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PMC3872579

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 .

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 .

INTRODUCTION MATERIALS AND METHODS Participants Instruments Procedure First step Second step Third step Forth step Interventions Fifth step (assessment) Data analysis RESULTS DISCUSSION CONCLUSION

PMC5330001

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 .

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 .

INTRODUCTION CASE REPORT DISCUSSION Declaration of patient consent Financial support and sponsorship Conflicts of interest

PMC4301082

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 .

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 .

Introduction Results and Discussion Conclusion Experimental Section

PMC4483387

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 .

[ 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 .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC2671006

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 ) .

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 ) .

Introduction Experimental Section Results and Discussion Conclusion

PMC3279530

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 .

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 .

RESEARCH DESIGN AND METHODS RESULTS Chronic high-fat feeding results in dramatic weight gain, whole-body insulin resistance, and altered substrate preference. Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 reverses diet-induced insulin resistance. Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 reverses diet-induced impairments in exercise capacity, which coincide with a restoration of whole-body oxygen consumption rates and inhibition of fatty acid oxidation. Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 reverses diet-induced impairments on heat production with no effect on animal activity. Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 has no effect on the obesity-associated increase of long-chain acyl-CoA and DAG accumulation in muscle, but significantly elevates TAG levels. Inhibition of de novo ceramide synthesis via pharmacological inhibition of SPT1 prevents the development of insulin resistance in leptin receptor deficient type 2 diabetes DISCUSSION Supplementary Material

PMC4989426

. 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 .

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 .

I M Subjects Mitochondrial DNA analysis Statistic analysis R Clinical features of patients Correlation between m.3243A>G mutation ratio and onset age Clinical symptom frequency of patients with different mutation ratio D Financial support and sponsorship Conflicts of interest

PMC2811487

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 .

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 .

OBJECTIVE RESEARCH DESIGN AND METHODS RESULTS CONCLUSIONS LIFESTYLE TRIALS IN PEOPLE WITH IGT TO PREVENT PROGRESSION TO TYPE 2 DIABETES LONG-TERM EFFECTIVENESS OF LIFESTYLE PREVENTION OF TYPE 2 DIABETES IN PEOPLE WITH IGT CLINICAL TRIAL EVIDENCE OF THE EFFECT OF LIFESTYLE FACTORS ON TYPE 2 DIABETES RISK COMMENT

PMC2870931

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 .

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 .

Introduction Materials and Methods Results Discussion

PMC1470015

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 .

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PMC3782582

the study protocol was approved by the institutional review board of kim 's eye hospital , seoul , korea . all participants provided informed consent prior to enrollment and all procedures conformed to the guidelines of the declaration of helsinki . from june 2006 to july 2011 , 134 eyes from 132 patients who underwent agv implantation surgery at kim 's eye hospital were analyzed retrospectively . each subject underwent a full ophthalmic examination , including visual acuity assessment , iop assessment with a goldmann applanation tonometer , anterior segment examination by slit - lamp biomicroscopy , fundus examination with a 90 diopter ( d ) lens , and 24 - 2 swedish interactive threshold algorithm standard automated visual fieldest ( humphrey visual field analyzer ; carl zeiss meditec , dublin , ca , usa ) before surgery as well as 1 day , 1 month , 2 - 3 months , 4 - 6 months , 11 - 16 months , and 17 - 24 months after surgery . the study included any patient who underwent agv implantation surgery at kim 's eye hospital performed using the same technique by 1 of 2 glaucoma specialists ( yhs and hkk ) with a minimum of 6 months follow - up . the exclusion criteria were : patients ' age less than 18 years or more than 80 years , agv implanted in inferior quadrants , previous seton surgery or trabeculectomy in the operated eye , inflammatory or ocular surface disease with severe conjunctival scarring , history of retinal detachment surgery with scleral buckle or vitrectomy in the operated eye , and history of endophthalmitis in the operated eye . among the 134 eyes from 132 patients , 44 eyes from 42 patients before surgery included : 50 eyes had neovascular glaucoma , 36 eyes had secondary open angle glaucoma , and 4 eyes had primary open angle glaucoma . the agv implantation selection was made by the surgeon based on conjunctival mobility and presence of previous surgical conjunctival scaring , limited subconjunctival space owing to previous ocular surgeries , scarred upper fornix , shallow fornix , and presence of small eyes . eyes implanted with the fp7 and fp8 agvs were assigned to the fp7 group and fp8 group , respectively . the procedures were performed in a similar manner in all subjects regardless of implant size . the conjunctival incision was made 4 mm posterior to the limbus and , following dissection of the conjunctiva and tenon 's capsule toward the fornix and limbus , bipolar cautery was performed . mitomycin - c ( 0.02% ) was applied with a large piece of soaked cellulose sponge under the conjunctival flap and over the episclera for 3 minutes followed by thorough irrigation of the area with a balanced salt solution . the plate of the implant was then secured to the sclera 8 to 10 mm posterior to the surgical limbus using 2 interrupted 7 - 0 silk sutures . the tube was trimmed to an appropriate length with the bevel facing anteriorly and , after injection of methyl cellulose into the anterior chamber via a clear cornea stab incision , the tube was inserted into the anterior chamber through a corneoscleral track created using a 23-gauge needle . the tube was fixed to the episclera with a 10 - 0 nylon mattress suture . a quadrangular donor scleral patch graft ( 4 7 mm ) was fashioned and secured over the exposed part of the tube using 10 - 0 nylon sutures . the conjunctiva and tenon 's capsule were repaired using 8 - 0 vicryl suture material in a running fashion . dexamethasone ( 4 mg ) and gentamycin ( 100 mg ) were injected subconjunctivally upon procedure conclusion . the postoperative regimen included topical levofloxacin ( 0.5% ) eye drops 4 times per day for 1 week and topical fluorometholone ( 0.1% ) eye drops 4 times per day that was usually tapered over 6 to 8 weeks . postoperative follow - up visits were scheduled for 1 day , 1 and 2 weeks , and 1 , 3 , 6 , and 12 months after the procedure and every 6 months thereafter . surgical success was defined as iop maintained below 21 mmhg regardless of the number of iop medications used during the final follow - up observation . the following observations made during follow - up were regarded as surgical failures : an iop greater than 22 mmhg at 2 or more consecutive follow - up visits , an iop less than 5 mmhg at 2 or more consecutive follow - up visits , additional glaucoma surgery was required , and loss of light perception . independent t - tests and chi - squared tests were performed to compare ocular and demographic variables between the 2 groups . to compare the success rate of surgery between the 2 groups , a log - rank ( mantel - cox ) test was performed the study protocol was approved by the institutional review board of kim 's eye hospital , seoul , korea . all participants provided informed consent prior to enrollment and all procedures conformed to the guidelines of the declaration of helsinki . from june 2006 to july 2011 , 134 eyes from 132 patients who underwent agv implantation surgery at kim 's eye hospital were analyzed retrospectively . each subject underwent a full ophthalmic examination , including visual acuity assessment , iop assessment with a goldmann applanation tonometer , anterior segment examination by slit - lamp biomicroscopy , fundus examination with a 90 diopter ( d ) lens , and 24 - 2 swedish interactive threshold algorithm standard automated visual fieldest ( humphrey visual field analyzer ; carl zeiss meditec , dublin , ca , usa ) before surgery as well as 1 day , 1 month , 2 - 3 months , 4 - 6 months , 11 - 16 months , and 17 - 24 months after surgery . the study included any patient who underwent agv implantation surgery at kim 's eye hospital performed using the same technique by 1 of 2 glaucoma specialists ( yhs and hkk ) with a minimum of 6 months follow - up . the exclusion criteria were : patients ' age less than 18 years or more than 80 years , agv implanted in inferior quadrants , previous seton surgery or trabeculectomy in the operated eye , inflammatory or ocular surface disease with severe conjunctival scarring , history of retinal detachment surgery with scleral buckle or vitrectomy in the operated eye , and history of endophthalmitis in the operated eye . among the 134 eyes from 132 patients , 44 eyes from 42 patients before surgery included : 50 eyes had neovascular glaucoma , 36 eyes had secondary open angle glaucoma , and 4 eyes had primary open angle glaucoma . the agv implantation selection was made by the surgeon based on conjunctival mobility and presence of previous surgical conjunctival scaring , limited subconjunctival space owing to previous ocular surgeries , scarred upper fornix , shallow fornix , and presence of small eyes . eyes implanted with the fp7 and fp8 agvs were assigned to the fp7 group and fp8 group , respectively . the procedures were performed in a similar manner in all subjects regardless of implant size . the conjunctival incision was made 4 mm posterior to the limbus and , following dissection of the conjunctiva and tenon 's capsule toward the fornix and limbus , bipolar cautery was performed . mitomycin - c ( 0.02% ) was applied with a large piece of soaked cellulose sponge under the conjunctival flap and over the episclera for 3 minutes followed by thorough irrigation of the area with a balanced salt solution . the plate of the implant was then secured to the sclera 8 to 10 mm posterior to the surgical limbus using 2 interrupted 7 - 0 silk sutures . the tube was trimmed to an appropriate length with the bevel facing anteriorly and , after injection of methyl cellulose into the anterior chamber via a clear cornea stab incision , the tube was inserted into the anterior chamber through a corneoscleral track created using a 23-gauge needle . the tube was fixed to the episclera with a 10 - 0 nylon mattress suture . a quadrangular donor scleral patch graft ( 4 7 mm ) was fashioned and secured over the exposed part of the tube using 10 - 0 nylon sutures . the conjunctiva and tenon 's capsule were repaired using 8 - 0 vicryl suture material in a running fashion . dexamethasone ( 4 mg ) and gentamycin ( 100 mg ) were injected subconjunctivally upon procedure conclusion . the postoperative regimen included topical levofloxacin ( 0.5% ) eye drops 4 times per day for 1 week and topical fluorometholone ( 0.1% ) eye drops 4 times per day that was usually tapered over 6 to 8 weeks . postoperative follow - up visits were scheduled for 1 day , 1 and 2 weeks , and 1 , 3 , 6 , and 12 months after the procedure and every 6 months thereafter . surgical success was defined as iop maintained below 21 mmhg regardless of the number of iop medications used during the final follow - up observation . the following observations made during follow - up were regarded as surgical failures : an iop greater than 22 mmhg at 2 or more consecutive follow - up visits , an iop less than 5 mmhg at 2 or more consecutive follow - up visits , additional glaucoma surgery was required , and loss of light perception . independent t - tests and chi - squared tests were performed to compare ocular and demographic variables between the 2 groups . to compare the success rate of surgery between the 2 groups , a log - rank ( mantel - cox ) test was performed among them , 66 eyes underwent fp7 implant surgery , and 24 eyes underwent fp8 implant surgery . table 1 summarizes the demographic and clinical characteristics of the study populations . the mean age was 57.6 11.4 years for the fp7 group and 61.0 9.9 years for the fp8 group ( p = 0.199 ) . preoperative iop was 41.9 9.2 mmhg for the fp7 group and 39.0 6.6 mmhg for the fp8 group ( p = 0.105 ) . there was no significant difference between the 2 groups in the following variables : sex , systemic disease distribution , glaucoma subtype , baseline visual acuity , number of preoperative antiglaucoma medications , previous ocular surgeries , laterality , and implantation site ( p > 0.05 ) ( table 1 ) . the mean follow - up period was 17.2 9.1 months for the fp7 group and 20.3 4.4 months for the fp8 group ( p = 0.273 ) . the postoperative visual acuity of the fp8 group was better than that in the fp7 group in some early postoperative periods ( 1 week , 2 to 3 months , and 7 to 10 months after the surgery ; p < 0.05 ) ; however , after 10 postoperative months , there were no significant differences in visual acuity between the 2 groups through the 3-year follow - up ( p > 0.05 ) ( table 2 ) . postoperative iop was not significantly different between the 2 groups ( p > 0.05 ) except for the iop on postoperative day 1 ( 11.4 8.4 mmhg for the fp7 group and 7.4 3.9 mmhg for the fp8 group , p = 0.031 ) ( table 3 ) . there were no statistically significant differences between the groups in terms of the number of glaucoma medications after surgery ( table 4 ) . in this study , surgical success was considered as iop maintained at less than 21 mmhg regardless of the medication used , no additional glaucoma surgery required , non - occurrence of light sense loss , and no consecutive follow - up visits with low iop . therefore , the cumulative success rate according to the log - rank test for kaplan - meier survival analysis was 79.2% for the fp8 group and 72.7% for the fp7 group at the 3-year follow - up ( p = 0.535 ) ( fig . 1 ) . the main reasons for failure were not significantly different between the 2 groups ( table 5 ) . it has been hypothesized that glaucoma drainage implants with large plates produces an increased surface area of encapsulation and a higher degree of iop reduction . in a prospective randomized clinical trial comparing single - plate and double - plate molteno implants , heuer et al . found a higher success rate and greater iop reduction with the double - plate implants than with single - plate implants , presumably because of the large surface area of the double - plate implants . on the contrary , seah et al . reported no significant difference in success rates , final iop , number of medications , and rates of complications between the 250 and 350 mm baerveldt glaucoma implants ( bgis ) . in another prospective study comparing the 350 and 500 mm bgis , lloyd et al . reported that there was no difference in success rates and visual outcomes between the different implant sizes . britt et al . also reported that the 500 mm bgi was not superior to the 350 mm bgi for iop control . these results are in agreement with the present study results : no differences were found in vision preservation , iop reduction , or decrease in number of glaucoma medications between the fp7 and fp8 agv implants in adult eyes with glaucoma . thus far , this is the only study comparing the surgical outcomes between fp7 and fp8 agv implants in adult eyes . similar surgical outcomes between the fp7 and fp8 groups imply that the plate area may not be the major factor in determining surgical success , and it is likely that there is a maximal surface area beyond which there is minimal improvement in iop control . kang and kee argued that there is an upper limit of the increase in the surface area at which the implants cease to have a beneficial effect on iop , and it is inferred that the effect of iop decrease does not depend on the area of filter cloth beyond a certain size . this phenomenon may be at least partly responsible for the lack of superior iop reduction of the fp7 agv implants compared to the fp8 agv implants . another possible explanation is the surgeon 's perceived a priori likelihood of failure or complications , because the choice of implant is to some extent influenced by the severity of the glaucoma . it is difficult to compare surgical success rates from different studies because of differences in study design , populations studied , types of implants used , and lengths of follow - up periods . variable success rates of agv implantation have been reported and range between 64.5% and 95.0% , with follow - up periods of 6 to 24 months . with fp7 after a follow - up of 6 months and 78% was reported by coleman et al . , with fp7 after a follow - up of 12 months . in addition , a success rate of 96% was obtained with the fp7 implant after a follow - up of 12 months . these results are similar to the results ishida et al . reported : a 94.2% success rate with fp7 implant at 12 months . in 40 eyes from 40 patients with different types of refractory glaucoma , a 92.5% success rate was reported with both fp7 and fp8 after a follow - up period of 6 months . unfortunately , this study did not report separate success rates between fp7 and fp8 groups . although materials vary , a 53.8% success rate was reported with the 96 mm agv ( model s3 , new world medical inc . ) and a 92.3% success rate was reported with the 184 mm agv ( model s3 , new world medical inc . ) after a follow - up period of 10 months . in the present study , the success rates were 79.2% for the fp8 group and 72.7% for the fp7 group at the 3-year follow - up , which are comparable with the success rates of other studies . in adult eyes , however , in conditions with conjunctival scarring or limited subconjunctival space , fp7 agv implantation is challenging . in addition , an oversized implant can result in various complications , including implant exposure , extrusion , discomfort , and motility defects . given that fp7 and fp8 agv implants did not show differences in surgical outcomes , we suggest that fp8 implantation may be a useful surgical option for iop control in adult eyes with insufficient conjunctival or subconjunctival areas . this option may be especially useful for special circumstances in asian eyes since small globes are common in this population . to compare surgical outcomes between fp7 and fp8 agv implants , randomization of subjects who underwent however , in the present study , the type of agv was chosen per the surgeon 's clinical judgment without randomization . other limitations include a relatively small sample size and limited follow - up period . long - term , prospective , randomized , patient- , and investigator - masked studies are needed to confirm the results of this study . in conclusion , fp7 and fp8 agv implants showed no difference in terms of preservation of vision , iop reduction , or decrease in the number of required glaucoma medications . the fp8 agv implant appears to be a viable option for the management of refractory glaucoma in adult eyes with conjunctival scarring or limited subconjunctival space owing to the previous ocular surgeries , inflammatory ocular disorders , or small eyes .

purposeto compare the success rates , complications , and visual outcomes between silicone ahmed glaucoma valve ( agv ) implantation with 96 mm2 ( fp8 ) or 184 mm2 ( fp7 ) surface areas.methodsthis study is a retrospective review of the records from 132 adult patients ( 134 eyes ) that underwent silicone agv implant surgery . among them , the outcomes of 24 eyes from 24 patients with refractory glaucoma who underwent fp8 agv implantation were compared with 76 eyes from 76 patients who underwent fp7 agv implantation . preoperative and postoperative data , including intraocular pressure ( iop ) , visual acuity , number of medications , and complications were compared between the 2 groups.resultsthere were no significant differences in baseline characteristics between the 2 groups ( p > 0.05 ) . the postoperative visual acuity of the patients in the fp8 group was better than that of the patients in the fp7 group in some early postoperative periods ( p < 0.05 ) ; however , after 10 postoperative months , visual acuity was not significantly different through the 3-year follow - up period ( p > 0.05 ) . postoperative iop was not significantly different between the 2 groups ( p > 0.05 ) except for iop on postoperative day 1 ( 11.42 mmhg for the fp7 group and 7.42 mmhg for the fp8 group ; p = 0.031 ) . there was no statistical difference in success rates , final iop , number of medications , or complication rates between the 2 groups ( p > 0.05).conclusionsthe fp7 and fp8 agv implants showed no difference in terms of vision preservation , iop reduction , and number of glaucoma medications required .

