , requiring the expertise of a senior pathologist and access to specialized techniques for handling frozen specimens for ache staining . this makes the diagnosis of hd difficult in centers where cases are infrequent , causing a delay in the treatment of the child . calretinin is proven to be highly sensitive for the presence of ganglion cells and nerve fibers . this protein is involved in calcium transport ; and its absence allows accumulation of cytoplasmic calcium , causing excess neuroexcitability and ultimate neurodegeneration . barshack , et al . , procured the colons of 10 patients with proven hd . calretinin ihc was performed on sections from the aganglionic zone , ganglionic zone , and transition zone of these colons . they identified calretinin staining in interstitial nerve fibres ( inf ) and ganglion cells in normal colon [ figures 1 and 2 ] and focal inf staining in 92% of transition zones . in contrast , there was no inf or ganglion cell staining in the aganglionic zone . it can be interpreted as positive or negative for immune reactivity reducing the ambiguity in diagnosis . in our study , in the rectal biopsy specimens , diagnosis was ambiguous in 15 patients on h and e examination . by using calretinin , immune positivity was demonstrated in 3 patients ( 3 for ganglion cells and 5 for nerve fibers ) ; thereby , ruling out hd in these patients . hematoxylin and eosin section with ganglion cell ( arrow ) ( h and e , 10 ) calretinin staining of ganglion cells ( h and e , 40 ) guinard - samuel , et al . , have studied the largest number of suction biopsies using calretinin . they have concluded that calretinin is accurate in proving the absence of ganglion cells , it is easy to interpret , and can replace ache to diagnose hd . they , however , recommend that it should be used as an aid along with h and e examination , especially in ultrashort segment disease and transition zone specimens . in our study , we found that calretinin correlated with h and e examination in both rectal biopsies and the resected bowel specimens . in the rectal biopsy specimens , calretinin also aided in the diagnosis of 15 patients with ambiguous findings . calretinin ihc is accurate in detecting the presence or absence of ganglion cells and holds several advantages such as follows : ( 1 ) it can be carried out on paraffin - embedded tissue sections ; ( 2 ) staining pattern is simple ; ( 3 ) binary pattern of interpretation ( negative or positive ) ; ( 4 ) it is cost effective . in the present study it can serve as a valuable cost - effective diagnostic aid in the centers where ache enzyme histochemistry is not available .

we have previously isolated -cop , the -cop interactor in copi of aspergillus nidulans , by yeast two - hybrid screening . to understand the function of -cop , the anea+ gene for -cop / anea was deleted by homologous recombination using a gene - specific disruption cassette . deletion of the -cop gene showed no detectable changes in vegetative growth or asexual development , but resulted in decrease in the production of the fruiting body , cleistothecium , under conditions favorable for sexual development . unlike in the budding yeast saccharomyces cerevisiae , in a. nidulans , over - expression of -cop did not rescue the thermo - sensitive growth defect of the -cop mutant at 42. together , these data show that -cop is not essential for viability , but it plays a role in fruiting body formation in a. nidulans . aim : to evaluate the efficacy of calretinin immunostaining in diagnosing hirschsprung 's disease ( hd).materials and methods : sixty cases were studied over a period of 1 year ( july 2010-june 2011 ) . there were 36 full - thickness biopsies and 24 resected specimens . calretinin processing was done on the paraffin - embedded blocks after routine histopathological examination.results:of the 36 biopsy specimens , in 19 cases hd was diagnosed by hematoxylin and eosin ( h and e ) staining earlier . in 2 patients , ganglion cells were seen and hd was ruled out . in 15 cases , there was a diagnostic dilemma and calretinin was used . ganglion cells were found in 3 specimens and nerve fibers in 5 . in all 24 resected specimens , calretinin correlated with the findings on h and e staining.conclusions:calretinin was extremely useful in solving the suspicious and indeterminate cases of hd . it can serve as a valuable cost - effective diagnostic aid in the centers where acetylcholinesterase enzyme histochemistry is not available .