Materials and Methods Participants Surgical procedures Statistical analyses Results Discussion

PMC4755996

the amateur wrestling community and maybe the entire sports community across the world were shocked by the deaths of 3 college wresters in the usa in six weeks in 1997 . the deaths of the athletes were attributed by autopsy to weight - loss performed in a short time the victims having undergone dehydration of up to 15%1 . following these deaths in 1997 , the national collegiate athletic association ( ncaa ) initiated and developed new safety precautions in order to prevent unsafe weight practices2 . after studies of this subject , ncaa decided that competition - weighing should be conducted nearer to the competitions and new weight classes should be determined by adding nearly + 3 kg to each weight class3 . besides , ncaa recommended that weekly weight - loss should not exceed 1.5% of body weight as a part of the wrestling weight certification ( wwc ) program4 . however , the most important reason why athletes lose weight is that they want to compete in the lowest weight - class possible , and they think that the time between weighing and competition ( ~16 hours ) is sufficient for rehydration after dehydration5 . nevertheless , studies report that this time period of ~16 hours is not enough to regain the body - weight lost6 and dehydration decreases athletes performances , too7 . it was reported that when athletes of particularly combat sports such as judo , karate , boxing and wrestling perform excessively severe dehydration in a short time ( 17 days ) , they undergo some hematologic changes8 , 9 . there would be changes not only in posm levels , but also in na , bun and glucose levels due to posm10 , 11 . it was reported that athletes who perform weight - loss before competitions undergo not only change their hydration status , but also experience health problems such as sleep disorders , learning and memory difficulties , anxiety , depression , irregular body temperature , vasoconstriction , low sexual performance and dysfunctions in skeletal muscles12 . skeletal muscle damage can be hematologically detected by analyzing serum levels of ck , and such indicators as ldh , ast and alt enzymes in the cells of many tissues13 . the most important indicator of skeletal muscle damage is serum ck level , because ck in skeletal muscle tissues is the enzyme that exists in higher amounts in the sarcolemma and mitochondrial cells of healthy muscle cells , and is primarily responsible for regulating anaerobic metabolism14 . observing serum ck and ldh levels may provide useful information about the status of the muscles and their adaptation to physical load because serum ck and ldh levels demonstrate the degree of metabolic adaptation of skeletal muscles to physical exercise . these values increase considerably after intense exercise15 . in physical tissue damage and other inflammatory conditions , c - rp is the main acute phase protein and a very sensitive and objective indicator16 , 17 . indeed , abramson and vaccarino reported that the c - rp level increased after one exercise dependent on the duration and intensity of the exercise18 . on the other hand , booth et al . reported that c - rp , which was one of the indicators studied in a 12-day military exercise , was 5.005.9 it was also reported that the serum c - rp level of athletes increased with one exercise protocol20 , but in prospective studies ; that the serum c - rp level of the athletes decreased21 . there are studies in literature of dehydration , skeletal muscle damage and inflammation among elite wrestlers22,23,24 . however , no study has been conducted to determine whether skeletal muscle damage or inflammation occur with dehydration . accordingly , the present study aimed to identify weight - loss and hydration levels before competitions among elite wrestlers and explore skeletal muscle damage and inflammation levels after dehydration . trainers and athletes will benefit from information on skeletal muscle damage and inflammation levels caused by dehydration . the study subjects were 72 volunteer elite wrestlers who competed in the turkish inter - university wrestling championship a division , who had at least 5 years sport experience and did at least one exercise on a daily basis . the participant athletes were asked not to use any kinds of medicine or ergogenic aids within 48 hours before competition - weighing in order to ensure standardization of the subjects and those who used medicines or ergogenic aids were excluded from the study . during the study period , no disease that could affect the blood values of the athletes was detected , but three athletes were excluded from the study because their ck levels were above 1,000 u / l ( hyper - responder ) and their values were considered as lost data . the study was completed with 69 elite wrestlers ( 22.512.49 years , 174.546.59 cm , 78.9815.87 kg and bmi 25.733.77 the details of dehydration protocols ( how long , between which time - periods , with what methods the elite wrestlers achieved weight - loss ) were datails of dehydration protocols . wrestlers reported that they lost weight 17 days before competitions through food and fluid restrictions , sauna and exhausting exercises . in the literature , euhydration is accepted to have a reference range of 280290 mosm / l . thus , wrestlers with posm 290 were assigned to the dehydrated group and those with posm > 290 to the not dehydrated group25 . dehydration was calculated using the formula below : posm = ( 2 * na)+(bun/2.8)+(glucose/18)26 . determined 1% of dehydration as satisfactory dehydration , > 13% of dehydration as mild dehydration , > 35% of dehydration as high dehydration , and > 5% of dehydration as severe dehydration27 . the percentage of body weight - loss ( pbwl ) calculation was performed using the formula below : body weight change=[(pre - body weight post - body weight)/pre - body weight ] 100 . with the help of specialists , 5 cc of blood was drawn from the forearm veins of the participant wrestlers into 8.5 ml tubes of a vacuatiner blood collection system at the competition place and at the weighing time ( one day before the competition , between 06:00 and 06:30 pm ) and transported to the laboratory for centrifugation . the blood samples were centrifuged with a nve nf-400 for 5 minutes at 4,000 rpm . for each athlete , serums extracted from blood was preserved in two different eppendorf tubes under 20 c until the time of analysis . on the day of the analysis biochemical analyses [ hydration indicators ( na , bun , glucose ) and analyses of skeletal muscle damage indicators ( ast , alt , ldh , ck ) ] were performed using a beckman coulter au2700 plus biochemical auto - analyzer with beckman coulter kits , while hormone analyses were roche hitachi cobas e601 auto - analyzer with roche kits . c - rp analyses were performed using a beckman coulter immage 800 nephelometer autoanalyzer with roche kits . the independent samples t test was used to compare pairwise group variables that followed a normal distribution , and anova was used for more than two groups . following variance analyses ; lsd multiple comparison test was employed in order to detect which measurement results caused the differences . the responses to the questionnaire show that 55.07% of the elite wrestlers underwent fast weight - loss just before the competition ( 17 days ) ( 4.55%1.87 ) . the wrestlers who underwent fast weight - loss had higher levels of posm ( 296.053.14 ) above the upper limit of reference value ( over 290 ) and suffered from dehydration . when intergroup hydration indicators were assessed , it was found that the na , bun and pbwl levels of the wrestlers who were dehydrated were higher than those of the wrestlers who were not dehydrated ( p<0.05 ) , but no differences existed between the groups in terms of glucose levels ( p>0.05 ; table 1table 1.comparison of hydration indicators of wrestlers in terms of hydration statusnhydration statusreference range na ( mmol / l)31dehydrated136146142.51.6**38not dehydrated137.52.7bun ( mg / dl)31dehydrated82016.43.4 * 38not dehydrated14.33.6glucose ( mg / dl)31dehydrated7410697.015.838not dehydrated97.112.5posm ( mosm / l)31dehydrated280290296.13.1**38not dehydrated285.35.2pbwl ( % ) 31dehydrated-4.61.9**38not dehydrated1.01.3*p<0.05 , * * p<0.01 , : mean , : standard deviation , na : sodium , bun : blood urea nitrogen , posm : plasma osmolarity , pbwl : percentage of body weight loss ) . * p<0.05 , * * p<0.01 , : mean , : standard deviation , na : sodium , bun : blood urea nitrogen , posm : plasma osmolarity , pbwl : percentage of body weight loss when skeletal muscle damage and inflammation differences of the elite wrestlers were examined in relation to hydration status , differences in ast , ldh and ck levels ( p<0.05 ) were found , whereas no differences existed between the groups in terms of alt and c - rp levels ( p>0.05 ) . although there were differences between the groups in terms of ast ( upper limit : 50 u / l ) and ldh ( upper limit : 248 u / l ) values , these values were within the reference ranges . however , ck levels ( upper limit : 171 u / l ) of both groups were higher than the reference range ( table 2table 2.comparison of skeletal muscle damage and inflammation indicators of the wrestlers in terms of posm levelsnposm ( mosm / l)reference range ast ( u / l)31dehydrated05024.96.3 * 38not dehydrated21.54.5alt ( u / l)31dehydrated05014.29.038not dehydrated14.07.3ldh ( u / l)31dehydrated0248207.237.2 * 38not dehydrated185.827.6ck ( u / l)31dehydrated0171421.0174.0**38not dehydrated175.880.7c - rp ( mg / dl)31dehydrated00.60.30.338not dehydrated0.30.3*p<0.05 , * * p<0.01 , ast : aspartate aminotransferase , alt : alanine aminotransferase , ldh : lactate dehydrogenase , ck : creatine kinase , c - rp : c - reactive protein , posm : plasma osmolarity ) . * p<0.05 , * * p<0.01 , ast : aspartate aminotransferase , alt : alanine aminotransferase , ldh : lactate dehydrogenase , ck : creatine kinase , c - rp : c - reactive protein , posm : plasma osmolarity according to the classification made by casa et al . when investigating skeletal muscle damage and inflammation indicators of the athletes . there were differences between the groups in terms of ast , ldh , and ck levels ( p<0.05 ) , but there were no differences between the groups in terms of alt and c - rp levels ( p>0.05 ; table 3table 3.comparison of skeletal muscle damage and inflammation indicators of the wrestlers in terms of pbwl classificationnpbwl ( % ) classification ast ( u / l)22% 0122.04.318% > 1319.95.019% > 3525.65.510% > 526.35.6alt ( u / l)22% 0113.65.918% > 1314.17.619% > 3515.311.910% > 513.13.2ldh ( u / l)22% 01182.624.118% > 13188.038.619% > 35203.225.810% > 5222.241.1ck ( u / l)22% 01191.689.618% > 13214.4125.119% > 35364.5170.810% > 5472.9226.3c - rp ast : aspartate aminotransferase , alt : alanine aminotransferase , ldh : lactate dehydrogenase , ck : creatine kinase , c - rp : c - reactive protein , pbwl : percentage of body weight loss ) . represents the differences among the groups . ast : aspartate aminotransferase , alt : alanine aminotransferase , ldh : lactate dehydrogenase , ck : creatine kinase , c - rp : c - reactive protein , pbwl : percentage of body weight loss generally , many athletes perform fast weight - loss just before competitions ( within 17 days ) so that they can adapt themselves to different weight classes or compete against rivals who are less strong and weaker than them in order to gain advantage . weight loss results in dehydration among the athletes by affecting their hydration levels negatively . studies of dehydration report that dehydration can be detected with urine specific gravity ( usg ) as well as serum posm2 , 28 . when the posm value is 290 , hydration is considered normal ( euhydration ) , when it is > 290 , hydration is lower than the normal level ( dehydration)25 . in the present study , 55.07% of the elite wrestlers underwent fast weight - loss just before the competition ( 17 days ) ( table 1 ) . the level of posm of the wrestlers who underwent fast weight - loss was above the reference range ( upper limit : 290 ) ( 296.053.14 ) and they suffered dehydration of 4.55% . these results of posm level of the wrestlers were similar to the results of usg29 , 30 . serum na and bun levels were higher among the dehydrated wrestlers due to the increase in posm , but no differences were seen in their glucose values ( table 1 ) . in the literature mmol / l . when the na concentration in a blood sample is 135 mmol / l it is defined as hyponatremia , and when it is 145 , it is defined as hypernatremia31 . it has been reported that the na+ concentration generally remains high due to long - term physical activities and excessive sweating and deficiency in all body fluids , and that na gradually increases with time32 , 33 . it has been recommended that during exercises , athletes should keep serum na concentration within the reference range through a controlled diet34 , otherwise nervousness , over - reaction , lethargy , muscle contraction , spasticity , convulsions , coma and even mortality may occur . it is our opinion that the differences in bun values resulted from the fact that wrestlers did long - term exhausting exercises and/or adhered to food and fluid restriction programs for dehydration . since bun is closely associated with the metabolic functions of the liver , and the filtrating and excreting functions of the kidneys , dehydration may increase35 . indeed , the study of mashiko et al . reported that according to measurements before and after a 20 day camp , rugby players lost weight and as a result their bun levels decreased owing to weight loss36 . the fact that there were no differences in the glucose values of the dehydrated wrestlers made us think that adrenaline increased by exercises converts glycogens stored in liver into glucose and glucose flows into blood with the help of glucagons and , as a result , regulates blood glucose levels . differences in na and bun values affect posm values of the athletes and change their hydration status . it is stated that dehydration of 23% causes cognitive disorders , irregular body temperature , cardiovascular dysfunctions as well as reduced endurance and weakens muscle strength37,38,39 . there were significant differences in terms of the damage to skeletal muscles between the dehydrated wrestlers and not dehydrated wrestlers . the differences between the hydration levels and the skeletal muscle damage was determined according to the classification of casa et al . , and in our study it was noted that there were significant differences in ast , ldh and ck , but no differences were found in alt values ( tables 2 and 3 ) between the two groups . in the study of nathwaniet et al . , it was reported that serum levels of ck , ldh , ast , alt increased following muscle damage13 . under normal conditions , these elevated levels may be tolerated by the athletes with nutritional and relaxation programs , but among the dehydrated athletes , metabolism consumes low levels of carbohydrate due to food and fluid restrictions and uses an energy combination of fat and proteins ; thus , glucose inhibition occurring with high plasma and free fat acids suggest that the damage to the muscles of the wrestlers would continue40 , 41 . in clinical practice , ck , ldh , ast , alt were commonly used for the diagnosis of skeletal muscle diseases and skeletal muscle tissue damage42 . the ldh level is accepted to be a specific indicator of fatigue44 , 45 . ast , being a cytoplasmic and mitochondrial enzyme , may increase in many clinical disorders , but alt has been reported to be a specific indicator of liver damage13 . in our study , serum ck and ldh levels , important damage indicators , were higher in the dehydrated group than in the not dehydrated group . among the dehydrated wrestlers , the high level of serum ck would have affected their performance negatively as well as restricted their movements because of muscle pains . it is also possible that the high level of serum ldh may cause wrestlers to be reluctant and/or unwilling for the next exercise . when aminotransferases were investigated , levels of ast and alt were increased . however , while there was a significant difference in ast levels , no significant difference was seen in alt level , which demonstrates that ast exists in higher amounts in skeletal muscles than alt . serum levels of all these damage indicators reduced after exercises and correlated with relaxation and feeding after exercises46 . all of the participant wrestlers reported that they did daily exercises and training . therefore ; it is our opinion that the high level of skeletal muscle damage among the dehydrated wrestlers was caused by food and fluid restriction following exercises . c - rp is a major acute phase reactant that increases acutely and quickly in response to tissue damage and infection47 . in a systematic study of c - rp , it was found that as an acute phase response , c - rp temporally increased after a single exercise protocol among trained athletes . however , in prospective studies , it was demonstrated that c - rp ( pretest - posttest ) levels of the exercise groups reduced . in other words : although physical activity increased c - rp levels , chronic physical exercises reduced c - rp levels48 . c - rp levels of the participant athletes of both groups were low and no difference existed between the groups . considering the fact that these athletes had a sportive experience of at least five years , it was expected that they would have increased tissue oxygenation because of having regularly exercised for years . uww ( united world wrestling ) shortened the resting - time in competitions of the wrestling championships , aiming at muscle endurance among the wrestlers . in a wrestling tournament , the qualifications , semi - finals and even finals , time between bouts will be short , and resting - time not be sufficient for full recovery , lowering the performance of wrestlers who lose weight . fast and/or higher levels of weight loss before a competition produced differences in wrestlers hydration indicator levels . damage in skeletal muscles of the dehydrated wrestlers was greater than in those hydrated , but no difference was found in the inflammation levels of the groups . if it is necessary to lose weight before a competition , athletes should do it in a way to achieve a gradual and extended weight loss over a period of time depending on the weight loss targeted . in addition , while athletes are losing weight , they should keep levels of hydration and skeletal muscle damage indicators within their reference ranges through ergogenic aids . thus , ergogenic aids will play a mediator role for wrestlers wishing to demonstrate maximum performance and to lead a healthy life .

[ purpose ] the present study aimed to identify weight - loss and hydration levels before competitions among elite wrestlers and determine the skeletal muscle damage and inflammation levels after dehydration . [ subjects ] seventy - two elite wrestlers who participated in the turkish wrestling championship . [ methods ] with the help of specialists , 5 cc of blood were drawn from the forearm veins of the wrestlers . laboratory analyses of na+ , bun , glucose , ck , ldh , ast , alt , c - rp levels were performed . using a mathematical formula for hydration the posm levels of the athletes were calculated . [ results ] the wrestlers were divided into two groups based on hydration status . there were significant correlations between hydration indicators of na+ , bun and pbwl values . there were significant differences between ast , ldh , ck values and skeletal muscle damage indicators of the two groups , but there were no significant differences between the inflammation levels and c - rp values of the groups . [ conclusion ] no differences existed in inflammation levels among the wrestlers , although dehydrated wrestlers suffered from higher level of skeletal muscle damage than wrestlers who were not dehydrated .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4499994

in japan , elderly individuals defined as people aged 65 years or more are known to account for more than 25% of the population . as people age , there is a concomitant decrease in muscle strength1 . this age - related change affects their ability to walk2 . as the ability to walk of the elderly decreases , there is an associated decrease in their balance ability3 , 4 , and independent daily living activities5 , and they show an increased risk of fall6 . therefore it is necessary to maintain the ability to walk of the elderly . ground reaction force is an external force involved in walking , which affects the acceleration of the body s center of mass7 . the vertical ground reaction force is often measured in gait analysis , and is regarded as a representative measure of walking8 . the two peaks of the vertical ground reaction force during the early stance and late stance phases reflect the support of the body s center of mass . the minimum value in the mid - stance indicates that the vertical force applied to the ground decreases , and thus , the reaction force from the ground to the body also decreases9 . the profile of the vertical ground reaction force correlates with many parameters of gait and functional performance , such as walking speed10 , the timed up and go test time and the functional reach distance11 in the elderly . it is also known that the vertical ground reaction force during walking is affected by aging . elderly people exhibit lower first peak and second peak forces , and a higher minimum value at mid - stance than young people12 . the vertical ground reaction force provides correlative information about the ability to walk of elderly people . several studies have reported that elderly individuals exhibit age - related alteration in their kinetic profiles of walking . hip and knee joint moments of the elderly are significantly lower than those of young people13 . moreover , devita and hortobagyi14 reported that elderly people showed different distributions of joint moments and powers compared to young individuals . support during the stance phase is achieved by a net extensor pattern of moments at the ankle , knee , and hip joint15 . age - related alterations in lower extremity joint moment suggest there is a difference in the support strategy between the elderly and the young . to date , it is unclear what causes the difference in the profile of the ground reaction force between the elderly and the young . the results of our previous study16 suggest that both men and women make use of different joint moments for the generation of antero - posterior ground reaction forces as they age , showing an age - related alteration in the motor pattern used to generate the antero - posterior ground reaction force . moreover , if elderly people exhibit an age - related alteration in support moment of the lower extremity , changes in joint moment may affect the magnitude of the vertical ground reaction force . by analyzing the relationships between vertical ground reaction force and lower extremity joint moments , it would be possible to clarify the difference in the support strategies of elderly and young people . therefore the purpose of this study was to examine the relationships between the vertical ground reaction force and the hip , knee , and ankle joint moments during walking by the elderly and young individuals . we hypothesized that the relation between the joint moments and vertical ground reaction force differs between the elderly and the young , similar to our previous study16 , and that there is an age - related change in the support strategy use for walking . forty community - dwelling elderly people , 65 years old or older ( mean age , 70.1 years ; age range , 6580 years ) , and 40 young people , aged 20 to 29 years ( mean age , 23.2 years ) , participated in this study . there were 20 males and 20 females in each group . the exclusion criteria included neurologic disorders , osteoarthritis , rheumatic arthritis , joint pain affecting walking , and a history of surgery to the lower extremities or spine . all procedures were approved by the hiroshima international university human research ethics committee and all participants gave their written , informed consent prior to enrollment . in the walking trials , all the participants walked barefoot to the end of a 7 m walkway at a self - selected preferred walking speed . a 3d motion analysis system with 8 infrared cameras ( vicon mx ; vicon motion systems ; oxford , uk ) and 8 force plates ( amti ; watertown , ma , usa ) was used to record kinematic and kinetic data at sampling frequencies of 100 and 1,000 hz , respectively . the force plate layout was designed to measure the ground reaction force of each limb using four force plates arranged in the longitudinal direction . participants were instructed to walk placing their foot on the left or right force plate , avoiding stepping on two force plates simultaneously . a total of 30 reflective markers were placed bilaterally , over the following landmarks of each participant : acromion process , olecranon , styloid process of the radius , anterior superior iliac spine , posterior superior iliac spine , greater trochanter , medial and lateral femoral condyles , mid - point between the greater trochanter and the lateral femoral condyles , medial and lateral malleoli , mid - point between the lateral knee joint line and the lateral malleolus , head of the first and fifth metatarsal , and the calcaneal tuberosity . these anatomical markers were used to construct anatomical coordinate systems for the pelvis , thigh , shank , and foot segments . the coordinates of each joint center were calculated following the description of a previous study17 . a 7-link segmental model was developed to calculate the kinematic and kinetic variables of the hip , knee , and ankle joints using the inverse dynamics technique of davis et al.18 and vaughan et al19 . anthropometric parameters for mass , position of the center of mass , and moment of inertia for the segments were obtained from the report by okada et al20 . we calculated the hip , knee , and ankle joint moments using a local coordinate system with the origin at the joint center . in this study , each joint moment was normalized to the subject s body mass ( nm / kg ) . walking speed ( m / s ) was calculated using the center of gravity ( cog ) of the whole body , and averaged over a 3-s data collection period . stride length ( m ) was measured as the antero - posterior distance between the left calcaneal tuberosity marker at one heel contact and at the next heel contact . initial contact was assumed to occur when the vertical reaction force exceeded 10 n , and toe off was assumed to occur in the first frame following the initial contact when the vertical reaction force fell below 10 n. to account for different builds , ground reaction force was normalized to each subject s body mass ( n / kg ) . first and second peaks of vertical ground reaction force , and the minimum value between the two peaks were extracted from the ground reaction force profile ( fig 1.graph representation of the vertical ground reaction forces and the joint moment of the hip , knee , and ankle during a gait cycle ) . peak values of each joint moment during the stance phase were extracted from the joint moment profile ( fig . graph representation of the vertical ground reaction forces and the joint moment of the hip , knee , and ankle during a gait cycle two - way analysis of variance was performed to examine the differences between the elderly and the young and the interactions between age and gender , with age , body height , body weight , body mass index ( bmi ) , and spatio - temporal parameters . two - way analysis of covariance was also performed to examine the difference between the elderly and the young , and the interactions between age and gender with the vertical ground reaction force , and peak values of each joint moment using walking speed as the covariate . step - wise linear regression was performed to determine the joint moments that predicted the amplitude of vertical ground reaction force in the elderly men , elderly women , young men , and young women . relationships among the peak values of the hip , knee , and ankle joint moments during the early stance phase and the first peak force , and the minimum value of the vertical ground reaction force , were investigated . relationships among the peak values of the hip , knee , and ankle joint moments during the late stance phase and the second peak force of the vertical ground reaction force were also investigated . the stepping - method criteria were : an f - value of 0.05 for inclusion in the segmental model , and 0.10 for removal from the model . the baseline demographic characteristics of the study subjects are presented in table 1table 1.characteristics of the subjectvariableelderlyyoungmales ( n = 20)females ( n = 20)males ( n = 20)females ( n = 20)age ( years)69.45 ( 4.80)*70.70 ( 4.62)*22.80 ( 2.80)23.50 ( 2.70)height ( m)1.64 ( 0.07)*1.49 ( 0.04)*1.71 ( 0.07)1.57 ( 0.05)weight ( kg)62.28 ( 9.78)52.09 ( 6.20)64.04 ( 8.34)52.91 ( 9.92)bmi ( kg / m)23.06 ( 2.96)*23.25 ( 2.69)*21.74 ( 2.63)21.21 ( 3.30)value : mean ( standard deviation : sd ) . * : significant difference from the young , p < 0.05 . : bmi : body mass index . the elderly participants had significantly shorter body height and higher bmi than the younger study participants , but there was no significant difference in their body masses . value : mean ( standard deviation : sd ) . * : significant difference from the young , p < 0.05 . : bmi : body mass index the spatio - temporal parameters and peak values of joint moment are presented in table 2table 2.spatio-temporal parameters and peak values of joint momentvariableelderlyyoungmales ( n = 20)females ( n = 20)males ( n = 20)females ( n = 20)walking speed ( m / s)1.20 ( 0.16)*1.15 ( 0.12)*1.26 ( 0.12)1.29 ( 0.10)stride length ( % body height)73.63 ( 6.79)*73.59 ( 5.48)*77.47 ( 5.64)81.68 ( 5.69)first peak force ( n / kg ) 10.57 ( 0.94)10.71 ( 1.06)10.96 ( 0.75)10.90 ( 0.79)minimum force ( n / kg)7.29 ( 0.58)7.52 ( 0.58)7.20 ( 0.58)7.00 ( 0.60)second peak force ( n / kg ) 10.43 ( 0.50)9.97 ( 0.61)*10.64 ( 0.52)10.74 ( 0.51)hipextension moment ( nm / kg ) 0.76 ( 0.31)0.58 ( 0.29)0.72 ( 0.19)0.64 ( 0.29)flexion moment ( nm / kg ) 0.67 ( 0.17)0.61 ( 0.17)0.71 ( 0.14)0.60 ( 0.13)kneeflexion moment during early stance ( nm / kg)0.23 ( 0.15)0.21 ( 0.11)0.08 ( 0.17)0.15 ( 0.11)extension moment during early stance ( nm / kg)0.58 ( 0.29)0.74 ( 0.26)0.71 ( 0.20)0.76 ( 0.27)flexion moment during terminal stance ( nm / kg)0.21 ( 0.09)0.10 ( 0.14)0.18 ( 0.12)0.19 ( 0.12)extension moment during terminal stance ( nm / kg)0.20 ( 0.07)*0.26 ( 0.12)*0.17 ( 0.08)0.17 ( 0.10)ankledorsiflexsion moment ( nm / kg)0.21 ( 0.10)0.24 ( 0.11)0.25 ( 0.08)0.21 ( 0.11)plantarflexion moment ( nm / kg)1.16 ( 0.22)*1.02 ( 0.16)*1.30 ( 0.11)1.22 ( 0.19)value : mean ( standard deviation : sd ) . * : significant difference between age groups , p < 0.05 . : significant difference between gender groups , p < 0.05 . the elderly males and females had significantly slower walking speeds , with shorter stride lengths than the young participants , but there was no significant different in cadence between the two age groups . the second peak of the vertical ground reaction force exhibited a significant interaction between age and gender , and the elderly females had a significantly lower second peak of vertical ground reaction force than the younger females . there was an approximately 7.5 percent difference between the elderly females and the young females groups . on the other hand , the extension moment during the terminal stance phase of the knee was higher in the elderly participants . there were approximately 14.5 and 30.0 percent differences between the elderly and the young groups of both males and females , respectively . although the flexion moment during the terminal stance phase of the knee exhibited a significant interaction between age and gender , there was no significant difference between the age groups , neither in malese nor in females . value : mean ( standard deviation : sd ) . * : significant difference between age groups , p < 0.05 . : vertical ground reaction force the results of step - wise linear regression analyses of vertical ground reaction force are presented in table 3table 3.models of the vertical ground reaction force by forward step - wise regression analysis.subjectindependent variableunstandardizedcoefficientstandardizedcoefficientadjusted rfirst peak forceelderly femalehip extension moment2.140.610.34young female knee extension moment during early stance1.350.470.22minimum value at mid - stanceyoung maleknee extension moment during early stance1.950.690.48elderly femaleknee extension moment during early stance1.310.510.26young femaleknee extension moment during early stance1.040.540.29second peak forceyoung maleknee extension moment during late stance2.670.450.21elderly female ankle plantarflexion moment2.220.570.33 * : p < 0.05 . : vertical ground reaction force , and the joint moments related to the vertical ground reaction force are shown in fig . 2fig . 2.the joint moments that can predict the amplitude of the vertical ground reaction force in each group . elderly males exhibited no statistically significant factors related to the vertical ground reaction force . * : p < 0.05 . : vertical ground reaction force the joint moments that can predict the amplitude of the vertical ground reaction force in each group for the young males and females , and elderly women groups , the knee extension moment during the early stance phase was related to the minimum value of the vertical ground reaction force at mid - stance . for young males , the knee extension moment during the late stance phase was related to the second peak of the vertical ground reaction force . on the other hand , for young females , the knee extension moment during the early stance phase was related to the first peak of the vertical ground reaction force . in contrast , for the group of elderly females , the hip extension moment was related to the first peak of the vertical ground reaction force , and the ankle plantarflexor moment was related to the second peak of vertical ground reaction force . this study examined the relationships between the vertical ground reaction force and hip , knee , and ankle joint moments during walking in young and elderly individuals . the analyses of this study were performed in consideration of gender as well as age . this was done because a previous report21 indicated that there are significant gender differences in walking speed , stride length , kinematics and kinetics . in this study , some parameters exhibited a significant gender difference and interaction between age and gender . thus , in order to maximize the ability to determine the effects of age on walking , the study subjects were divided by gender . the first peak and the minimum value at mid - stance of the vertical ground reaction force were not significantly different between the elderly and the young . on the other hand , the second peak of the vertical ground reaction force of the elderly women was significantly lower than that of the young women . yamada and maie11 reported that elderly people have lower first peak and second peak forces , and a higher minimum value at mid - stance than younger people . on the other hand , larish et al.22 reported that there was no significant difference in the first peak force , but a significant difference in the second peak of the vertical ground reaction force . because the amplitude of the peak vertical ground reaction force is affected by cadence rather than stride length23 , there may be no difference between the elderly and young people in the first peak of the vertical ground reaction force . both the first and second peaks of the vertical ground reaction force correspond to the upward acceleration of the body center of mass8 . the elderly females in this study had low upward acceleration of body center of mass at push - off . this may increase the load on the front limb when producing upward acceleration during the double support phase of elderly females . the plantar flexion moment of the ankle of the elderly was smaller by approximately 14.5 percent than that of the young people . therefore , the plantarflexor muscles of the elderly exhibited an age - related functional decline . knee extension moment during early stance had a negative relationship with the minimum value at mid - stance , and the knee extension moment during late stance had a positive relation with the second peak of the vertical ground reaction force the young males . the knee extension moment during early stance had a positive relation with the first peak force , and a negative relation with the minimum value at mid - stance of the ground reaction force of the young females . these results suggest that the vertical ground reaction force may be controlled by knee extension in young people . in our previous study16 , the knee extension moment during late stance was associated with the anterior component of the ground reaction force of young males , and the knee joint moment was associated with the antero - posterior component of the ground reaction force of young females . the knee extension moment of young people is important for decreasing the reaction force from the ground to the body at mid - stance , and increasing the ground reaction force during weight acceptance and the push off phase . it is necessary to have muscle strength in order to increase the joint moment during walking . brown et al.25 reported that there was a relationship between quadriceps strength and walking ability , especially in young people rather than in the elderly . therefore knee joint function is important for support during walking by young people . in the elderly females of this study , the extension moment of the hip had a positive relationship with the first peak of the ground reaction force . in our previous study16 , the extension moment of the hip was associated with the posterior component of ground reaction force in elderly females , and the present result is consistent with the result of that previous study . the elderly females had a large hip extension moment , and their ground reaction force during the early stance phase was relatively high . although the knee joint moment was associated with the first peak of the vertical ground reaction force in the young females , the hip joint moment was associated with the first peak of the vertical ground reaction force in the elderly females . the elderly and young females in this study made use of different joints to control of the vertical ground reaction force . these results are mostly consistent with our hypothesis that there is an age - related change in the support strategy of walking . support for the body is provided by a combination of knee extensors and hip extensors during the early stance phase26 . while walking , moreover , elderly people had more forward trunk lean , and a large hip flexion angle during early stance14 . because the function of the muscles surrounding the knee joint reduces with aging in addition to age - related alternation in the attitude of walking , weight acceptance during the early stance phase of the elderly is accomplished by the hip joint . for weight acceptance in the initial stance , therefore , it is necessary for elderly females to develop a hip extensor . the extension moment during the early stance phase of the knee had a negative relationship with the minimum value of the ground reaction force at mid - stance in the elderly females . this result was similar to the one found for the young females in this study . in order to reduce the mechanical load exerted on the body at mid - stance , it may be important for elderly females to strengthen the quadriceps muscles , and generate a knee extension moment during early stance . moreover the plantarflexion moment of the ankle had a positive relationship with the second peak of the ground reaction force of the elderly females . winter15 reported that the ankle plantarflexion moment was the major contributor to support during push - off . the rate of decline in maximal voluntary isometric force with aging is greatest in the plantar flexors28 . it is probables that because the elderly females had a lower plantarflexion moment of the ankle than that of young females , the second peak of the vertical ground reaction force was smaller in the elderly females in this study . a previous study reported the importance of plantarflexor muscles of the ankle during walking by the elderly29 . therefore it is important to strengthen the plantarflexor muscles of the ankle of elderly females in order to enhance support during late stance . in contrast , no factors related to the first peak and second peak forces and the minimum value of the vertical ground reaction force of the elderly males were found . it has been reported that the contribution of joint moment to the antero - posterior ground reaction force is small in elderly males16 . because individual variation was greater among the elderly males , factors related to the vertical ground reaction force were found not to be significant , similar to the antero - posterior ground reaction force . the changes in physical function with aging are not equal in males and females30 , and physical characteristics , lifestyle , and social background are different between males and females . therefore there are gender differences in the age - related changes of walking characteristics , and elderly males make use of various joint moment patterns to the control of the vertical ground reaction force . the relationship between the vertical ground reaction force and lower extremity joint moments during walking in both elderly and young subjects were investigated . however , step - wise linear regression analysis did not find a factor related to the vertical ground reaction force in several models , because we analyzed the data divided by age and gender . using mechanical analysis using equations of motion , kepple et al.26 showed that support of the body was provided by the ankle plantarflexor moment during single limb support , and by a combination of ankle plantarflexors , knee extensors , and hip extensors during double - limb support in younger people . therefore in order to clarify the age - related changes in walking mechanism of elderly people , it will be necessary to perform detailed analyses of dynamic equations of motion . furthermore , we were unable to identify the causative factor of age - related changes in the support strategy of walking . further studies are required to determine the causes of changes in strategy with aging .

[ purpose ] this study examined the relationships between joint moment and the control of the vertical ground reaction force during walking in the elderly and young male and female individuals . [ subjects and methods ] forty elderly people , 65 years old or older ( 20 males and 20 females ) , and 40 young people , 20 to 29 years old ( 20 males and 20 females ) , participated in this study . joint moment and vertical ground reaction force during walking were obtained using a 3d motion analysis system and force plates . stepwise linear regression analysis determined the joint moments that predict the amplitude of the vertical ground reaction force . [ results ] knee extension moment was related to the vertical ground reaction force in the young males and females . on the other hand , in the elderly females , hip , ankle , and knee joint moments were related to the first peak and second peak forces , and the minimum value of vertical ground reaction force , respectively . [ conclusion ] our results suggest that the young males and females make use of the knee joint moment to control of the vertical ground reaction force . there were differences between the elderly and the young females with regard to the joints used for the control of the vertical ground reaction force .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4493289

hepatitis e virus ( hev ) is a nonenveloped , single stranded rna virus which belongs to the hepeviridae family . hev is a causative agent for acute hepatitis in one - third of the world 's population and fulminant hepatitis in pregnant women . the virion is relatively resistant to environmental conditions and remains infectious even in rough situation such as sewage . therefore the major route of hev transmission is the ingestion of the fecal contaminated water ; however , hev can be spread zoonotically and by blood transfusion especially in industrialized countries . although there is an inclusive debate on the parental route of transmission , available evidence seems to prove the ability of the virus to cause congenital infections . hev infection is a significant public health concern especially in developing countries , where large outbreaks as a result of poor sanitation and lack of sewage infrastructures have been reported . there is also a growing support for the claims that seroprevalence of hev infection in industrialized countries is increasing . patients with chronic liver disease , travelers to endemic areas , and people working with animals like pigs , cows , sheep , and goats are at high risk of hev infection [ 5 , 810 ] . pregnant women infected in third semester develop fulminant hepatic failure particularly in the endemic areas of hev infection [ 11 , 12 ] . iran is an endemic country for hepatitis e infection [ 7 , 13 ] , but hev prevalence has not been determined among general population in all parts of this country . most conducted studies in iran have reported the hev prevalence in specific groups and studies on hev prevalence in general populations are limited . hev prevalence information in general population can be a better indicator of the public health and hygiene . ahvaz is a large city in the south - west of iran with a population of about 1.18 million inhabitants that consists of two ethnic groups : arab and farsi . ahvaz is located in the banks of the karun river , which is the main river in this area . no data is available so far on the prevalence of hev among general population of ahvaz city ; therefore this study was conducted to determine the prevalence of hev among adults in south - west of iran . this cross - sectional study was approved by the ethical committee of ahvaz jundishapur university of medical science with research project number 91112 . to estimate the prevalence of anti - hev igg and igm antibodies in the general population of ahvaz city , 510 blood samples from the adult population of ahvaz city were collected randomly using the multistage cluster sampling method from february to july 2014 . ahvaz is a large city in the south - west of iran that consists of 8 districts and has 94 public health centers . in the first stage , 4 public health centers were selected randomly from each district . in the next stage , the family registry code was used to randomly select 16 households within each public health center . from each family , one subject the trained interviewers visited the subjects in their homes and completed a questionnaire containing information of age , gender , and race / ethnicity for each individual . the subjects who refused to participate in the study were replaced with the next random participants . the serum samples were tested in duplicate for anti - hev igg and igm antibodies by using dia.pro hev ab elisa kit and hev igm elisa kit ( dia.pro , italy ) according to the manufacturer 's instructions . statistical analyses were performed using spss 17 package program ( spss inc . , chicago , il , usa ) and p values of less than 0.05 were considered statistically significant . data were analyzed and compared by descriptive statistics and chi - square test or fisher 's exact test . out of 510 study subjects , 206 ( 40.4% ) were male and 304 ( 59.6% ) were female . the average age of participants was varying from 18 to 81 years while the mean age sd was 45.89 14.63 years . the subjects were classified into six age groups : 1830 , 3140 , 4150 , 5160 , 6170 , and over 71 years . 70 ( 13.7% ) subjects were between 18 and 30 years old , while 135 ( 26.5% ) were between 31 and 40 years old , 135 ( 26.5% ) were between 41 and 50 years old , 80 ( 15.7% ) were between 51 and 60 years old , 55 ( 10.8% ) were between 61 and 70 years old , and 35 ( 6.9% ) were older than 71 years . based on race / ethnicity , 53.7% ( 274 ) of cases were arab and 46.3% ( 236 ) were farsi . of the 510 subjects , 235 ( 46.1% ) are shown to be positive for anti - hev igg antibody by dia.pro hev ab elisa kit , while 275 ( 53.9% ) were negative . with regard to gender and race , 86/206 ( 41.7% ) in the male group and 149/304 ( 49% ) in the female group were positive for anti - hev igg antibodies . 134/274 ( 48.9% ) in the arab group and 101/236 ( 42.8% ) in the farsi group are shown to be positive for anti - hev igg antibody . however , the seroprevalence was higher among arab and female groups ; hev seropositivity was not statistically associated with gender ( p = 0.106 ) and race ( p = 0.168 ) . meanwhile , there was statistical difference in anti - hev igg seroprevalence rate between the subjects grouped according to age ( p < 0.001 ) , so that seroprevalence of hev increased with age from 14.3% ( 10/70 ) in subjects aged 1830 years to 71.4% ( 25/35 ) in persons over 71 years old , with a peak among 6170 year - olds ( 90.9% , 50/55 ) . the highest rate of anti - hev seroprevalence was seen in subjects aged 6170 years ( table 1 ) . when we evaluated anti - hev igm antibody seroprevalence rate in the gender and race groups , no significant differences were observed between the subjects regarding gender ( 1% in females and 1.9% in males , p = 0.448 ) and race ( 2.2% in arab and 0.4% in farsi , p = 0.130 ) . however , with regard to age , 4/70 ( 5.7% ) in the age group 1830 years and 3/135 ( 2.2% ) in the age group 3140 years were positive for anti - hev igm antibodies . there was a significant difference between the age groups regarding hev seropositivity ( p = 0.012 ) . the highest rate of anti - hev seroprevalence was observed in subjects aged 1830 years ( table 2 ) . overall , 7 blood samples ( 1.4% ) are shown to be positive for hev - specific - igm antibodies , while 503 samples ( 98.6% ) were negative . hepatitis e infection is a worldwide public health concern , which causes large outbreaks of acute hepatitis in developing countries especially asia , middle east , and africa and also sporadic cases of the infection in developed countries such as south america and europe . although hev is mainly transmitted via the fecal - oral route especially contaminated water in endemic areas , transmission via the blood transfusion has also been suggested according to the high prevalence of anti - hev igg among blood donors [ 4 , 13 , 14 ] . epidemiological studies in different parts of the world show the wide variation in hev prevalence patterns , though the hev seroprevalence rates are higher among less developed countries . high prevalence rates are often reported from south asia , egypt in the middle east , and the far east except japan , and low rates are often found in europe and the americas . iran is an endemic country for hepatitis e infection [ 7 , 13 ] , since hev seroprevalence in general population is above 5% . ataei et al . in 2005 reported hev seroprevalence rate of 3.8% among general population in isfahan province , iran . assarehzadegan et al . in 2005 reported hev prevalence rate of 11.5% among blood donors in khuzestan province . study , hev prevalence was 9.3% in general population of tehran . in another study by nazer et al . , the prevalence of hev was reported to be 7.8% in khorramabad city in 2009 . regarding hev prevalence among the general population of other countries , the overall hev prevalence rate was reported to be 22.5% among general population in bangladesh by labrique et al . , about 3.20% in french blood donors by boutrouille et al . , 13% in the general population in england by ijaz et al . , 1.9% in the general population in netherlands , and 5.3% in the general population of japan . in the present study we investigated the hev seroprevalence among adult population in ahvaz city and found that anti - hev igg and igm seroprevalence were 46.1% and 1.4% , respectively . the result of the current study is considerably higher than that reported among adults in other parts of iran : 9.3% in nahavand , 8.1% in isfahan , 7.8% in western iran , 7.3% in sari , and 7.915% in tehran ; it is also higher than that reported among adult population of some other countries : 3.9% in united kingdom , 16.8% in germany , 7.3% in spain , about 20% in korea , 23% in thailand , 3942% in usa , and 5.9% in turkey ; however , it is lower than that reported among rural population older than four years in egypt ( 5178% ) [ 16 , 34 ] , pregnant women in nile delta , egypt ( 84% ) [ 16 , 35 ] , general population older than 11 years in central malaysia ( 5067% ) , tribes population ( 50100% ) and adult population ( 1677% ) in andaman islands , india , and homeless children in cochabamba city , bolivia ( 66% ) . however , a part of this difference may be due to differences in the used elisa detection kits , the time of sampling , and the demographics and size of studied population . overall , our results compared with the previous studies from iran indicate that the geographic distribution of hev infection is different even within a specific country , which most likely reflects different levels of exposure to infection over time due to different living conditions in different regions and fecal - oral transmission of hev . in the current study , the hev seroprevalence rate significantly increased with age from 14.3% in people aged below 31 years to 90.9% in persons aged 6170 years . improvement of public health and hygiene results in decreased exposure to the virus over time . however , exposure to hev increases with age . this is consistent with most studies which reported a significant association between age and higher anti - hev positive values , since the prevalence of the disease increases with age [ 26 , 29 , 31 , 36 ] . similarly high seroprevalence was found among adult population older than 60 years in china ( 7080% ) , adult population older than 80 years in bangladesh ( 67% ) , and adult population older than 80 years in hong kong ( 5260% ) . similar to the results of previous studies [ 5 , 13 , 17 , 29 , 37 ] , our results show that the presence of anti - hev igg and igm antibodies is not associated with gender ; also we did not find any association between race / ethnicity and hev seropositivity . our data showed that the anti - hev igg prevalence rate among adult population in ahvaz is 46.1% , the highest rate reported in different parts of iran . the implication is that ahvaz city is a highly endemic area for hev and the main route of hev transmission in this city is most likely karun river . evidence for this claim is that the drinking water source of the city is supplied from karun river and this river is commonly used for swimming , fishing , and other household needs by inhabitants . since the major transmission route of hev is most often the fecal contaminated drinking water and also this virus is relatively resistant to environmental conditions and remains infectious in sewage , the river can be considered as the water source for hev infection . however more studies are required to confirm this hypothesis . therefore , type e hepatitis is more common among adult population of ahvaz city compared with other parts of iran and this finding should be considered in the differential diagnosis of hepatitis infections and also prediction of possible outbreaks . in conclusion , high anti - hev igg seroprevalence of 46.1% was observed among the adults population living in ahvaz city of iran . determination of hev prevalence in different regions can be used for the purpose of hev epidemiology by developing a prevalence map on the base of hev geographical distribution . in addition to epidemiological purposes , hev prevalence information is important in evaluating the public health and hygiene and in identifying the major route of hev transmission in iran .