drug abuse is considered a critical health related social and economic problem in most countries . in the last three decades , the world has experienced shocking figures expressing drug abuse prevalence in societies , esp . among teenagers and the youth . united nation 's office of crime and drug abuse prevention has recently reported 185 million drug consumers world - wide and an increasing treatment demand all over the world . iranian drug abuse prevention headquarter has estimated 4.5 million opium consumers in iran by the end of 2004 . in a study conducted by serajzadeh and feyzi in 21 iranian state universities , 5.8% of the interviewed drug consumers claimed to be drug dependent , while 3.1% claimed a high drug dependency , making a total of 8.9% of a drug dependent university students . regarding the harmful pharmaceutical , social , legal , health and economic effects of drug abuse , the need for serious preventive action one divides drug abuse prevention into three comprehensive , selective and obligatory categories . other preventive strategies include life skills training ( lst ) , information improvement , affective education , social influence and drug abuse resistance education . life skills are abilities needed to provide the groundwork for effective stress management and presentation of positive behaviors . these skills enable an individual to accept his social role responsibilities and to face others demands and expectations and daily interpersonal problems effectively without hurting himself or others . the term life skills involves a big class of socio - psychological and interpersonal skills which help an individual to take conscious decisions , communicate effectively , improve his interactive and self - management skills and adopt an active healthy life - style . life skills can organize personal , interpersonal and environmental actions in a way to lead to better health , which in turn leads to more physical , psychological and social comfort . lst program aimed at drug abuse prevention is a new preventive strategy which primarily focuses on socio - psychological factors leading to drug abuse and mainly emphasizes the promotion of personal and social skills . this program was first planned by gilbert and botvin from kernel university to prevent smoking abuse among teenagers in 1997 and was then used for alcohol and drug abuse prevention . researchers have confirmed the positive influence of lst on drug abuse reduction , effective use of intelligence capacity , furthering self - confidence and ego improvement , prevention of aggressive behaviors and suicide and aids prevention . lots of other investigations have also been conducted on other individual and social problems and their solution through lst . studied the impact of lst on middle schoolers knowledge , insight and ability to adopt a healthy life - style . findings reported a lower rate of smoking among those who attended the program compared with those who refused to attend . eisen et al . showed a significant difference between pre- and post - test scores of 6239 students . in this study , drug abuse rate among the intervention group was significantly lower than the norm compared to the control group . another study by botvin and griffin claimed the positive effect of lst on addiction prevention . the same survey tested 4466 students of new york schools for 3 years to investigate the effectiveness of the cognitive - behavioral program of drug abuse prevention . those who received at least 60% of the intervention program showed a significant drug abuse reduction . furthermore , qaderi indicated that training courses could reduce drug abuse tendency and affect addicts attitude . one other survey was carried out by nazarpoor et al . on 243 students of tabriz medical university to investigate how lst affects an individual 's opinion toward drug abuse prevention . besides , there was a change or rise in knowledge , insight and assertiveness skills rate after attending lst workshops . a significant relationship was also observed between participants change of attitude toward drug abuse and the rise in social skills due to participation in workshops . furthermore , a significant relationship was detected between major and gender and social skills promotion rate ( a greater change was observed among boys ) . they concluded that due to its impact on cognitive skills development , attending lst workshops is necessary for all university students . results of a survey in feredrick university in germany not only demonstrated the preventive effect of lst on non - smokers and non - drinkers , but also its great impact on smokers and drinkers . moradi et al . investigated the effect of drug abuse resistance and prevention skills training on 181 workers of asalooyeh petrochemical company under two intervention and control groups . results indicated the positive impact of training on the intervention group 's knowledge and insight , drug abuse resistance skills , self - efficacy and decision - making in preventive actions . in their study , barati et al . showed the positive role of lst in the reduction of abstract norms encouraging drug abuse among university students . this can prove the positive impact of