background . knowledge regarding prevalence of hev in general population can be an indicator of the public health and hygiene . therefore , this study was conducted to evaluate the prevalence of hev among adults in south - west of iran . methods . blood samples were taken from 510 participants , 206 ( 40.4% ) males and 304 ( 59.6% ) females from february to july 2014 . detection of anti - hev igg and igm antibodies was carried out by elisa test . results . the overall anti - hev igg and igm prevalence rates were 46.1% and 1.4% , respectively . anti - hev igg and igm seropositivity were not statistically associated with gender and race / ethnicity . meanwhile , there were significant differences between the age groups regarding hev igg and igm seropositivity . hev igg seroprevalence increased with age from 14.3% in subjects aged 1830 years to 71.4% in persons over 71 years old , and considerably individuals aged 61 to 70 years had the highest hev prevalence ( 90.9% ) . also , 5.7% in the age group 1830 years and 2.2% in the age group 3140 years were positive for anti - hev igm antibodies and the highest rate was observed in subjects aged 1830 years . conclusion . in conclusion , high hev igg seroprevalence of 46.1% was observed among adults in south - west of iran .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusion

PMC3396243

with the announcement on 26 october 2011 that eli lilly and company ( indianapolis , in , usa ) was withdrawing drotrecogin alfa ( activated ) from the worldwide market , we witnessed the end of the only drug specifically approved for sepsis . the move was prompted by the failure of prowess - shock , a large international study , to confirm the benefit reported from the original trial , prowess ( protein c worldwide evaluation in severe sepsis ) , 10 years ago . in the aftermath , many questions will be raised . here , , i should declare that i led the long - term follow - up and cost - effectiveness studies accompanying prowess and served on the data safety and monitoring board of prowess - shock ( see acknowledgments for full disclosure ) . drotrecogin was approved largely on the basis of a single phase 3 trial , which was stopped early for efficacy . most drugs are approved after two positive phase 3 trials , but exceptions are made when there are impressive supporting data , there is a compelling unmet clinical need , or the trial results are particularly impressive . although an external us food and drug administration ( fda ) advisory panel was split on whether to approve , the fda nevertheless felt that these conditions were met . it seems , therefore , that the decision to approve , though based on only one phase 3 trial , was consistent with the procedures and habits of regulatory bodies around the world . for all new drugs typically , the drug has been administered to only a few thousand patients , in highly controlled situations , with limited long - term follow - up . second , a longer time to generate more evidence raises the drug development costs for pharmaceutical companies while shortening the post - approval patent life ( when a company recoups its investment ) , thus worsening the risk - reward ratio and potentially choking overall investment in drug development . because approval occurs while uncertainty persists , a variety of post - approval surveillance activities are performed in case the decision must be reversed . in the five years leading up to drotrecogin 's approval in 2001 , the fda approved 597 new therapies . in other words , there is a low , but non - zero , rate of drug withdrawal . a lower rate would be preferable , but without major changes to patent laws or to the science and costs of drug development , the chilling effect of a more stringent approval process on dwindling drug pipelines would likely be considered intolerable . so , while we might lament that a sepsis drug was one of the unlucky ones , the fundamental drug approval process that led to drotrecogin approval does not seem too lenient , wrong , or unreasonable . that said , it is a shame that prowess was stopped early , something outside the control of the fda , as early stopping biases toward an overestimate of treatment effect . and it is a shame that the costs and logistics of running two concurrent phase 3 trials in critical care seem to be insurmountable obstacles in the drug development process . cheaper and easier trials could allow us to generate greater certainty without compromising drug pipeline . the usual reason for withdrawal is determination of a previously unknown yet highly undesired side effect . these studies generally reported mortality benefits similar to that seen in prowess [ 6 - 12 ] . the studies also provided greater information about bleeding risks , which led to further label restrictions . however , somewhat unusually , the withdrawal in this instance was a voluntary decision based not on safety but on failure to confirm efficacy . numerous human and animal studies suggest that it modulates coagulation and inflammatory pathways and interacts with endothelial function in the midst of intense innate immune responses to challenges such as sepsis . a previous simulation exercise of theoretical anti - tumor necrosis factor ( anti - tnf ) anti - body trials in sepsis demonstrated that modest differences in the distribution of unmeasured variables such as host genotype and pathogen characteristics could lead to trials in which the same drug produces opposing results , even when patients meet the same clinical criteria . prowess - shock attempted to enroll patients ideally suited for drotrecogin . in the original prowess study , the largest reduction in absolute mortality was noted in patients who appeared to be sicker ( for example , those presenting with septic shock ) . a subsequent trial - administration of drotrecogin alfa ( activated ) in early stage severe sepsis , or address - targeted patients with lower severity of illness and could not demonstrate efficacy . thus , we now have an original trial suggesting benefit in both low and high severity risk , with a constant relative risk reduction but variable absolute reduction , and two subsequent trials mimicking the low and high ends of the original trial but failed to repeat the positive findings . provided that all trials were conducted well , the results of prowess - shock and address raise doubt about the prowess results , but , equally , the prowess results raise doubt about those of prowess - shock and address . when data from multiple trials are synthesized , such as in a meta - analysis , a negative study does not trump a positive study of similar quality . rather , the results from both trials are combined to give an overall estimate of treatment effect . given the potential for differences in important but unmeasured variables at baseline in sepsis to change the result , such an approach seems wise . however , it seems possible that the result will be a point estimate in favor of drotrecogin over placebo but of a magnitude far smaller than in the original trial and probably no longer statistically significant . if the combined estimate from the literature is , say , a non - significant 2% to 3% reduction in mortality , would we be interested in confirming whether such an effect was significant ? that would normally depend on the cost of one more ( much larger ) confirmatory trial , our desire to tolerate non - fatal bleeding side effects , and the resulting financial impact ( both per - patient cost - effectiveness and global increase in spending ) of adopting the drug should the benefit be confirmed . such quandaries could be formally estimated in a value - of - information analysis - how valuable is it to know the answer with greater certainty ? however , the decision was taken out of our hands . eli lilly and company no doubt considered the potentially very large cost of another trial , the fact that regulatory agencies and the field of critical care might be so skeptical as to threaten the ability to conduct another trial or adopt its results into practice or both , and the likelihood that the trial would be negative . taking these factors together , the company presumably decided that to go forward was just too risky . whether the action of eli lilly and company has left a drug that could cut sepsis mortality by 2% to 3% ' on the table ' is something we will now likely never know . we will all see the details of prowess -shock published in the coming weeks or months . first , should this story reinvigorate our drive to complement clinical enrollment criteria with biomarkers that better select patients likely to benefit from a given immunomodulating agent ? second , should we ask of ourselves what we as a community might have done differently ? certainly , greater engagement in clinical trials would seem to be an obvious first step - a huge part of the costs of clinical trials is that they take a long time and enroll only a tiny fraction of all patients with severe sepsis . our ability to make wiser choices about drugs would be enhanced if we could conduct larger trials more rapidly . much time , energy , and emotion were devoted to forming opinions about prowess and about drotrecogin , often with strongly voiced opinions about both the drug and eli lilly and company . one can hope that decisions about future drugs will be made in environments richer in data and poorer in opinion . if we believe that there is a role for pharmacomanipulation of critical illness , our partnership with the pharmaceutical industry is a prerequisite . thus , we must think about whether the partnership works properly in terms of open , efficient , rapid , and rigorous science for the optimal benefit of all concerned , especially our patients . address : administration of drotrecogin alfa ( activated ) in early stage severe sepsis ; fda : us food and drug administration ; prowess : protein c worldwide evaluation in severe sepsis . the author declares that he was principal investigator on grants received by the university of pittsburgh from eli lilly and company for the conduct of the long - term follow - up and cost - effectiveness studies accompanying prowess . he also received consulting fees and speaking honoraria from eli lilly and company between 1996 and 2004 . he received compensation from eli lilly and company for serving on the data safety and monitoring board for prowess - shock . ( salt lake city , ut , usa ) , which are also engaged in sepsis research . the author gratefully acknowledges shamly austin for her assistance generating data on fda approval and withdrawal rates discussed in this article .

following the failure of prowess - shock to demonstrate efficacy , eli lilly and company withdrew drotrecogin alfa ( activated ) from the worldwide market . drotrecogin was initially approved after the original trial , prowess , was stopped early for overwhelming efficacy . these events prompt consideration of both the initial approval decision and the later decision to withdraw . it is regrettable that the initial decision was made largely on a single trial that was stopped early . however , the decision to approve was within the bounds of normal regulatory practice and was made by many approval bodies around the world . furthermore , the overall withdrawal rate of approved drugs remains very low . the decision to withdraw was a voluntary decision by eli lilly and company and likely reflected key business considerations . drotrecogin does have important biologic effects , and it is probable that we do not know how best to select patients who would benefit . overall , there may still be a small advantage to drotrecogin alfa , even used non - selectively , but the costs of determining such an effect with adequate certainty are likely prohibitive , and the point is now moot . in the future , we should consider ways to make clinical trials easier and quicker so that more information can be available in a timely manner when considering regulatory approval . at the same time , more sophisticated selection of patients seems key if we are to most wisely test agents designed to manipulate the septic host response .

None First, was the initial decision to approve drotrecogin wrong? Second, was the decision to withdraw drotrecogin too hasty? Next steps Abbreviations Competing interests Acknowledgements