assertiveness training program on modification of beliefs and abstract norms of the youth and also its great influential role in planning and performance of drug abuse prevention training programs , especially in universities . the present study was aimed at investigating the effect of lst on promotion of drug abuse preventive behaviors among university students and stability of the results after a 4-year follow - up . this field trial study was conducted using pre- and post - experimental design with the control group . samples were 60 university students ( 50% male and 50% female ) entering university in different majors , selected through quota random sampling and assigned randomly into two equal intervention and control groups after matching . the selection criterion included factors as : being a student of gonabad medical universityentering the university in the second semester of 2007giving consent and being able to attend workshops . being a student of gonabad medical university entering the university in the second semester of 2007 giving consent and being able to attend workshops . the intervention group took part in two one - day lst workshops held weekly . before the first workshop four years after intervention ( in 2011 ) , all examinees except for 9 were tested again using the same instrument ( 4 from the intervention group and 5 from the control group ceased to continue with the study due to attrition ) . to control all the confounding variables all participants were under inspection during this period by researchers . data were collected by the use of a researcher made questionnaire including : ( a ) demographic information : age , gender , major , father 's and mother 's educational level , father 's job and habitation . ( b ) drug abuse preventive behaviors including : self - awareness , interactive skills , decisiveness , ability to say no , problem - solving , ability to resist others illogical demands , stress management , familiarity with drug abuse side - effects and negative attitude toward drug abuse . the questionnaire included the following items : regarding self - awareness , items such as familiarity with the components of self - awareness , self - evaluation skills , knowledge of self - strengths and weaknesses , positive thinking abilities , positive ego development skills , relationship between self - esteem and drug abuse , self - esteem and endurance , features of tolerant people and factors affecting those features were investigatedthe domain of interactive skills investigated issues such as : knowledge of definition and objectives of communication , components of communication , communication barriers , ineffective interaction , effective listening , interpersonal relations and their importance and ways to improve themregarding decisiveness , saying no to others and resistance to others illogical demands , items such as : familiarity with basic concepts of assertiveness , interactive styles , types of assertiveness , cognitive barriers to assertiveness , crucial steps to behavior change , steps of assertive behavior , some advice on how to say no and some special techniques of assertive behavior were discussedproblem - solving domain paid to issues as familiarity with basic concepts in problem - solving , problem - centered coping , emotion - centered coping and steps to the problem - centered copingdecision - making skill involved familiarity with decision - making process , factors affecting decision , different styles of decision - making and steps to logical decision - makingcritical thinking domain studied items as knowledge of basic concepts in critical thinking , components of critical thinking and thinking instrumentsin the stress management domain , factors such as knowledge of the concept of stress and related factors , a model of application of coping strategies , stress symptoms and stress management strategies and their descriptions , self - cooling and adaptive introspections were investigateddrug abuse side - effects focused on types of drug , their mechanism and side - effects , risk factors and preventive factorsattitude toward drug abuse included items as general attitude toward addiction and drug abuse , social opinion about addiction , drug abuse as a solution to problems , addiction avoidance and addicts . regarding self - awareness , items such as familiarity with the components of self - awareness , self - evaluation skills , knowledge of self - strengths and weaknesses , positive thinking abilities , positive ego development skills , relationship between self - esteem and drug abuse , self - esteem and endurance , features of tolerant people and factors affecting those features were investigated the domain of interactive skills investigated issues such as : knowledge of definition and objectives of communication , components of communication , communication barriers , ineffective interaction , effective listening , interpersonal relations and their importance and ways to improve them regarding decisiveness , saying no to others and resistance to others illogical demands , items such as : familiarity with basic concepts of assertiveness , interactive styles , types of assertiveness , cognitive barriers to assertiveness , crucial steps to behavior change , steps of assertive behavior , some advice on how to say no and some special techniques of assertive behavior were discussed problem - solving domain paid to issues as familiarity with basic concepts in problem - solving , problem - centered coping , emotion - centered coping and steps to the problem - centered coping decision - making skill involved familiarity with decision - making process , factors affecting decision , different styles of decision - making and steps to logical decision - making critical thinking domain studied items as knowledge of basic concepts in critical thinking , components of critical thinking and thinking instruments in the stress management domain , factors such as knowledge of the concept of stress and related factors , a model of application of coping strategies , stress symptoms and stress management strategies and their descriptions , self - cooling and adaptive introspections were investigated drug abuse side - effects focused on types of drug , their mechanism and side - effects , risk factors and preventive factors attitude toward drug abuse included items as general attitude toward addiction and drug abuse , social opinion about addiction , drug abuse as a solution to problems , addiction avoidance and addicts . the questionnaire was made by the use of related references , texts and expert panel . eight experts on the field commented on the face and qualitative content validity of the questionnaire . having taken the required permits , validation of the aims of the study , emphasizing the confidentiality of the results and attracting the trust and consent of the participants , data were collected through distribution of the questionnaires among the target group before and after lst workshops . as the study was of a pre- and post - test design , the questionnaires were encoded , so the examinees could be tested both before and after workshops and 4 years after educational intervention . furthermore , to ensure one - way blindness of the intervention , pre - test and post - test were administered by an experienced test - taker who was unaware of the type of intervention . workshops were held by four experts including two psychiatrists and two clinical psychology masters who had passed lst courses . training techniques included lecturing , discussion , question and answer , role - play , modeling and handing out educational booklets . chicago , il , usa ) . and descriptive and analytical analyses such as frequency , mean , standard deviation , t - tests and chi - square test ( p = 0.0.5 ) permission to data collection was gained from the research ethics committee of gonabad university of medical sciences . their responses to the questionnaire were anonymous and all respondents participated in the study voluntarily . permission to data collection was gained from the research ethics committee of gonabad university of medical sciences . their responses to the questionnaire were anonymous and all respondents participated in the study voluntarily . 51.6% of the samples were between 17 and 19 years and 38.3% were between 20 and 22 years and 10.2% were 23 - 24 . 50% of the participants were studying in public health , 26.7% in nursing and 23.3% in the operation room and anesthesia . regarding father 's educational level , 10% had a primary school education , 18.3% middle school education , 35% diploma , 20% aa , 10% ba and 6.7% ma or higher . as to mother 's educational level , 13.3% had primary school education , 38.3% middle school education , 30% diploma , 10% aa , 8.3% ba or higher . considering father 's job , 8.3% were jobless , 33.3% workers and farmers , 26.7% employees and 31.7% were self - employed . nearly 66.7% of the participants resided in towns ( urban ) while 33.3% lived in villages ( rural ) [ table 1 ] . descriptive data for demographic variables the independent t - test results indicated no significant difference between pre - test and post - test drug abuse preventive behaviors of control and intervention groups before intervention ( p < 0.36 ) , but a significant difference was observed immediately after intervention ( p < 0.001 ) . in other words , lst could successfully promote drug abuse preventive behaviors or reduce risk factors leading to drug abuse [ table 2 ] . comparison of drug abuse preventive behaviors based on independent t test before and after intervention and follow - up test administered also , paired t - test showed no significant difference between the control group 's pre- and post - intervention insight toward drug abuse prevention ( p > 0.05 ) , but regarding the same factor , intervention group showed a significant change before and after intervention ( p < 0.001 ) , which remained stable in the follow - up test administered after 4 years ( p < 0.01 ) [ table 3 ] . comparison of drug abuse preventive behaviors based on paired t test before and after intervention and follow - up test administered chi - square test results detected no significant relationship between factors such as gender , age and major with drug abuse preventive behaviors ( p > 0.05 ) , but demonstrated a significant relationship between father 's educational level and promotion of drug abuse preventive behaviors ( p < 0.05 ) in a way that father 's higher educational level led to participant 's more awareness of these behaviors . also , participants whose fathers were clerks or employees thought more of tobacco as harmful than those whose fathers were workers or jobless , though the difference was not statistically significant ( p > 0.05 ) . those inhabiting towns had more knowledge about decisive behaviors compared to villagers , but this difference was n't significant either ( p > 0.05 ) . results showed that lst could either promote participant 's knowledge about drug abuse preventive behaviors or decrease risk factors leading to drug abuse as a significant post - test increase in the intervention group 's drug abuse preventive behaviors mean scores was observed . the findings of this study is in line with the other studies , e.g. , zollinger et al . , botvin and griffin , botvin et al . , moradi et al . which confirmed the positive impact of lst on promotion of the intervention group 's insight , drug abuse resistance skills , self - efficacy and decision - making balance in preventive activities also , barati et al . showed the effectiveness of lst on the reduction of abstract norms encouraging drug abuse among university students which can affirm the positive impact of training on modification of beliefs and abstract norms of the youth . also , the mentioned factors of this study results show that are directly related to psychological capacity which can be improved through lst . the findings of this study are also in line with those of bohler in feredrick university which demonstrated not only the preventive role of lst among non - smokers and non - drinkers but also its great impact on smokers and drinkers , and smith et al . who also claimed the significant impact of lst on leadership and management skills of the youth . findings also confirm the results of the studies by samari and laalyphase who asserted that lst can promote one 's interactive skills and social acceptability which can in turn affect drug abuse preventive behaviors and valyany et al . who emphasized the importance of lst workshops as they can reduce drug abuse tendency . the study results indicated that the intervention group 's observed pre- and post - intervention difference remained stable even in the follow - up test conducted 4 years after the main treatment . this is in line with the findings of botvin and griffin who suggested that their 6-year - long educational program confirms the effectiveness of lst on drug abuse prevention and nazarpoor et al . which claimed a promotion in the participants knowledge and social skills and also a meaningful increase in the change of outlook toward drug abuse after attending workshops . it seems as if the change in the outlook can in the long run reduce drug abuse tendency . our study proved a significant positive relationship between father 's educational level and children 's knowledge of drug abuse preventive behaviors . this confirms the findings of babayi that showed students whose parents had a higher academic level had better information about drug abuse . probably families with higher academic levels have more potential skills and use various techniques to promote their children 's insight toward drug abuse . this proves the crucial role parents play in forming a proper insight toward drug abuse and in turn in the formation of drug abuse preventive behaviors in their children . our study included some limitations as some samples ceased to go on with the study or were not available for the follow - up test . lst can promote interactive , decision - making , problem - solving , critical thinking and stress management skills and lead to more social acceptability , which in turn reduce drug abuse tendency . it is recommended to plan and perform constant lst workshops as effective tools of drug abuse prevention in universities . q fever is a zoonotic disease that is caused by coxiella burnetii , a microorganism that frequently infects domestic ungulates , as well as wild mammals in many genera ( 1 ) . in animals , the disease is transmitted to humans incidentally by inhalation of aerosols from infected cattle and sheep ( 1 , 2 ) . in humans , c. burnetii infection may be asymptomatic , acute , or chronic . chronic q fever is rare , with endocarditis presenting as the most common complication ( 1 , 3 ) . q fever has been reported in almost every country , except new zealand ( 4 ) . in canada , burnetii antibodies ( 5 ) . in japan , 60 to 84% of cattle with reproductive disorders are seropositive ( 6 ) . a study conducted in southern france showed that 5 to 8% of endocarditis cases in humans were due to c. burnetii , and the prevalence of acute q fever was 50 cases per 100,000 inhabitants ( 7 ) . researchers have suggested that the incidence of q fever is chronically underestimated because clinical manifestations of the disease are often nonspecific or even absent . therefore , concerns with the disease focus on the importance of detection ( 1 , 3 , 8) . in korea , there is a little information concerning the epidemiology of c. burnetii infection in either animals or humans . a few cases of acute q fever in humans have been reported ( 9 , 10 ) . we examined the