PMC4908242

dkk1 is a 29 kda secreted protein belonging to the dickkopf ( dkk ) family , which comprises four main glycoproteins in vertebrates ( dkk1 - 4 ) . dkk1 has been identified as a potent inhibitor of the canonical wnt signaling due to its ability to bind to the wnt coreceptor lrp5/6 , thus blocking the canonical wnt/-catenin pathway . canonical wnt pathway activation is initiated by the direct binding of the wnt glycoprotein to frizzled ( fz ) membrane receptor and to the lrp5/6 coreceptor [ 46 ] . in the absence of wnt wnt - mediated assembly of the activated fz - lrp5/6 receptor complex is followed by the recruitment of the axin - gsk3 to the plasma membrane , resulting in the reduction of the phosphorylation and degradation of -catenin [ 9 , 10 ] . stabilized -catenin accumulates in the cytoplasm and translocates to the nucleus , where it interacts with dna - bound tcf - lef proteins and activates the transcription of target genes . the wnt pathway is involved in many stages of invertebrate and vertebrate development and in adult tissue homeostasis [ 8 , 12 ] . dysfunction within the wnt/-catenin signaling cascade has been associated with many human pathologies [ 8 , 13 ] , such as cancer [ 1417 ] and bone disease [ 18 , 19 ] . lrp5-activating mutations are mainly associated with high - bone mass , while loss - of - function mutations on lrp5 are linked to bone degeneration and osteoporosis [ 20 , 21 ] . the inhibition of wnt signaling by dkk1 has been related to bone degeneration processes and reduced bone mass . in the central nervous system , dkk1 has been associated with the pathophysiology of neuronal degeneration in alzheimer disease ( ad ) [ 2326 ] . dkk1 expression is increased in cortical neurons exposed to -amyloid peptide ( -ap ) , where its aberrant expression is responsible for hyperphosphorylation of tau in neurons challenged with -ap . moreover , increased dkk1 expression was also observed in degenerating neurons in brain samples of ad patients . dkk1 was shown to be neurotoxic when locally infused into brain regions where neurodegenerative processes associated with brain ischemia or ad normally take place . moreover , dkk1 is also associated with neuronal death in cellular and animal models of excitotoxic / ischemic neuronal death , and the treatment of ischemic animals with dkk1 antisense oligonucleotides protects hippocampal neurons against ischemic damage and cultured cortical neurons against nmda toxicity . for these reasons , the dkk1-lrp6 interaction can be considered as a potentially interesting therapeutic intervention point and dkk1 a potential drug target for the treatment of bone and neurodegenerative disorders . a small molecule ( nci8642 ) has been described as an inhibitor of the interaction between dkk1 and one of its receptors ( lrp5 ) , as well as an inhibitor of dkk1 activity in reducing wnt/-catenin signaling activation . in this study , we sought to further characterise nci8642 activity , using biochemical and biophysical approaches , and to extend its characterization in relation to lrp6 , given the prominent expression in brain of lrp6 and its relevance to the neurodegenerative diseases field . human embryonic kidney cells ( hek293 ) were obtained from the german collection of microorganisms and cell cultures . wnt3a all cell lines were cultured in dmem supplemented with fbs and glutamax ( gibco ) . the monoclonal antibody against -catenin was obtained from bd biosciences , the polyclonal goat anti - dkk1 antibody from r&d , while the fluorescently conjugated secondary antibodies were all from invitrogen . the dna encoding the full - length human lrp6 sequence was cloned into the pcdna3.1/zeo(+ ) expression vector ( invitrogen ) . the dna encoding the human dkk1 sequence was cloned into pcdna6.2/clumio - dest ( invitrogen ) . the sequence encoding the secreted alkaline phosphatase from clontech was amplified by pcr and inserted at the c - terminus of dkk1 sequence into the pcdna6.2/clumio - dest - dkk1 plasmid . the luciferase reporter plasmid p4tcf - luc comprises four copies of a tcf - responsive element upstream of a tata element luciferase coding sequence transcriptional unit . all cell lines were cultured in dmem supplied with 10% fbs , 2 mm glutamax , 100 units / ml penicillin , 100 g / ml streptomycin , and the appropriate selection antibiotic ( 50 and 100 g / ml zeocine for pc12-tcfluc and hek293-lrp6 , resp . , 10 g / ml blasticidine for hek293-dkk1 , 0.4 mg / ml g418 for wnt3a l - cells , 20 g / ml hek293-ap - dkk1 ) . for the generation of the stable cell lines , hek293 or pc12 cells were transfected with fugene 6 ( roche ) and subjected to antibiotic selection 24 hours after transfection . clones were selected using immunofluorescence ( hek293-dkk1 , hek293-lrp6 ) , ap enzymatic assay on the culture medium ( hek293-ap - dkk1 ) , or measurement of luciferase activity upon stimulation with wnt3a cm ( pc12-tcf - luc ) . for the production of dkk1 and ap - dkk1 conditioned media ( cm ) , hek293 cells stably expressing the appropriate construct were seeded into a 150-cm flasks at half confluence . the day after media were collected 96 h after seeding , sterile filtered , and stored at 20c in aliquots . the control conditioned optimem was obtained in the same way using the parental hek293 cells . for the functional studies ( -catenin translocation and tcf - luc reporter assay ) , wnt3a cm was used at a 1 : 2 dilution . each batch of dkk1 cm was functionally titrated in order to determine the volume of cm that gives about 50% inhibition of wnt signaling . from dot blot analysis ( data not shown ) , the concentration of dkk1 in the dkk1 cm was estimated to be about 1 g / ml . for the quantitative ap - dkk1 binding assay , ap - dkk1 cm was diluted to obtain a phosphatase activity of 20 mu / ml . for the immunofluorescence detection of cell - bound dkk1 , lrp6 - 293 cells were seeded onto poly - d - lysine - coated coverslips 24 hours before the experiment and incubated with dkk1 or ap - dkk1 cm diluted in cell culture medium for 2 hours at 4c . when indicated , 100 m nci8642 ( or 1% dmso ) was added alongside dkk1 cm . after washing , the cells were fixed in 3% paraformaldehyde ( 15 min , room temperature ) , blocked with pbs/0.1% bsa , and incubated with the primary goat - anti - dkk1 antibody , and fluorescent secondary antibody diluted into blocking solution . images were collected using a zeiss lsm 510 meta confocal microscope . for the quantitative ap - dkk1 binding assay , the ap activity in the cell lysate was measured with attophos ap fluorescent substrate , according to the manufacturer 's instructions . spr was performed using a biacore t100 ( ge healthcare ) . where not specified , experiments were conducted at 25c . kinetic constants were calculated by nonlinear fitting to the association and dissociation curves according to the manufacturer 's instructions . apparent equilibrium dissociation constants ( kd ) were then calculated as the ratio of kd / ka . all immobilization steps were performed at a flow rate of 5 l / min using hbs - ep+ buffer ( 10 mm hepes buffer , ph 7.4 , containing 0.15 m nacl , 3 mm edta , and 0.05% ( v / v ) p20 surfactant ) . full - length human lrp6-fc tagged ( r&d systems ) was immobilized via amine coupling on a flow cell of a cm3 sensor chip . 3000 resonance units ( rus ) were measured at the end of the immobilization procedure . for the indirect capture of lrp6 , protein a was covalently coupled via amine immobilization on cm3 chip . lrp6-fc capture on protein a coated surface was performed by injecting the protein for 180 sec at the concentration of 0.5 g / ml in hbs - ep+ with a flow rate of 10 l / min . control sensorgrams , obtained on an empty flow cell where the coupling reaction had been conducted in the presence of coupling buffer alone , were always subtracted from binding responses . this allowed subtraction of nonspecific binding , which might be generated by residual charged negative groups , which had not been neutralized by the coupling procedure . bovine serum albumine ( bsa ) was immobilized via standard amine coupling procedures on a flow cell of a cm3 sensor chip to obtain the nonrelated control flow cell . for binding and kinetic assays , dkk1 ( r&d systems ) , diluted in hbs - ep+ at concentration ranging from 3 to 54 nm , was injected for 180 sec at the flow rate of 20 l / min over lrp6 either directly immobilized or captured via protein a as described before . for nci8642 binding and kinetic assays , the compound was diluted in hbs - ep+ buffer at concentration ranging from 3 to 50 m and injected over lrp6 surface for 60 sec at the flow rate of 30 l / min . 0.2% dmso was added to hbs - ep+ when performing the experiments with nci8642 compound , and dmso correction module of the software was used to compensate for refractive index change induced by dmso . for inhibition analysis , dkk1 ( 54 nm , diluted in hbs - ep+ buffer ph 7.4 ) was incubated with nci8642 at concentration from 5 to 20 m . after 1 hour at 25c , dkk1 was injected over immobilized lrp6 for 240 s at the flow rate of 10 l / min . pc12-tcf - luc cells were seeded at the density of 100,000 cells / well into black - walled clear - bottom 96-well plates . after 24 hours , culture medium was replaced with 50 l dkk1 cm diluted as appropriate in optimem . after one - hour incubation ( 37c ) , the cells were stimulated by adding 50 l of wnt3a cm ( 50% of the final volume ) and supplemented with either dmso or nci8642 at the indicated final concentration ( final dmso concentration 1% ) . after 24 hours of incubation , the luciferase activity was measured with steady lite substrate ( perkin - elmer ) , according to the manufacturer 's instructions . for each condition 3 , l - cells were seeded into black - walled clear - bottom 96-well plates at a density of 10,000 cells / well . after 24 hours , culture medium was replaced with 50 l dkk1 cm diluted as appropriate in optimem . after one - hour incubation ( 37c ) , the cells were stimulated by adding 50 l wnt3a cm supplemented with either dmso or nci8642 at the indicated final concentration ( final dmso concentration 1% ) . after two - hour stimulation ( 37c ) , the cells were fixed in 3% paraformaldehyde ( 10 min , room temperature ) , permeabilized ( 0.2% triton x-100 in pbs , 10 min , room temperature ) , and blocked with 0.1% bsa in pbs . the cells were then incubated for 2 hours at room temperature with the primary anti--catenin antibody ( 1 : 500 ) , followed by the secondary alexafluor 555 goat - anti - mouse antibody ( 1 : 1000 ) . the ratio of the nuclear and cytoplasmic intensity of -catenin staining was calculated for each cell and averaged within the well . nci8642 was originally characterized as an inhibitor of the lrp5/dkk1 interaction . in order to determine if nci8642 was also able to disrupt the binding of dkk1 to lrp6 , a recombinant hek293 cell line stably expressing lrp6 was generated ( hek293-lrp6 ) . an additional recombinant hek293 cell line was established for the production of human recombinant dkk1 fused to the v5 tag at the c - terminus ( hek293-dkk1 ) . when conditioned medium ( cm ) containing dkk1 was incubated on cells expressing lrp6 and the cell - bound dkk1 was detected by immunofluorescence , a strong membrane signal was observed in hek293-lrp6 cells ( figure 1(a ) , panel b ) , while it was almost absent on the parental hek293 cells ( figure 1(a ) , panel a ) . when dkk1 cm was supplemented with 100 m nci8642 , the binding of dkk1 ( figure 1(a ) , panel d ) was reduced compared to the dmso - treated control ( figure 1(a ) , panel c ) . in order to obtain a quantitative measure of the dkk1/lrp6 interaction , a construct coding for human dkk1 fused to a secreted domain of alkaline phosphatase was generated ( ap - dkk1 ) and used to establish a stable hek293 cell line secreting ap - dkk1 in the culture medium . the ap tag at the c - terminus does not impair dkk1 binding to lrp6 , as shown by immunofluorescence of cell - bound ap - dkk1 on hek293-lrp6 ( figure 1(a ) , panels e and f ) . the binding of ap - dkk1 to hek293-lrp6 , quantified by enzymatic ap assay , was reduced when ap - dkk1 was incubated in the presence of increasing concentrations of nci8642 . the binding of dkk1 was reduced in a concentration - dependent manner compared to dmso sample ( figure 1(b ) ) . the calculated ic50 was 14.6 m ( pic50 = 4.8 , stdev 0.2 , n = 10 independent experiments ) . when flowed over the surface of the sensor chip coated with recombinant lrp6-fc , dkk1 bound to immobilized receptor both specifically and in a concentration - dependent manner ( figure 2(a ) , kon 5.3 10 m s , koff 1.8 10 s ) . indirect capturing of lrp6-fc on recombinant protein a and direct lrp6-fc covalent coupling via amine groups produced similar interaction with recombinant human dkk1 ( kd of 8.4 and 3.5 10 m , resp . ) , demonstrating that the covalent coupling of the receptor to the chip surface does not influence its binding site . the interaction constants obtained are in agreement with the literature data [ 1 , 34 , 35 ] . the lrp6-dkk1 interaction was also verified using recombinant dkk1 cm instead of commercial recombinant dkk1 ( figure 2(b ) ) . when we investigated whether nci8642 was able to bind to the lrp6 receptor , we observed a consistent and effective binding signal that is not present in the control flow cell . the kinetic analysis of the interaction by injection of an nci8642 dilution series ( from 3 m to 50 m ; figure 2(c ) ) allowed the determination of a kd of 4.7 10 m for nci8642 on lrp6 . we also analyzed the effect of nci8642 on the binding of dkk1 to lrp6 , to determine whether the compound could interfere with the interaction between dkk1 and lrp6 . we injected dkk1 and nci8642 over lrp6 previously immobilized . when dkk1 was injected together with nci8642 after 1-hour incubation at 25c , we observed a consistent reduction of dkk1 binding on lrp6 with 5 m and 10 m compound , and even a complete binding inhibition at the concentration of 20 m ( figures 2(d ) and 2(e)-upper panel ) . on the contrary , sequential injections of 20 m nci8642 and 54 nm dkk1 did not inhibit the dkk1-lrp6 interaction . nci8642 was not able to displace dkk1 when injected after it and dkk1 could still bind to lrp6 when injected after nci8642 at the concentration of 20 m ( figure 2(e)-middle and lower panels ) . to investigate the functional activity of nci8642 , we tested the compound in a tcf reporter - based assay in pc12 cells ( neural crest - derived rat pheochromocytoma cells ) . tcf , similarly to lef-1 , is a downstream target transcription factor of the canonical wnt - signaling pathway . a stable pc12 cell line expressing a tcf - dependent luciferase reporter was established ( pc12-tcf - luc ) . wnt3a - mediated response was reverted by dkk1 cm in concentration - dependent manner ( figure 3(a ) ) . for the purpose of compound testing , the dilution factor of dkk1 cm was chosen in order to have about 50% inhibition of wnt signaling ( figure 3(b ) , open symbols ) . in the presence of exogenously added dkk1 the effect was concentration - dependent and was reproducibly and significantly different from dmso treatment at 25 m and 50 m , although higher concentrations of the compound were toxic to the cells and an ic50 value could not , therefore , be calculated . nci8642 was also tested in an assay measuring the modulation of -catenin intracellular accumulation and nuclear translocation . the assay was performed in l - cells , an established model system for studying wnt signaling . upon treatment of l - cells with wnt3a cm , the intracellular amount of -catenin increases , with an enrichment in the nuclear compartment and a concomitant increase in the nuclear / cytoplasmic ratio of -catenin intensity . in the sample treated with control cm , -catenin is hardly detectable , with a nuclear / cytoplasmic ratio close to one . the wnt3a - mediated effect is reverted by dkk1 cm in a concentration - dependent manner ( figures 4(a ) and 4(b ) ) . when nci8642 was added to the cells together with dkk1 , and then the cells were stimulated with wnt3a the measured ic50 of the compound is 12.6 m ( pic50 = 4.9 stdev 0.2 , n = 5 independent measurements ) , in good agreement with the binding data and the reporter assay ( figures 5(a ) and 5(b ) ) . nci8642 is a small molecule that has been recently identified as a specific inhibitor of lrp5-dkk1 interaction . given the role of the wnt signaling in many pathophysiological contexts , modulation of the wnt pathway has become a valuable target in drug discovery . the modulation of the pathway at the level of the cell surface poses the problem of targeting a protein - protein interaction , which might be challenging . recently , the lrp5-dkk1 interaction has been targeted with monoclonal anti - dkk1 antibodies [ 39 , 40 ] . these antibodies were effective in increasing bone density in vivo , both in nave normal growing female mice and in a model of postmenopausal osteoporosis . however , the lack of blood - brain penetration of the antibodies limits their application to peripheral indications such as osteoporosis . on the contrary , a small molecule blocking dkk1 inhibition would have the advantage of being potentially useful also for the treatment of cns pathologies , such as neurodegeneration . our study confirmed that nci8642 acts as a dkk1 inhibitor , and we extended the finding to the interaction of dkk1 to lrp6 . in fact , the small molecule caused the displacement of dkk1 from lrp6 overexpressed in hek293 cells , as shown both by immunofluorescence and quantitative enzymatic assay . the displacement was also demonstrated in a cell - free context by using spr technology , where nci8642 blocks the binding of dkk1 to the immobilized lrp6 . interestingly , the compound was able to block dkk1 binding only when injected together with dkk1 , and not when injected sequentially after dkk1 , which is probably due to the high affinity of dkk1 for lrp6 compared with the micromolar affinity of nci8642 for lpr6 ( figures 2(c ) and 2(e ) ) . spr technology also showed that nci8642 interacts with the immobilized lrp6 with a reproducible and concentration - dependent binding in the absence of dkk1 ( kd 4.7 10 m ) . the interaction between nci8642-lrp6 is highly specific , because no significant binding was observed on two control flow cells . when immobilized on the sensor chip , dkk1 lost the ability to bind lrp6 , suggesting that the immobilization caused a modification of dkk1 structure - function , thus the direct interaction of nci8642 to dkk1 was not measured . nci8642 is also functionally active in the inhibition of dkk1 activity on wnt signaling mediated by lrp5/6 as demonstrated by the tcf - luc and -catenin assays in two different cellular backgrounds . the compound ic50 measured in the tcf - luc and -catenin assays , where lrp5/6 is expressed at endogenous , physiological levels , is comparable with the one obtained in the binding assay on the overexpressed lrp6 . in the tcf - luc assay , we observed a slight increase of the wnt3a - mediated activation of the reporter gene , even in the absence of dkk1 . the hypothesis that it could be due to the presence of endogenous dkk1 in these cells was not confirmed , since we could not detect it by western blot or by quantitative rt - pcr ( data not shown ) . nevertheless , this was not observed in the -catenin translocation assay ( data not shown ) , suggesting that it could be an artifact of the reporter assay , or a cell - line - specific effect . although nci8642 has been reported as a dkk1/lrp5 inhibitor , our data suggest that it is also effective on dkk1/lrp6 interaction . in fact , nci8642 blocked dkk1 binding on lrp6 , both in lrp6 overexpressing cells and in spr analysis . this was also confirmed by functional studies , since only lrp6 , but not lrp5 , is expressed in pc12-tcf - luc cell line , and lrp6 mrna concentration is about 7 times that of lrp5 in l - cells , as assessed by qpcr ( data not shown ) . our results suggest that it is possible to antagonize the dkk1-lrp6 interaction with a small molecule , and this leads to an enhancement of the wnt signaling pathway . although nci8642 micromolar potency might be too low for its application in in vivo experiments , this compound can be considered as a reference small molecule for further drug development aiming at the inhibition of this therapeutically important protein - protein interaction .

background . dkk1 antagonizes canonical wnt signalling through high - affinity binding to lrp5/6 , an essential component of the wnt receptor complex responsible for mediating downstream canonical wnt signalling . dkk1 overexpression is known for its pathological implications in osteoporosis , cancer , and neurodegeneration , suggesting the interaction with lrp5/6 as a potential therapeutic target . results . we show that the small - molecule nci8642 can efficiently displace dkk1 from lrp6 and block dkk1 inhibitory activity on canonical wnt signalling , as shown in binding and cellular assays , respectively . we further characterize nci8642 binding activity on lrp6 by surface plasmon resonance ( spr ) technology . conclusions . this study demonstrates that the dkk1-lrp6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with dkk1 dysregulation .

1. Background 2. Methods 3. Results 4. Discussion

PMC5040587

this case is more unusual , as the patient was a known case of recurrent patellar dislocation and presented with an atraumatic locked and vertically rotated patellar dislocation . this type of presentation has never been reported in literature to the best of our knowledge . a 14-year - old healthy male child with previous history of recurrent lateral dislocation of patella presented to accident & emergency department with complaints of inability to walk or bear weight on his left lower limb after he spontaneously dislocated his patella while running on uneven ground . radiographs revealed a laterally displaced and vertically rotated patella along its long axis with the medial patellar edge locked and dipping into the lateral gutter . open reduction was performed along with lateral patellar retinacular release with medial patellar retinaculum plication , to achieve satisfactory patellar stability and patellofemoral tracking . we would recommend that in the settings of patella being vertically dislocated and locked , open reduction would be the management of choice , as these types of dislocations are difficult to relocate by closed reduction . open reduction not only yields better outcomes but also allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in cases of prior patellar instability . a case of locked patellar dislocation with vertical axis rotation was first described in literature by cooper in 1844 . since then only a handful of cases about the condition intra articular patellar dislocations can be superior or inferior or can be horizontal or vertical . locked vertical dislocation of patella is a rare entity and poses a therapeutic challenge in which closed reduction often requires a general anaesthetic or , may require an open procedure . there have been no published reports on recurrent patellar instability culminating in a vertically rotated and a locked patellar dislocation . we report an unusual case of lateral recurrent dislocation of patella in a 14-year - old healthy child who presented with vertically rotated and locked patella without any history of trauma , which required an open reduction along with proximal extensor apparatus realignment procedure . a 14-year - old healthy male child presented to accident & emergency department with complaints of inability to walk or bear weight on his left lower limb after he spontaneously dislocated his patella while running on uneven ground . upon further inquiry , the patient gave a history of similar episode 10 months back following a fall . at that time , his mother pushed the patella back after which he was taken to the hospital where radiographic images showed that patella was reduced with no associated fracture of patella , tibial tuberosity or femoral condyles . he was given a full length leg cast for 1 month after which he was able to resume his daily activities without any difficulty . previous hospital records revealed that he had history of anterior knee pain with a positive apprehension sign indicating lateral patellar instability . on examination , the knee was markedly swollen with obvious deformity in the lateral aspect in the form of tenting of the skin and soft tissue by underlying patella without any contusion or bruise . the knee was locked in 15 degrees of flexion and patient was unable to perform any movements at the knee joint . standard anteroposterior and lateral plain radiographs were taken which showed a laterally displaced and vertically rotated patella along its long axis with the medial patellar edge locked and dipping into the lateral gutter ( fig . 1 ) . there was no evidence of associated fracture or signs of osteochondral damage . clinical signs of genu valgum , patella alta , tibial torsion or trochlear dysplasia were absent and the quadriceps ( q ) angle on the normal limb was within normal range ( 11 degrees ) . pre - operative radiographs ; antero - posterior and lateral radiographs of the knee joint of the patient showing vertically rotated and locked patellar dislocation in the lateral femoral gutter . an attempt to closed reduction was made under conscious sedation but was not successful . a decision for open reduction the patella was found to be locked with its articular surface facing laterally and the medial edge of patella locked past the lateral femoral condyle and wedged into the lateral gutter . the medial patellar retinaculum was found stretched and attenuated but no tear or defect was noticed . a small incision was given along the lateral retinaculum , the index finger inserted was through it and the wedged medial patellar edge was freed from the lateral femoral condyle ; and at the same time lifting the patella out of the lateral gutter , it was relocated to its anatomical position . the stability and tracking of the patella was checked and it showed a tight lateral retinaculum along with a lax medial retinaculum . release of the lateral retinaculum was done along with medial patellar retinaculum plication using multiple interrupted prolene sutures ( fig . intra - operative image ; image obtained intraoperatively showing reduction of patella with lateral retinaculum release and medial retinaculum plication . post - operative radiographs ; anter - oposterior and lateral radiographs of the knee joint of the patient showing relocated patella to its anatomical position . quadriceps setting exercises and active straight leg raising were started in the second post operative week . progressive active and passive range of motion exercises were started after 3 weeks and full range of motion was achieved by 7 weeks . patient resumed his sporting activities by 6 months post operative with no recurrence or any symptoms at the time of final follow up of 18 months . lateral dislocation of patella is a common entity in adolescents , whereas rotational dislocation of patella which involves rotation of patella along the horizontal or vertical axis is quite rare . in vertical dislocation , the patella rotates along its long axis with the articular surface of the patella facing laterally and gets locked into the lateral gutter . vertical patellar dislocations pose a therapeutic challenge to the orthopaedic surgeons and are difficult to reduce by closed methods . repeated attempts at manipulation can lead to osteochondral fractures , which necessitates open methods to reduce the vertical dislocations . most of the patellar dislocations are seen with direct blow to the medial aspect of patella with knee in near extension or due to indirect force causing sudden contraction of quadriceps muscles while the knee is stretched in valgus and the tight quadriceps act as a bow string and prevents relocation . pre - disposing factors are anatomical variations such as shallow inter condylar groove , patellar hypermotility , patella alta and tibial torsion . in this report , we review a case of lateral dislocation of patella which is of interest ; in that it was vertically rotated locked dislocation of patella which was atraumatic i.e with no direct trauma to the patella in an individual with a history of recurrent dislocation of patella . there was no clinical evidence of medial patella - femoral ligament tear or any radiological evidence of trochlear dysplasia , patella alta , or shallow inter condylar groove . similar cases have been reported previously in the literature but with a concomitant history of trauma . hackl et al reported a locked dislocation with bony avulsion of medial structures after fall . corso et al reported a lateral dislocation with vertical rotation by laterally directed blow to patella during wrestling . gidden and bell reported a vertically rotated irreducible patella due to medial border force in femur as a result of motor bike accident . only a handful of cases have been reported in literature in whom there was no history of direct trauma . gann and nalty reported a vertical patellar dislocation in a 10 year old girl without any trauma . michels et al reported a locked patellar dislocation in a 16 year old girl while she was dancing . this case report is rare in the sense that there was no history of direct trauma preceding the dislocation and the mode of dislocation was running over uneven surface . although from literature review it is apparent that these types of dislocations are mainly traumatic and result from direct blow to medial side of knee , it is to be assumed naturally that the medial attachments of the knee joint would be torn in such circumstances ; but nevertheless , a locked dislocated patella may occur in atraumatic patients with prior history of dislocation without any damage to attachments along the medial border which provides the major support in preventing lateral dislocations . during surgery also , we found that the medial retinaculum to be stretched and lax with no defect or apparent tear , the lateral retinaculum was found to be tight and that the intra operative tracking of patella was abnormal . following the proximal patellar realignment procedure in the form of medial retinacular plication and lateral retinacular release , the patella was found to be tracking well in the trochlear groove . the tight lateral retinaculum with lax medial patellofemoral ligament could be related to previous episodes of recurrent patellar instability and one episode of lateral dislocation of patella in our case . hence , we would recommend that in the settings of patella being vertically dislocated and locked , open reduction would be the management of choice as these types of dislocations are difficult to relocate by closed reduction . open reduction not only yields better outcomes but also allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in a setting of prior patellar instability . open reduction avoids osteochondral damage associated with repeated attempts of closed reduction and allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in a setting of prior patellar instability .

introduction : locked vertical patellar dislocations are rare and pose a therapeutic challenge . this case is more unusual , as the patient was a known case of recurrent patellar dislocation and presented with an atraumatic locked and vertically rotated patellar dislocation . this type of presentation has never been reported in literature to the best of our knowledge.case presentation : a 14-year - old healthy male child with previous history of recurrent lateral dislocation of patella presented to accident & emergency department with complaints of inability to walk or bear weight on his left lower limb after he spontaneously dislocated his patella while running on uneven ground . radiographs revealed a laterally displaced and vertically rotated patella along its long axis with the medial patellar edge locked and dipping into the lateral gutter . open reduction was performed along with lateral patellar retinacular release with medial patellar retinaculum plication , to achieve satisfactory patellar stability and patellofemoral tracking.conclusion:we would recommend that in the settings of patella being vertically dislocated and locked , open reduction would be the management of choice , as these types of dislocations are difficult to relocate by closed reduction . repeated attempts of closed reduction may cause osteochondral damage . open reduction not only yields better outcomes but also allows the surgeon to perform patellar realignment procedures in order to prevent further patellar dislocations in cases of prior patellar instability .