prevalence of antibodies to c. burnetii in dairy cattle nationwide and in people for health screening in a rural area of korea , and used the data to evaluate the impact of q fever in both animals and humans in korea . upon agreement with dairy owners , serum samples from 414 dairy cattle were collected on 31 farms from march to june , 2001 . serum was collected from people who visited kangwon national university hospital for health examinations between april and december 2002 . the subjects were interviewed to confirm the absence of symptoms of respiratory tract infection during the preceding two weeks . informed consent was obtained from all people for health screening and the animals are treated by the ethical guidelines of kangwon national univesity . coxiella burnetii phase ii antigen ( nine mile whole - cell antigen ) was prepared as previously described at the national institute of infectious diseases ( niid ) in tokyo , japan ( 12 ) , and dotted onto teflon - printed glass slides . each serum sample was diluted 1:16 with phosphate - buffered saline ( pbs ) , overlaid on the antigen dots , and incubated for 45 min at 37 in a moist chamber . the slides were subsequently washed twice for 5 min in pbs plus 0.05% tween-20 and then incubated with a 1:1,400 dilution of fluorescein isothiocyanate ( fitc)-conjugated rabbit anti - bovine igg ( sigma - aldrich , st . louis , mo , u.s.a . ) or fitc - conjugated rabbit anti - human igg ( dakocytomation , glostrup , denmark ) for 45 min at 37 in a moist chamber . the slides were again washed twice using the same method and examined using fluorescence microscopy ( axioskop 2 , zeiss , germany ) at 200 magnification . all sera that produced positive or equivocal reactions at 1:32 were further analyzed using 2-fold serial dilutions up to 1:4,096 . approximately 10% of the sera were divided and tested concurrently at kangwon national university and at the niid for quality control of test reproducibility ; there was greater than 95% concordance between the results from the two laboratories . upon agreement with dairy owners , serum samples from 414 dairy cattle were collected on 31 farms from march to june , 2001 . serum was collected from people who visited kangwon national university hospital for health examinations between april and december 2002 . the subjects were interviewed to confirm the absence of symptoms of respiratory tract infection during the preceding two weeks . informed consent was obtained from all people for health screening and the animals are treated by the ethical guidelines of kangwon national univesity . coxiella burnetii phase ii antigen ( nine mile whole - cell antigen ) was prepared as previously described at the national institute of infectious diseases ( niid ) in tokyo , japan ( 12 ) , and dotted onto teflon - printed glass slides . each serum sample was diluted 1:16 with phosphate - buffered saline ( pbs ) , overlaid on the antigen dots , and incubated for 45 min at 37 in a moist chamber . the slides were subsequently washed twice for 5 min in pbs plus 0.05% tween-20 and then incubated with a 1:1,400 dilution of fluorescein isothiocyanate ( fitc)-conjugated rabbit anti - bovine igg ( sigma - aldrich , st . louis , mo , u.s.a . ) or fitc - conjugated rabbit anti - human igg ( dakocytomation , glostrup , denmark ) for 45 min at 37 in a moist chamber . the slides were again washed twice using the same method and examined using fluorescence microscopy ( axioskop 2 , zeiss , germany ) at 200 magnification . all sera that produced positive or equivocal reactions at 1:32 were further analyzed using 2-fold serial dilutions up to 1:4,096 . approximately 10% of the sera were divided and tested concurrently at kangwon national university and at the niid for quality control of test reproducibility ; there was greater than 95% concordance between the results from the two laboratories . each province had a relatively high prevalence , ranging from 8.9 to 59.3% ; the overall national prevalence was 25.6% . of the positive sera ( n=106 ) , 80 ( 75.5% ) had high antibody titers ( 1:256 ) . the mean age of the subjects was 43.7 yr ( standard deviation 15.9 , range 19 - 82 ) , and the male - to - female ratio was 1:0.86 ( n=110:95 ) . of these , one had a serum sample with a high antibody titer ( 1:512 ) , and two had samples with low antibody titers ( 1:64 ) ( table 2 ) . this study examined the seroprevalence of c. burnetii infection in cattle and people for health screening in korea . the sera of cattle showed a high prevalence ( 25.6% ) of anti - c . burnetii antibodies , with 75.5% of the positive sera having high titers ( 1:256 ) . by contrast , the sera of healthy people showed a relatively low prevalence ( 1.5% ) of anti - c . the sera of dairy cattle were collected regardless of the disease status of each animal , which is a limitation of this study . since we could not find an association between a history of reproductive failure and seropositivity of the cows because of the absence of the data on the disease status , no prediction concerning seropositive status and reproductive problems can be made . however , c. burnetii infection is prevalent in all areas of korea ; every region had a seroprevalence above 8% , and the