Introduction: Case presentation: Conclusion: Introduction Case Presentation Discussion Conclusion

PMC3866000

between 1903 and 1906 , oswaldo cruz , director - general of public health , had devoted his energies to improving sanitary conditions in rio de janeiro , a government priority under the administration of francisco de paula rodrigues alves ( 19026 ) . if yellow fever put a damper on the country s business prospects and image abroad , malaria made it virtually impossible to build the infrastructure needed to connect the country s inland regions to its ports . to control it , researchers new to the ranks of the instituto soroterapico federal ( renamed the instituto oswaldo cruz in 1908 and also known as the instituto de manguinhos ) pulled together the knowledge that had been amassed on the disease , creatively adapting it to the realities they encountered in the field . carlos chagas , a young doctor recently admitted to the institute , had been dispatched in 1905 to fight malaria in itatinga , in so paulo state , where a dam was under construction to provide power for the port in santos , then the largest exporter of coffee . chagas identified which species of anopheles were present in the region , describing some that were new to science . he chose to administer quinine preventively , drain and landfill wetlands , and provide collective protective measures for the workers in the form of screens in their lodgings , while keeping the chronically ill in isolation and providing treatment for acute cases . in early 1907 , chagas and another researcher recently admitted to the institute , arthur neiva , were sent to the vast swampy plains near rio de janeiro , known as baixada fluminense . chagas stayed there just three months before being sent to to fight malaria in minas gerais , where the central do brasil railway was being extended . there , he came across a new human trypanosomiasis , which was later named after him : chagas disease . when they reached the baixada fluminense plains , they found almost all the workers suffering from malaria and a few work sites already shut down . spanish and portuguese immigrants were taken straight from the port to the work site by rail to prevent alarming news of the state of affairs from reaching them and scaring them off . much of the land where pipes were being laid was swampy , and the workers were often up to their waists in water . by 1908 , there were close on four thousand workers there . many brought their families to the region , which also attracted travelling salesmen , merchants and other migrants . a wooden hospital protected by mosquito screens was built at the end of the railway . the general inspectorate of public works was responsible for all water treatments ; insect larvae were killed by the spraying of oil , water tanks in houses were covered , and larva - eating fish were introduced . as in railway construction projects and scientific and military expeditions across malaria zones , quinine was the mainstay of the campaign , being used for both treatment and prevention . the workers soon protested against having to take quinine for prophylactic purposes , prompting neiva to impose a strict , compulsory system : either the workers took the alkaloid or they would lose their jobs . quininisers , who had to make sure they had actually swallowed the capsules , rather than palming them or hiding them under their tongue . the daily dose of 30cg recommended in the manuals by patrick manson , botto scheube and carl mense had already proved insufficient in itatinga . from february to august 1907 , the workers at xerm started to be given 50cg of quinine hydrochloride , produced by merck , every three days , which yielded promising results . however , from late august and into september , neiva started to see a relatively large number of first - time infections amongst the workers who had taken the higher dose of quinine preventively . he first put it down to higher individual susceptibility , since the cases then being recorded were of workers who had failed to follow protocol regarding the taking of quinine . the morbidity rate was just 0.59% , which seemed to suggest they were on the right track , even though the protocol could not be applied to workers families and other migrants , amongst whom the number of cases was very high . the natives of the region , carriers of chronic infections , were a constant source of contamination from anopheles . much as robert koch had seen in africa , neiva encountered infected children whose only pathological manifestation of the disease was a dilated spleen . there was something else that drew his attention : workers who had taken quinine regularly and had no symptoms in xerm were struck by malaria when they visited rio for a few days without taking the alkaloid . from september 1907 onwards , such occurrences became increasingly commonplace . so it was that in october neiva upped the dose to 50cg every other day . the cases of first - time infection vanished wherever quinine administration was strict , but the change in dosage did not halt the rise in the number of workers who fell ill when they were away from the worksite temporarily . in november 1907 , first - time infections started to appear amongst people who had taken quinine preventively every two days . in response , neiva started administering it every day at the same dose of 50cg . this seemed to work , but now the disease struck those who were taking lower doses . and even amongst those who were taking the full daily dose , there were still cases of workers who would fall ill or have a relapse when they went to the capital . by january 1908 , the four thousand workers were receiving medication either every day or every other day , depending on where they were : in some locations , wrote neiva , we have never , for various reasons , managed to arrange things so that we could provide safe quinine administration. the only safe form of treatment was , then , 50cg taken without the exception of even a single day. even so , if treatment was interrupted for one or two days , malaria would strike . neiva himself , upon moving away from the region and stopping taking quinine preventively , was struck with a benign tertian fever at his home in rio de janeiro . in may 1908 , he was dispatched to head an anti - malaria campaign in the region to be crossed by the brazilian north - western railway , which stretched from the main coffee production area of so paulo across the mato grosso until the border with bolivia and paraguay . the article he published two years later shows , from its somewhat muddled structure and imprecise data , that he had not had enough time to test experimentally the deductions he had made in the months he was working on the problem . neiva wrote that he had the opportunity to observe a great number of people and thus a large quantity of human material in conditions that virtually corresponded to those of laboratory research . perfect contrast : the workers , who took quinine , and the women , children and other migrants almost as numerous as the workers who took no quinine and were totally attacked by malaria. it is likely that part of this second contingent took poor quality medications , including ones based on quinine . meanwhile , there were differences amongst the workers because of the different dosages and frequencies with which they took the drug , depending on where they were based , and also because some workers had relapses , showing the group to be less homogeneous than it was first believed because there were infected people in its midst . neiva s interpretation of the situation was that the malaria haematozoon had developed resistance to quinine . it says that when exposed to the constant action of quinine over a more or less lengthy period of time , the parasite became adaptation opportunity for the haematozoa as they fed on blood with different degrees of chemical richness. resistance was the result of the haematozoa s adaptation to organic media containing different quinine levels , which ultimately made them resistant , so that , over the generations , they acquired the capacity to be differentiated clearly in quinine - resistant races. in a third explanatory hypothesis , neiva highlights the relationship between non - infected individuals and victims of relapses in a situation of homogeneous chemical richness , supposing that the haematozoa got accustomed to the alkaloid when they encountered it in high concentrations throughout the whole man the resistant plasmodium races were formed by their cultivation in a quinine - rich environment , both in man and in vectors : as such , growing from gametes accustomed to living in media where quinine was always present , the haematozoa in another cycle , from ookinete to the final feeding that released the sporozoite in the bloodstream , already continued to develop even in the presence of quinine . as such , growing from gametes accustomed to living in media where quinine was always present , the haematozoa in another cycle , from ookinete to the final feeding that released the sporozoite in the bloodstream , already continued to develop even in the presence of quinine . while this may not have been the first time such a phenomenon had been observed by tropical medicine , it seems to have been the first theory in the scientific literature on malaria to explain it . from his experience in xerm , neiva concluded that constant quinine administration amongst malarial population groups , when it is not administered equally to all the inhabitants , will give the haematozoa the means to acquire specific resistance to the point of splitting into different races. in other words , he believed resistance to be a phenomenon which , from a clinical perspective , was manifested in the failure of quinine to cure patients , but whose origin lay in the development of parasite strains resistant to the drug . the publication of neiva s theory is related to oswaldo cruz s trip to the amazon on the service of the madeira - mamor railway , or the devil s railway , as it became known for the many thousands of people who lost their lives to the disease during its construction . there , the director of the instituto de manguinhos noted that huge doses of quinine , up to 3 g a day , still failed to prevent malaria from breaking out amongst people working on the railway . oswaldo cruz reached the region where the tracks were being laid in july 1910 , after the first section of the railway had been opened . in september , he delivered a report to the company containing an analysis of the sanitary conditions and his recommendations for their improvement . notwithstanding the range of diseases that plagued the workers , the main culprit for crippling the madeira - mamor works was malaria . it was so prevalent that it inverted the ratio between normal and pathological : the people , wrote oswaldo cruz , have no idea what a healthy state is. morbidity was high , but mortality was far lower . the most widespread form of malaria was the most serious : aestival - autumnal , or tropical ( 70% ) , caused by plasmodium falciparum . as in the campaigns run by chagas and neiva , emphasis was put on using quinine and providing protection against mosquito bites . oswaldo cruz considered the doses used in xerm and minas gerais to be ineffectual in such circumstances , and proposed the daily administration of 23 g , even though he noted that a daily dose of over 0.75 or 1 g produced signs of toxicity . in his report to the company building the railway , oswaldo cruz endorsed neiva s theory about the formation of quinine - resistant races of the plasmodium . some patients continued to contain parasites in their blood 24h after an intravenous dose of 6 g of the alkaloid ! despite noting this , he did not make any progress in his theoretical formulations about this biological phenomenon , which would go on to intrigue german researchers with broad experience in malaria prevention and treatment . german researchers followed closely the scientific theories and innovations produced on the other side of the atlantic . the intense commercial and diplomatic relations between germany and brazil , and the increasing flow of german emigrants to a country long ruled by a monarchy linked to the habsburgs created favourable conditions for scientific interchange . the archiv fr schiffs- und tropen - hygiene [ naval and tropical hygiene archive ] frequently commented on or summarised articles published by brazilians in local or foreign journals . one of the progeny of german science was adolpho lutz , an important actor in the institute of microbiology and medical zoology in brazil . and oswaldo cruz himself , when studying medicine in the 1880s , belonged to a group of germanists who would meet up to learn the language in order to keep up with the latest medical breakthroughs published in german journals . the successful campaign against yellow fever led by oswaldo cruz in rio de janeiro attracted two researchers from hamburg s institute for maritime and tropical diseases in 1904 . their mission was to observe the methods used to fight the disease and to study as yet obscure aspects of its aetiology and epidemiology . in the brazilian capital , hans moritz otto and rudolf otto neumann kept in close contact with the manguinhos institute s staff . the expedition was sponsored by shipping companies that connected hamburg and south america and coffee exporters like theodor wille : the hanseatic port was one of the main destinations for brazil s most important export . later , otto and neumann would apply the methods used in the brazilian capital to control yellow fever in togoland , then a german protectorate in west africa . henrique da rocha lima , a founding staff member at the instituto de manguinhos and oswaldo cruz s right - hand man , had specialised in microbiology and pathology in berlin from 1901 to 1903 , and in munich from 1906 to 1908 . in 1909 he was hired by the hamburg institute and from then until his death in 1956 , rocha lima worked actively as a mediator of german , he helped organise brazil s exhibits at the exhibition appended to the 14th international congress of hygiene and demography held in berlin in 1907 . the brazilian delegation presented materials illustrating the campaign against yellow fever , histopathology collections related to tropical diseases and other innovations , such as the technology used to produce bubonic plague serum and a description of the exoerythrocytic cycle of the protozoan haemoproteus columbae , an important breakthrough in malaria studies made by henrique arago . these contributions earned brazil a gold medal , awarded by the german empress , and awakened the curiosity of more german scientists about the research being done there . henrique arago and alcides godoy , another researcher at the instituto oswaldo cruz , both did postgraduate studies in germany in 1907 . the following year , the brazilian institution received two researchers from the hamburg institute , the protozoologist stanislas von prowazek and the chemist gustav giemsa . in their six - month stay , they did research and trained young scientists at the instituto oswaldo cruz . in 1910 , max hartmann , then on the staff of berlin s institute for infectious diseases , also spent some months there . two years later , hermann duerck helped to consolidate the anatomical pathology department at the instituto oswaldo cruz . the period in which prowazek and giemsa were working at the instituto oswaldo cruz coincided with neiva s malaria studies and his campaigns in inland brazil . neiva and prowazek even undertook a joint expedition to the tiet river in the state of so paulo to study its flora and fauna and also the epidemiology of malaria in the region . during prowazek and giemsa s sojourn at the instituto oswaldo cruz , the institution was also visited by ernst rodenwaldt , an army physician also connected to hamburg s institute for maritime and tropical diseases . he was journeying along the coast of south america as a ship s doctor on a vessel owned by the hamburg it is therefore more than likely that giemsa , prowazek and rodenwaldt learnt of neiva s observations of quinine resistance in rio de janeiro s swampy lowlands before he systematised and published his findings in the first volume of memrias do instituto oswaldo cruz . the fact that this and other articles were published in both german and portuguese in the institute s journal shows not only germany s leading position at the time in biomedical research , but also the close dialogue maintained by german and brazilian specialists . it was interrupted during the first world war but was soon re - established , along with an exchange of merchandise and capital and an influx of german migrants to brazil . the point we want to stress is that brazil s involvement in the production of knowledge on tropical diseases was more complex than is suggested in traditional representations of its role in international capitalism as a mere exporter of primary goods and destination of capital and manufactured products from europe and north america . scientific relations between brazil and germany in the field of tropical medicine were very dynamic , with knowledge and innovations being produced on both sides . not beholden to an imperial power , brazil had developed a strong tradition in medical research that focused on local diseases that threatened both the imperial and the republican elites nation - building and modernisation projects . this tradition created the institutional and cultural basis for its reception of cultural and scientific initiatives from the empire of wilhelm ii , including those from its chemical and pharmaceutical industry . in hamburg , the director of the institute for maritime and tropical diseases , bernhard nocht , and the head of the institute s clinical department , heinrich werner , would have the chance to observe resistance amongst the german workers who had been repatriated to that hanseatic city , as mentioned before . in an article also published in 1910 , they stated that they had been prepared for some quinine resistance in the malaria contracted in inland brazil , but they had been taken aback by its strength . the reason nocht and werner were so surprised at the failure of quinine in patients from the madeira - mamor region was simply that the treatment they had used until then had been successful . since the founding of the hamburg institute and the sailors hospital , the disease that had been most studied and treated there had been malaria . nocht had developed a new method for administering quinine that differed from the method suggested by koch , which consisted of administering 1 g of the drug on the ninth and tenth day of the disease . inspired by the italian method of fractionated dosage , nocht was in favour of administering a total of 1 g every day in five separate doses for eight days running , after which time he would increase the gap between the doses ( for instance , the same five doses a day for just two days ) until the patient was cured . this method had proved successful until the influx of workers from the madeira region of brazil . according to the tropical medicine specialist ernst rodenwaldt , amongst the specialists with experience of working in the tropics or on board ship , the way to treat malaria was taken for granted by all. the success of their methods had been proven by the constant improvement in morbidity and mortality rates amongst europeans in the colonies . several colleagues , wrote rodenwaldt in 1919 , will still remember the time when a malaria diagnosis in the tropics or at home would somehow lift our spirits about the patient , for we were certain we could help and promise a definite cure. amongst the workers being repatriated from the amazon , outbreaks of fever were exacerbated by symptoms of diarrhoea , dysentery and repeated relapses . in 90 cases observed by nocht and werner ( a number that included the patients and their relapses ) , 56 had tertian fever , 15 had tropical fever and 19 had mixed infections . unlike previous experience , the tropical form was more likely to relapse than the tertian forms , which normally was caused by a species of plasmodium that tended to produce more obstinate relapses . as they observed these anomalies , nocht and werner progressively raised the dose of quinine they administered twofold and even more . the initial dose rose to 2 g and , in the complementary treatment phase , between pauses of increasing length , they would administer quinine for two or three days so that the first pause would last two days , the second three , the next four and so on until eight days break was reached . however , the higher dose did nothing to improve the results : neither the frequency nor the seriousness of the relapses diminished . this quite unusual circumstance led them to experiment with other products . compounds containing arsenic had been tested by patrick manson in 1902 and by peter mhlens a year later . koch observed the effect atoxyl had on the malaria parasite , but as it also affected eyesight , its use in treating the disease was not pursued . the usefulness of another compound , methylene blue , had first been reported in 1891 by paul ehrlich and paul guttmann , and had been recommended afterwards for children . miguel couto also recommended it for brazilian malaria , which encouraged neiva to test it in xerm . he did find it to have some effect against the plasmodium , but its action was very slow . incontestable the effect of ehrlich hata preparation 606 ( salvarsan ) in five cases of quinine - resistant malaria . werner tested the preparation again on brazilian malaria , but it failed to prevent relapses in any case . werner also sought to demonstrate how the amazonian variant of the disease differed from other regions in the world . with nocht , he had observed several clinical peculiarities , especially the more frequent disturbance of the intestine . in no patients they also found it notable that the nervous system was affected in two serious cases , both with symptoms of beriberi . and the spleen , while only slightly dilated in several very serious cases of malaria , was found to be alterations of the lungs were also a characteristic of patients from brazil : fourteen had more or less serious cases of bronchitis , which disappeared as the malaria treatment progressed . the doctors from hamburg also reported temperature variations that had not previously been seen . in six cases , shortly after the fever had been abated by quinine , the patients temperature had risen to for several days without any parasites being detected in the peripheral blood . pseudo - relapse. faced with these singularities , nocht and werner started to wonder whether the species of parasites involved in brazilian malaria were different , not just in their virulence and resistance , but also morphologically . the parasites in the tertian form were observed to be slightly different to the ones found in other parts of the world . in an article published in 1910 , the tropeninstitut researchers did not put forward any explanation for the development of quinine resistance . only the following year did werner discuss it theoretically , differentiating two forms of resistance : that acquired during the passage of the plasmodium through warm - blooded animals that is human hosts and that acquired in the passage through insects . the first form , resulting from the repeated effect of quinine on the plasmodium , was correlated to the prolonged use of the alkaloid in malaria treatment . this would partially explain the resistance seen in the cases from the amazon , including the peruvian amazon , where quinine had been used to fight malaria for centuries . meanwhile , werner thought it unlikely that the second form during the passage through the insect existed . for neiva , the high levels of quinine absorbed by the insects as they fed on human blood were enough to assure the continued or even increased resistance of the parasites in their passage through the vector . according to werner , research by gustav giemsa and heinrich schaumann had demonstrated that the level of alkaloid in human blood was too low for a new generation of resistant parasites to develop from the fertilisation of the insect . the hamburg researcher believed it was impossible to reach any firm conclusions about the phenomenon of resistance based on deductions that were primarily theoretical. the tests made by nocht and werner with methylene blue and salvarsan show that they were attuned with the chemotherapy developed by paul ehrlich and his collaborators . the resistance of trypanosomes to atoxyl studied by ehrlich from 1907 onwards influenced in large measure the analysis of the phenomenon now observed with malaria parasites . according to christoph gradmann , ehrlich was interested in the trypanosomes resistance to atoxyl not so much for its clinical importance as for the fact that it could furnish evidence about the existence of cell receptors and to support his theory about the drug s action on the cell , which he compared with the relationship between toxins and antitoxins . both bernhard nocht and ernst rodenwaldt wrote that the results obtained in experiments with protozoan parasites of the blood like trypanosomes could not be extrapolated to intracellular parasites . according to nocht ( 1919 ) , the trypanosomes resistance to drugs was stable , and was maintained after many passages , while quinine resistance disappeared in plasmodia after prolonged interruption of medication in the same host . rodenwaldt was not so sure of this , but added that the disappearance of alterations in the malaria plasmodia was in part due to the fact that their asexual reproduction was more complex than that of trypanosomes . new theories emerged during the first world war , where the havoc wreaked by malaria in the balkans , turkey and syria made resistance to quinine a top priority . the action of quinine often left much to be desired , both therapeutically and prophylactically, wrote the army physician wilhelm hoffmann as he weighed up the impact of malaria on german troops . the initial optimism after the discovery of its aetiology and transmission and the success of experiments to control the disease in specific geographical contexts was badly shaken during the first world war , forcing the specialists in the field to review the epidemiological and scientific studies on the disease . in macedonia in particular , british , french and german troops were struck by particularly devastating epidemics and caught off - guard by the inefficacy of the classic preventive and therapeutic methods . the infection rate amongst the local people was 6095% and the very high density of mosquitoes made it impossible to control the vector . in late 1916 , french army doctors were alarmed to find that sixty thousand men had lost their lives to malaria in the region , half of the french military contingent . malaria starts to appear as a serious sanitation issue in german medical records in early 1916 . let us turn to the testimony of ernst rodenwaldt : first of all , prevention seemed to fail , then the cure . everything indicated that quinine was not the specific medication one had hoped for , or was being administered incorrectly , or that there must be interferences that reduced or prevented its expected efficacy. some doctors even defended the idea that in regions like macedonia , albania or the taurus mountains there was a different form of malaria that could not be treated with quinine . its prophylactic use , considered by the doctors working in the tropics to be essential , started to be seen with increasing scepticism by the doctors working on the front . they argued that it fostered a certain resistance and worsened the chances of treatment with the alkaloid when infection hit . the drug s side effects queasiness , weakness , bleeding under the skin , impaired sight and hearing , chest pain and blackwater fever made it hard to get the soldiers to stick to the treatment . for this reason , peter mhlens , head of the clinical section at the tropeninstitut , recommended , as neiva had in the baixada fluminense lowlands in brazil , strict supervision by the sanitation officials to make sure they actually ingested the quinine . the servicemen would often hide the drug and later exchange it for cigarettes or alcohol . certain authors suggested that many supposed cases of ( biological ) quinine resistance were not just down to human resistance and the inadequate use of the drug , but also to the general state of the patients , who suffered from malnutrition , stress and fatigue because of the physical and emotional impact of the war . at the time of the war , few german doctors were familiar with the clinical aspects of malaria and how to treat and prevent it , because , according to rodenwaldt , having been eliminated from germany half a century ago , except for small enclaves , malaria was no more than a curiosity in medical teaching before the war. this explains why so many of them were so little prepared for dealing with it during the conflict . to make matters worse , it seems that the military sanitation organisation failed to give clear , standard guidelines on how to fight the disease . nevertheless , the new circumstances provided an unprecedented opportunity for verifying findings from previous , more sporadic observations of malaria on a far larger scale , albeit with little control over the conditions . ultimately , the war served as a great experimental field for malaria research and especially for evaluating the prophylactic and therapeutic efficacy of quinine. whenever quinine was taken preventively , the doses used varied greatly . in some regions around 1 g quinine was administered a day , while in others the troops would receive 1.8 g in ten days . for rodenwaldt , treatment had to be continued for two to three weeks after the malaria - infested region was left , warned mhlens , but this rarely took place . there were also disagreements over the best way of using quinine : whether in tablets , in a solution , or in subcutaneous or intramuscular injections . apart from the hypothesis that there was a specific form of the disease in the balkans and turkey , other theories were also put forward to account for the failure of treatment or prevention using quinine . in a bid to make some sense of the arguments and opinions that abounded , martin mayer distinguished two forms of resistance : chininabstmpfung dulling or insensitivity to quinine due to factors within the patient s body that made the individual unresponsive to the alkaloid and chiningewhnung adaptation to quinine referring to what took place inside the parasite . in the former case , physiological processes were responsible for the drug s diminished effectiveness , witnessed by the reduced excretion of quinine in the urine or its reduced concentration in the blood . gustav giemsa and joseph halberkann , from the hamburg institute , questioned these results : the excretion of quinine did not prove that it had failed to exert any action against the parasite in the organism . however , the products of the alkaloid s decomposition could be involved in the process , and this was something giemsa turned his attention to . nocht felt that the inefficacy of ever larger doses of quinine was not related to the emergence of strains of quinine - resistant parasites , as neiva had suggested . rather , he attributed the diminished or non - existent effect of quinine to the weakening of the organism s defences and the prolonged and inappropriate use of the drug . the fact is that the parasite s or human organism s resistance to quinine involved complex issues that were beyond the grasp of medical science at the time . indeed , several aspects of the epidemiology and physiopathology of malaria would remain obscure . resistance to quinine became a polysemic concept , employed in different circumstances depending on the author s principles or procedures . if before the war neiva and werner spoke of a property developed in the parasites , during the war the phenomenon started to be related to the inefficacy of the alkaloid caused by incorrect prophylaxis , organic factors like immunity , the social resistance of troops ascribable to the drug s side effects , and so on . in an attempt to avoid the ambiguity suggested by the word resistance , martin mayer , from the hamburg institute , proposed a distinction between insensitivity to quinine , referring to the patient s organism , and quinine adaptation , a characteristic developed by the parasite . the hypothesis of direct action was refuted by laboratory evidence that the concentration of the alkaloid in the blood was far lower than that needed to kill the microbe . repulsion theory , proposed by julius morgenroth in 1917 , was that quinine would build up inside erythrocytes and would there repel the parasites by chemostatic action , preventing them from penetrating the cell . free in the blood stream , their life cycle was interrupted and they were destroyed . however , if they were only repelled by erythrocytes , argued rodenwaldt , it was hard to imagine how these plasmodia would adapt to a drug that had no direct effect on them . morgenroth viewed the action of quinine in the light of the principle of selective action proposed by ehrlich , with whom he had worked . when it came to the life cycle of the plasmodium , the process by which it resisted immune reactions and the effects of quinine , remaining quiescent in the organism until the disease relapsed , was still a mystery . it was not known where in the body it remained , nor whether it was capable of being maintained inside other cells than erythrocytes . the asymptomatic carriers of parasites and the chronically ill were held accountable for the introduction of the disease to areas prone to epidemics . incite the manifestation of hidden infections , using techniques that included submitting individuals suspected of being infected to temperature shocks or physical exertion followed by hot and cold baths . in a chapter on malaria in the respected handbuch der tropenkrankheiten [ handbook on tropical diseases ] by carl mense , published between 1917 and 1918 races in certain regions like brazil , which could have existed previously or have developed in response to the overuse of the drug . finally , the third form was indirect resistance via the failure of the patients defences . ziemann had summarised the different explanations , but had made no progress in developing a theory , thereby corroborating nocht and mhlens statements that the abundant literature on malaria published after the war discussed specific problems concerning the epidemiology , physiopathology , treatment and prevention of the disease , but failed to put forward any satisfactory solution for most of the enigmas the doctors on the front line had been faced with . neiva and werner s theoretical speculations continued to be the most comprehensive attempts to explain quinine resistance , even if they were riddled with inaccuracies and gaps . however , the idea that the parasite acquired resistance to the alkaloid during its life cycle