national prevalence was 25.6% . moreover , the high prevalence of high - titer sera provides evidence that the disease might be very active in this country . in neighboring japan , the seroprevalence of c. burnetii in healthy cattle ranges from 2 to 46% ; in cattle with reproductive disorders , the range was 60 to 84% ( 6 ) . reported that bovine placentitis was highly associated with the presence of c. burnetti ( 13 ) . in addition , 9% of abortions in goats are reported to be caused by this microorganism ( 14 ) . this is the first report to provide data that suggest that c. burnetii infection might be one of the important causes of reproductive problems in cattle in korea . further studies based on the isolation of c. burnetii are needed to elucidate the etiologic role of this microorganism in the reproductive problems of cattle in this country . furthermore , the high seroprevalence of c. burnetii among cattle suggests the possibility of contamination of the environment around farms . since c. burnetii is widely distributed in wild animals and ticks and causes q fever to humans ( 1 , 3 ) , it is necessary to evaluate the environmental hazards associated with c. burnetii infection that may threaten public health in the near future . in addition , the survey on the high - risk group such as farmers and butchers is strongly needed . the finding that people in a rural area demonstrated a relatively low seroprevalence of c. burnetii , as well as lower titers , suggests that infection with this microorganism seems to be relatively low in the rural area of korea . in a previously published report ( 11 ) , less than 1% of both healthy people and patients with fever of unknown origin tested positive for anti - c . another report showed none of the sera from 70 healthy people was reactive for c. burnetii antigen ( 15 ) . recent data showed that 11 out of 448 healthy people were reactive ( 16 ) . on the other hand , our group previously reported that only one of 88 patients with community - acquired pneumonia was diagnosed with q fever with an ifa assay using both phase i and ii antigens ( 18 ) . despite the low seroprevalence of c. burnetii in humans , we can not exclude the risk of q fever to individuals with high - risk occupations , such as farmers , veterinarians , and meat - processing workers , considering the high rate of infection observed in cattle in this study . previous korean report showed two of 46 stock breeders had an antibody titer of 1:20 ( 15 ) , and 5 sera from 202 abattoir workers reacted with phase ii antigen ( 16 ) . in conclusion , c. burnetii appears to be a highly prevalent pathogen in cattle in korea and , accordingly , the studies on the high - risk groups are needed to evaluate the seroprevalence for this organism in korea .

background : drug abuse is now - a - days one of the gravest social harms . recent years have experienced a drastic rise in drug abuse among school and university students . thus , the need for special attention to the issue is deemed important . the present study was conducted with the aim of assessing the impact of life skills training on promotion of drug abuse preventive behaviors.methods:this field trial experimental study was conducted on 60 students of gonabad medical university selected through quota random sampling and assigned randomly into two intervention and control groups . data were collected through a questionnaire , including two sections of demographic information and drug abuse preventive behaviors . the questionnaire was first assessed as to its validity and reliability and then administered both before and after educational intervention and also as a follow - up 4 years after intervention data were then analyzed using t - tests and chi-square.results:comparison of post - test mean scores of drug abuse preventive behaviors of both groups showed a significant difference ( p < 0.01 ) which remained stable 4 years after intervention . there was a significant relationship between father 's educational level and drug abuse preventive behaviors ( p < 0.01).conclusions : life skills training is effective in the promotion of drug abuse preventive behaviors of university students . we report results on the seroprevalence of antibodies to coxiella burnetii in cattle and healthy people in korea . upon agreement with dairy owners , serum samples from 414 dairy cattle were collected between march and june 2001 and samples from 205 people for health screening were collected between april and december 2002 . the sera were analyzed for the presence of anti - c . burnetii phase ii antibodies using an indirect microimmunofluorescence test ; strong fluorescence at a 1:32 dilution was regarded as positive . the overall seroprevalence of c. burnetii in cattle in korea was 25.6% , with regional variation from 8.9 to 59.3% . of the positive serum samples , 75.5% had antibody titers 1:256 . by contrast , only 1.5% of people in a rural area were seropositive , and most of the positive samples had low antibody titers . in conclusion , this study showed that relatively high seropositivity of c. burnetii in dairy cattle , accordingly , the studies on the high - risk groups are needed to evaluate the seroprevalence for this organism in korea .