in humans and/or in mosquitoes , and that it was able to transmit resistance to its offspring , was refuted by ernst rodenwaldt , who in 1919 wrote one of the most robust works on the subject , presented as a thesis to the university of heidelberg . why have our skills failed in the balkans and turkey if before the war tropical medicine had so many successful experiences? he wondered . let us , then , examine the ideas he galvanised to explain the complex biological process by which the malaria parasites became resistant to quinine . writing in his memoirs about the german retreat from asia minor , where he had worked as a hygiene adviser , rodenwaldt relates that he took advantage of the six - week wait for a passage to europe to study wilhelm johannsen and erwin baur s theories of heredity . upon reaching germany , he decided to enrol at the medicine department of heidelberg university , where he wrote a thesis about the question that had intrigued him since he had been in africa : the resistance of the malaria plasmodium to quinine . in his memoirs he adds that in his thesis he took pains to apply the notions of genetics to the issue of infection , being at the time a staunch believer in august weissmann s theories of heredity . a defender of darwin s theories and critic of the inheritance of acquired characteristics , an idea associated with lamarck but admitted by darwin himself , weissmann , a german biologist , proposed the almost absolute constancy and autonomy of what was known as the germinative plasma , an element responsible for carrying the hereditary factors in a way that was completely immune to the influence of the sum , the rest of the body , which merely transmitted the germinative plasma from one generation to the next . germinative plasma would only change under exceptional circumstances , so that any alteration the organism went through during its life would not be transmitted by hereditary means . the blending of weissmann and mendel s conceptions in the early years of the twentieth century meant that heredity was seen as a fixed condition that was inherent to the biological characteristics of individuals. but rodenwaldt s formulations about quinine resistance were more strongly influenced by wilhelm johannsen and erwin baur , whose insights contributed towards a reworking of gregor mendel s ideas from the early 1900s onwards , especially concerning the emergence of species . different theories were expounded to explain the mechanisms by which different variants and therefore new species emerged , as well as the processes involved in transmitting these variations through successive generations . mutationists , who rejected the role of natural selection as a mechanism of evolution , holding that only mutations responsible for major modifications could have some role in the emergence of new species ; and the biometricians , like karl pearson , who not only recognised natural selection as the principal driving force for evolution , but also the idea that small , continuous variations led to the development of new species . in the midst of this debate came the experiments by a dane , wilhelm johannsen , on pure lines of beans , showing that natural selection was only induced by genetic differences , not environmental ones . johannsen distinguished the genotype , or the set of factors responsible for heredity , from the phenotype , which were the observable characteristics of individuals . individuals with pure lineages that is , with the same genetic material , resulting from self - fertilisation could have different phenotypes in response to different environmental conditions , but their genotype would remain the same . he concluded that an organism s appearance was the result of the influence of mendelian traits and also a response to its environment . mendelian factors only determined the breadth of an organism s reactions to its environment . like johannsen , baur distinguished environmentally - induced changes from heredity . he conceded that mutations could modify genetic traits , but not on the scale proposed by the mutationists. they took place on a small scale , resulting in such subtle changes that they often went unnoticed . these small mutations were important in the formation of new races , but not of new species . in einfhrung in die experimentellen vererbungslehre [ introduction to the experimental doctrines of inheritance ] , a book published in 1914 and re - edited in 1919 , baur mentions the resistance of the plasmodium to quinine . he did not see this property as being an inheritance of acquired modifications : in this case , as in others , it is naturally far more reasonable to concede that isolated races of malaria parasites had differing resistances to quinine , and that by treating an infected person with the drug , a more resistant race that already existed was simply cultivated in the pure form . in this case , as in others , it is naturally far more reasonable to concede that isolated races of malaria parasites had differing resistances to quinine , and that by treating an infected person with the drug , a more resistant race that already existed was simply cultivated in the pure form . in a similar vein and also drawing on weissmann , johannsen and baur s ideas , rodenwaldt sought to refute neiva and werner s lamarck - inspired interpretation . they both held that the parasite developed resistance to quinine over several generations , maintaining or increasing its resistant features in its passage through mosquitoes . for neiva , the quinine the mosquito absorbed when it fed on blood was in some way implicated in the process , although this is an idea that werner ruled out as he thought that the quantity of alkaloid in the blood was too small for this . for rodenwaldt , none of these hypotheses explained the failure of quinine in brazil , the balkans or turkey , nor the contrast with successful cases of tropical medicine in the colonies . neither did they explain the failure of the prophylaxis , nor the way the preparations were used , nor the circumstances of war , nor factors relating to the human organism , nor even the existence of a specific form of malaria in the combat zones . rodenwaldt believed that plasmodium strains that had different levels of susceptibility to quinine , but the same physical features , emerged spontaneously in nature . most of the parasites quickly succumbed to the action of the alkaloid , while a few races were selected . it is not a matter of the creation or cultivation of quinine - resistant strains , as most authors would have us believe , but pre - existing quinine - resistant strains that are selected. selection took place when quinine was used in too small quantities in regions where malaria was widespread and where the arrival of non - immune people provoked more virulent forms of the disease . this process could take place in regions where quinine had been used for centuries , such as the south american mountain ranges , or over a short period of time where a large population group was submitted to the same pattern of administration of the drug . the incomplete prevention or cure of patients contributed to the development of chronic malaria , as in the regions where growing resistance to quinine had been observed . this dependence of the selection of quinine - resistant races on the greater or lesser efficacy of therapeutic treatments explained why the problem did not occur in regions like germany , where malaria had been endemic for a long time , or italy , which had had a systematic process for fighting the disease for some time : the main consequence of treatment in a country with doctors and where the people are used to the therapy until the disease is eradicated was that the selection of quinine - resistant races was prevented , because the use of high doses of quinine until the parasites were killed made it impossible for epidemic levels to be reached . the main consequence of treatment in a country with doctors and where the people are used to the therapy until the disease is eradicated was that the selection of quinine - resistant races was prevented , because the use of high doses of quinine until the parasites were killed made it impossible for epidemic levels to be reached . in the tropics , selection did not take place because the native population had a degree of immunity to the disease and almost never took quinine , while europeans were generally treated correctly . there , the rare relapses and quinine resistance occurred only in people who had the bad luck of being infected with an already existing strain that had not passed through selection . but although there were signs that the plasmodia from regions where there was quinine resistance presented morphological particularities , this was not essential for proving that there were different races of the parasite , rodenwaldt argued . his acceptance of johannsen s views is evident in the distinction he makes between genotype and phenotype to interpret the phenomenon of resistance . alterations to the phenotype did not in any way alter the genotype , whose constitution remained constant . therefore , the alterations could not be transmitted by hereditary means : in a morphologically identical population of plasmodia , but with a different genotypical behaviour towards quinine , it is possible that quinine - resistant races can be isolated by selection ; that is , plasmodia that have the same phenotypical appearance , but which behave differently towards quinine , could lead to the selection of a quinine - resistant race with common phenotypical and genotypical features . in a morphologically identical population of plasmodia , but with a different genotypical behaviour towards quinine , it is possible that quinine - resistant races can be isolated by selection ; that is , plasmodia that have the same phenotypical appearance , but which behave differently towards quinine , could lead to the selection of a quinine - resistant race with common phenotypical and genotypical features . if rodenwaldt owed johannsen the distinction between variations in visible characteristics and variations in hereditary material , the core of his theory was based on baur s deductions . the environment that selected quinine - resistant strains was understood in a broad sense as being an ecological and social environment . in the balkans and turkey , the environment was war - ravaged , crossed by people who were physically and psychically weakened , where troops came into contact with villages rife with chronic malaria and other diseases , and where army doctors had little familiarity with treating malaria , a key ingredient in view of the importance rodenwaldt gave to the correct administration of preventive and therapeutic quinine in his theory . similar experiments to johannsen s would have to be done on pure lines of plasmodia , that is ones with homogeneous genotypes . in 1919 , in the archiv fr schiffs- und tropen - hygiene , rodenwaldt published an article about the plasmodium s resistance to quinine . the following year , werner rosenthal ( 1920 ) , from gttingen , author of tierische immunitt [ animal immunity ] , praised the heidelberg professor for his contribution , but criticised how he had presented the concepts of immunity and resistance in malaria . rodenwaldt s response leads us to a new ramification of the debates and investigations about this disease that experience in the war had made such a moot scientific and epidemiological issue , here approaching it not from the perspective of genetics , but immunology , a field of knowledge that went on to gain ground in experimental and practical medicine . the material researched for this paper is not yet enough to analyse these discussions about the problem of quinine - resistant malaria in any great depth . basically , rosenthal accused rodenwaldt of mistaking a non - specific form of immunity observed in people from a region with chronic malaria with the specific elements of the defence mechanisms activated by the organism in its interaction with the pathogen . europeans were prone to malaria in the tropics because they did not have this non - specific resistance and because they were weakened by the unfamiliar climatic conditions . unlike immunity in bacterial infections , he went on , malaria immunity was developed very slowly through continued exposure to the parasite , that is in the chronic form of the disease . he further stated that it did not produce long - lasting effects , as seen in bacterial diseases , and could be dissipated by internal or external factors ( other illnesses , physical debilitation , etc . ) . unstable , were exposed to a new infection , they tended to exhibit far milder symptoms which would subside more readily under drug - based therapy . rodenwaldt was involved in another far less gentlemanly exchange of views with another former student of the hamburg school , viktor schilling , who visited brazil in 1912 . as a physician and hygiene adviser in turkey and syria , schilling had observed several cases of malaria that showed resistance to mounting doses of quinine . it appears that as early as 1917 he had written a communiqu about this subject which was not published because of strict censorship at the time . his comments only became public two years later in the archiv fr schiffs- und tropen - hygiene and deutsche medizinische wochenschrift , at the same time that rodenwaldt was publishing his thesis . according to schilling : the conditions that neiva , nocht , werner , mhlens and others have presented as being propitious for the development of quinine - resistant races are valid . the parasites have always found a way to partially escape quinine , increase their virulence in new ground and adapt in some way to the alkaloid . . . generally speaking , i am less inclined to accept that the parasites develop hereditary resistance to quinine , and deem it more likely that that there is selection and increased virulence in particularly virulent , resistant strains . the conditions that neiva , nocht , werner , mhlens and others have presented as being propitious for the development of quinine - resistant races are valid . the parasites have always found a way to partially escape quinine , increase their virulence in new ground and adapt in some way to the alkaloid . . . generally speaking , i am less inclined to accept that the parasites develop hereditary resistance to quinine , and deem it more likely that that there is selection and increased virulence in particularly virulent , resistant strains . later , in 1921 , schilling modified his view slightly , bringing it more into line with rodenwaldt s : i have reached the idea of parasite races that are resistant to quinine by practical means , in a severely affected population which received an unreliable supply of the alkaloid , [ with the process observed not being due ] to developed and inherited resistance , but rather to selection and increased virulence , especially in more lethal and resistant parasite strains . rodenwaldt later tried to make selection the theoretical basis for this process , exactly in the same sense i gave to him . i have reached the idea of parasite races that are resistant to quinine by practical means , in a severely affected population which received an unreliable supply of the alkaloid , [ with the process observed not being due ] to developed and inherited resistance , but rather to selection and increased virulence , especially in more lethal and resistant parasite strains . rodenwaldt later tried to make selection the theoretical basis for this process , exactly in the same sense i gave to him . rodenwaldt reacted to this claim , which he believed diminished his contribution to the debate , thus : it is far easier , with so many and such broad possibilities , for the person to defend the idea as their own later , when one of these lines becomes more likely. he went on to reiterate his belief in the selection of pure lineages of already existing resistant strains in which , furthermore , nothing is altered , there is no increase or diminishment of virulence , and they do not adapt to anything at all. in his rebuttal , schilling accused rodenwaldt of having appropriated his ideas , which he had learnt about in smyrna , a province in south - western turkey , where they had met in 1918 . even so , schilling acknowledged that the way rodenwaldt had developed baur s ideas was an interesting , independent theoretical accomplishment which far outstrips my scant experiences derived from practice. rodenwaldt s reply ( 1923b , 404 ) was curt and was accompanied by a note by the archiv editors : the tone of the text had been moderated , as it had involved accusations of a personal nature , and the objective discussion of the matter was now closed . today , the resistance of the plasmodium to antimalarial drugs is one of the big problems facing international health care , prompting the continued development of different therapeutic compounds and combinations of drugs . in this study , we show that quinine resistance was already recorded in the early decades of the twentieth century in some regions of brazil , giving rise to different theories to explain it . at the time , the alkaloid was practically the only option available for preventing and treating outbreaks of malaria . the theory of the development of quinine - resistant races of the plasmodium put forward by arthur neiva would appear to be the first scientific work in the literature about malaria that attempted to give an explanation for the persistence of the disease even when the alkaloid was taken in ever higher doses . these preliminary hypotheses were never verified theoretically or experimentally by neiva , the researcher from the oswaldo cruz institute , whose career took a different turn . meanwhile in brazil , the supremacy of preventive approaches which focused primarily on the vector of malaria ( adopted in the campaigns started by the rockefeller foundation after the first world war ) put strategies designed to address the parasite , and the preventive and therapeutic use of quinine on the back burner . neiva s hypothesis does not seem to have inspired any particular interest amongst brazilian specialists . neiva s observations about the malaria parasite s resistance to quinine reveal the interdependency between laboratory and field work in overcoming public health problems and producing new knowledge about tropical diseases . vast field of observations and experiments. this interdependency is characteristic of the institutionalisation of experimental medicine during the brazilian belle poque . the anti - malaria campaigns were a key part of the collaborative efforts of doctors keen to make their names as scientists in conjunction with the public and private clients that sought their assistance , through both the old and the new biological and medical institutions in which they worked . they did research , ran projects and ultimately made important advances in the most pressing problems of the day , affecting not just public health , but also engineering , farming and resource exploitation . field research was essential for understanding what linked human population groups to parasites , with their complex life cycles and multiple vectors , in intricate chains of interdependency in the natural and social ecosystems . in a manner that was not unlike the scientists working for the administrations of european colonial possessions , brazilian researchers like neiva saw the unexplored brazilian hinterland as a potential repository of new scientific data . as helen tilley reports about britain s african colonies , while they [ the colonial scientists ] evoked the authority of laboratory knowledge , they simultaneously challenged the physical boundaries and natural validity on which that authority was based. the wetlands in the fluminense lowlands were not exactly a laboratory because they presented the biological phenomena in all their complexity in a space that was partially immune to the manipulations and controls inherent in experimental research . however , the field was a fine observation context for malaria because at the time the different plasmodium species related to it could not be grown artificially . according to leo slater , by different routes from those of neiva and other tropical medicine specialists , researchers working on new drugs for malaria did their best to reconstitute the complex connections between parasite , host and vector in the laboratory . in brazil s vast unexplored inland regions , researchers like arthur neiva and carlos chagas obtained new knowledge for tropical medicine . as had been the case when the theory of quinine resistance had been formulated , it was also in a campaign to fight malaria ( in itatinga , in 1905 ) that chagas formulated his hypothesis about domestic infection by anophelines , which would be called on years later to support the campaign to kill malaria vectors by using ddt inside homes . while he was engaged in a campaign against malaria in the worksites for the central do brasil railway , chagas identified the clinical , etiological and epidemiological phenomena of a new human trypanosomiasis which was later named after him . just as it had been propitious for the study of quinine resistance , the exchange between brazil and germany contributed to the communication of the discovery of chagas disease . it was in the archiv fr schiffs- und tropenhygiene that the brazilian scientist first published his findings , identifying trypanosoma cruzi , its vector , and the clinical aspects of the disease , which was so different from the well - known sleeping sickness . the recognition of the brazilian discovery came in june 1912 , when chagas was awarded the schaudinn prize by the hamburg institute of maritime and tropical diseases as the best work in the field of protozoology . interestingly , the presence of german researchers at the instituto oswaldo cruz contributed towards the theoretical research of the new trypanosomiasis , as reported by magali s . malaria , chagas disease and ancylostomiasis formed the triad of diseases which , during and after the first world war , galvanised debate in brazil about the need to extend public health services into the inland areas of the country . sanitation movement was spearheaded by intellectuals , writers and scientists , including chagas and neiva , who drew attention to the prevalence of disease amongst the people who were left at the mercy of god when it came to medical care. according to neiva , one of the factors that exacerbated malaria amongst these population groups was the incorrect use of quinine , which was sold mainly in the form of a sulphate that was effective neither for prevention nor for treatment . as head of public health in so paulo from 1916 to 1920 , neiva faced some serious malaria epidemics in inland and coastal areas of brazil s richest state . alongside measures to attack the mosquitoes , he recommended treating the sick with quinine . the difficulty of importing it during the first world war prompted him to start quinine production at the butant institute , run by the so paulo public health service . from the 1920s onwards , neiva started working mainly in the organisation and administration of scientific institutions and , from 1930 , as a politician . even though he no longer researched malaria , it continued to be a topic of interest to him , albeit now in the arena of debates and political action . and despite having witnessed quinine resistance first - hand , neiva held by the opinion that it was the most reliable method for preventing and treating the disease . in a letter he wrote in 1935 to a fellow public health expert , he criticised the aggressiveness with which the german pharmaceuticals industry was marketing its new antimalarial agents : i am sure that quinine will valiantly withstand all these tests , but responsible work must be done under strict scientific determinism , and these new products manufactured by german laboratories after experiments using bird haematozoa and launched on the market under treacherous commercial conditions must be kept at bay . i am sure that quinine will valiantly withstand all these tests , but responsible work must be done under strict scientific determinism , and these new products manufactured by german laboratories after experiments using bird haematozoa and launched on the market under treacherous commercial conditions must be kept at bay . one of the people involved in marketing the new malaria drugs was peter mhlens , director of the clinical section of the institute for tropical diseases in hamburg . in august 1925 this drug had been developed that year by werner schulemann , fritz schonhfer and august wingler in the laboratories of ig farben by modifying quinoline , one of the main components of quinine . in 1930 , ig farben also brought out atebrine , developed by hans mauss and fritz mietzsch . these synthetic malarial agents were presented as being more effective and incurring fewer side effects than quinine . mhlens deployed his network of scientific and political contacts to get testing approved in balkan nations like romania and bulgaria , and also in latin america , especially in 1931 , when he was in venezuela , guatemala and mexico . he was also in mexico in late 1926 , when he faced some resistance to the promotion of the new malaria drugs being supported by the german foreign ministry as part of its cultural diplomacy efforts . the mexican press warned mhlens that the public references to plasmoquine compromised his medical standing . a mexican military doctor also censured him for behaving not as a scientist , but as a sly travelling marketeer. in a series of articles published not just in german , but also in english , spanish , french and italian , mhlens set forth the advantages of plasmoquine and atebrine over quinine . the resistance of the plasmodium to the alkaloid was cited as one of its main disadvantages . mhlens also enumerated the main side effects of quinine as being blurred vision , nausea and ringing of the ears , and associated it with blackwater fever and quinine idiosyncrasy. in a text written with bernhard nocht , mhlens did admit that the malaria parasite may come to develop resistance to plasmoquine analogous to the so - called quinine - resistance , but went on to say that the veracity of this hypothesis remains for the present a debatable point. a publication from the following year shows that he saw resistance differently from neiva and rodenwaldt , which suggests that the latters hypotheses had not left the theoretical realm : my personal views on the matter have always been that the apparent resistance to medicine was less a characteristic of the parasite itself or even of certain strains of parasites than of the infected individual , in whom it represented a lowering of the protective powers of the organism . my personal views on the matter have always been that the apparent resistance to medicine was less a characteristic of the parasite itself or even of certain strains of parasites than of the infected individual , in whom it represented a lowering of the protective powers of the organism . in a chapter on malaria in the renowned clinical medicine handbook edited by klemperer he claimed that many cases of tropical malaria ( caused by p. falciparum ) were found to be resistant to treatment using drugs because the treatment was inadequate : this is why during the war and in the following years so much was said about the malaria parasites resistance to quinine. but this resistance was down to weakened immunity caused by factors such as malnutrition or other diseases . without doubt , the strength of the organism s defences exerts a fundamental role in any therapy . the medication alone is not capable of leading to a cure. this was equally true for quinine and plasmoquine , whose action on the plasmodium was indirect . when the national socialist party came to power in 1933 , mhlens competed for the top position at the institute of tropical medicine in hamburg . however , he was up against another key figure in this narrative , ernst rodenwaldt , the nazi party s favourite for his staunch anti - semitism and his involvement in the study and communication of theories of racial hygiene . from 1921 to 1934 , when he was working in dutch colonies in south east asia , primarily combating malaria , rodenwaldt published dozens of papers about racial hygiene or eugenics . his involvement with the problem of quinine resistance certainly influenced the campaign strategies adopted in asia . this attempt to apply modern heredity theories to the study of an infectious disease could be seen as a milestone in the development of ideas that ultimately led to his being well known as a racial hygienist. according to heiner fangerau , the combination of mendel and weissmann s ideas that formed the groundwork for rodenwaldt s analyses of resistance represented a watershed for the theories of racial hygiene , it becoming clear how predictable was the inheritance of isolated features. rodenwaldt s trajectory took a similar turn to one of his mentor s , erwin baur , the ideologue behind eugenics and author , with eugen fischer and fritz lenz , of the manual that inspired hitler s racial conceptions set forth in mein kampf ( fangerau , 2000 ) . die mestizen auf kisar ( 1927 ) , about the hereditary transmission of the biological features of a mixed - race population , earned rodenwaldt an eminent position in the international league of racial hygiene . during the years of national socialism , he was the main link between tropical medicine and the much - touted racial conceptions of the day . in the words of wolfgang eckart , notable protagonist of anthropology and racial hygiene in germany in the 30s and 40s , fervently defending racial segregation . as shown by christoph gradmann with regard to trypanosomes and arsenic compounds , the analysis of quinine resistance on the part of the plasmodium shows that this became an object of investigation far before the phenomenon gained the full attention of the medical community because of bacterial resistance to antibiotics starting in the 1960s . but unlike gradmann s findings concerning trypanosomes , the plasmodium s resistance to quinine was closely related to the frequency with which it occurred as a clinical phenomenon . years after the debates about the resistance of the malaria plasmodium to quinine , the resistance of bacteria to antibiotics prompted equally heated discussions in the field of genetics . angela creager suggests that the researchers involved in bacterial genetics in the 1940s and 50s rejected the conceptions of lamarck , by which microbes adapted to drugs and transmitted this property to their progeny . however , the bacteriologists had the chance to replicate resistance in the laboratory as they cultivated bacteria in the presence of antibiotics , unlike the researchers working on malaria resistance at the beginning of the century . the role of mutations in the emergence of variations that granted bacteria their resistance was discussed . from the perspective of mendelian genetics and darwinism , resistance was seen as the selection of mutated lineages that had developed randomly from a population of bacteria that were sensitive to the antibiotic . experimental studies and theoretical speculations , creager continues , led to the identification of the means by which the genetic inheritance was transmitted without involving the chromosome core , ultimately revealing that in the end , adaptation versus mutation turned out to be a false dichotomy. the interpretations of bacterial resistance to antibiotics were applied to the analysis of the similar phenomenon seen amongst malaria parasites : spontaneous mutations conferring reduced sensitivity to a given drug or class of drugs . nevertheless , from the 1950s onwards , the drug that drew specialists attention when it came to resistance was not quinine , but chloroquine , the most widely used drug in malaria treatment since its development . its proven efficacy considerably restricted the use of quinine , and chloroquine was itself succeeded by other drugs , including primaquine , mefloquine and proguanil . this fact helps explain why neiva , nocht , werner and rodenwaldt s observations had such a limited impact and became reduced to mere footnotes in narratives about malaria treatment . and yet , echoes of these authors deductions can be heard in the way researchers currently address the plasmodium s resistance to chloroquine and other drugs : population movements induce the introduction of resistant parasite strains , while the widespread , large - scale use of the drugs operates as a selective pressure in that it removes populations of susceptible protozoa and maintains the resistant ones . the ways the leading actors and conceptions in malariology interacted in different contexts in the twentieth century are not clear - cut and beg further exploratory study . the scientific and geopolitical cartography that emerged after the second world war was a far cry from the context when neiva , nocht and rodenwaldt were active in their fields . this war fostered appropriations of scientific ideas and actions that are not always clear or obvious . the alliance between german researchers and the german chemicals and pharmaceuticals industry , which was strengthened during the weimar republic , became tarnished during the nazi regime and the second world war . not only was rodenwaldt an active promoter of racial hygiene theories , for which he was put on trial at nuremberg , but , when the germans retreated in the face of the advancing allied troops , he was also instrumental in the destruction of italy s sanitation engineering system , which had ensured the control of malaria in the country , resulting in a significant upsurge in the proliferation of the disease . it is also important to remember that the potentialities and limitations of the work of neiva and other brazilian scientists were also related to the fact that it was produced in a country which by many was considered subaltern to europe s centres of civilisation. however , as we follow the research and disputes on both sides of the atlantic , we can see not just how internationalised tropical medicine was in those early decades of the twentieth century , but also how the interaction between the researchers on both sides took such complex and convoluted paths and dynamics . brazilian doctors like arthur neiva , miguel couto and oswaldo cruz did not restrict themselves to adopting and adapting theories and practices that circulated through publications , correspondence , conferences , exchanges of patients and biological materials , but actually formulated new concepts and theories that had an impact on the central social formations . the study of the historical records about the resistance of the malaria parasite to quinine demonstrates how important the research into the topic was to individual careers and to the formation of collective ideas , which , despite being involved in international research programmes , followed national styles in the way they studied and fought the disease , a prototypical tropical disease ,

this article addresses the discussion about quinine - resistant malaria plasmodium in the early decades of the twentieth century . observed by arthur neiva in rio de janeiro in 1907 , the biological and social resistance of malaria sufferers to preventive and curative treatment with quinine was corroborated three years later by oswaldo cruz during the construction of the madeira - mamor railway in the brazilian amazon . likewise in 1910 , ailing german workers were transferred from brazil to hamburg s institute for maritime and tropical diseases , where quinine resistance was confirmed by bernard nocht and heinrich werner . when the first world war saw failures in treating and preventing malaria with quinine along with violent outbreaks of the disease on the turkish and balkan fronts , resistance to this alkaloid became the topic of the day within the field of experimental medicine in germany . new attempts were made to account for the resistance , especially by the physician ernst rodenwaldt , who explored the topic by applying modern theories on heredity . the present article offers a preliminary survey and analysis of pronouncements about quinine resistance , shedding new light on the circulation of knowledge in the field of tropical medicine .

Malaria Campaigns in Brazil and Resistance to Quinine German Medical Doctors Prove Malaria Resistance Malaria During the First World War: Quinine Resistance on the German Medical and Scientific Agenda Ernst Rodenwalt and Quinine Resistance in the Light of Modern Genetics Concluding Remarks

PMC4363823

posterior vitreous detachment ( pvd ) is a common phenomenon frequently related with aging of ocular structures . the presence of persistent vitreomacular adhesions exerting tractional forces ( vitreomacular traction , vmt ) may be associated with the development of macular hole ( mh ) [ 2 , 3 ] . these alterations in the symptomatic phase may cause visual disturbances , including photopsia , metamorphopsia , blurred vision , and decreased visual acuity , which in addition of causing visual - related problems may affect negatively the patient 's health - related quality of life . the introduction of optical coherence tomography ( oct ) has allowed a more accurate visualization of the macular anatomy and better knowledge of the pathophysiology of the process , including measurement and assessment of mh characteristics [ 57 ] , facilitating treatment decision - making . the vitreous gel is responsible for the stabilization of the eyeball through collagen fibers ( mainly type ii collagen ) . collagen fibers are running in an anteroposterior direction through the vitreous center , convering in the anterior vitreous base , and inserting into the posterior vitreous cortex . spaces between the collagen fibrils are maintained by the protein opticin and the glycosaminoglycan chondroitin sulphate . spaces between the collagen fibrils are mostly filled with water ( 98% of the vitreous gel component ) and hyaluronic acid , which provides the gel - like consistency of the vitreous . the vitreoretinal interface is a complex anatomical structure composed by the union between the retina and the vitreous . densely packed collagen fibrils of the posterior vitreous cortex ( 100300 m in thickness ) lie over the macula and are superficially inserted into the internal limiting membrane ( ilm ) of the retina by means of adhesion molecules , such as laminin , fibronectin , and proteoglycans , which interact with opticin in the vitreous gel . adherences are more firmly attached to the retina at the vitreous base , optic disc , and fovea , as well as along the major retinal blood vessels . the vitreomacular junction has an annular shape , with a diameter of 3 - 4 mm . the set of events that occur as the eye ages are associated with a series of physiological changes in the vitreous gel , with progressive liquefaction ( at the age of 80 , around 50% of the vitreous gel has been liquefied ) and gradual destruction of the collagen - hyaluronic acid network . this occurs as a result of the development of fluid - filled pockets beginning in front of the macula , which over the time coalesce and enlarge , resulting in a weakened adhesion between the vitreous and the retina . this gradually predisposes to pvd , defined as separation of the posterior cortex from the ilm of the retina , which represents the final step of the normal vitreous aging process [ 11 , 12 ] . pvd is an insidious process that occurs over the course of months or years , being asymptomatic in many cases until complete separation of the vitreous from the macula and optic nerve , which is the final stage . however , the anterior attachment to the vitreous base is very strong and remains for a long time . acute symptoms of complete pvd include photopsia ( by vitreous traction on the peripheral retina ) and floaters by condensation of the vitreous collagen , glial tissue , or blood around the optic nerve . studies in healthy adults have shown that focal perifoveal pvd occurs in 50% of subjects aged between 30 and 39 , whereas complete pvd is found in 50% of subjects aged 70 years or older [ 13 , 14 ] . in addition to advanced age , pvd is more frequent in postmenopausal women by the effects of decreased estrogens on the connective tissue ( within the vitreous gel ) , as well as in the presence of myopia . the normal process of pvd due to vitreous aging may be complicated by the presence of vitreomacular adhesions between the cortex and the macular area , resulting from vitreous syneresis . these adherences may be focal or extensive , affecting the foveola only or a wide region of the macular area and the optic disc . simple vitreomacular adhesion ( vma ) is not associated with distortion of the macular architecture . however , these adherences may exert traction forces on the macula ( vmt ) , increasing secondarily during ocular saccades . this may cause retinal distortion and foveal detachment . on the other hand , continuous anteroposterior traction by vitreous contraction may cause alterations , such as cystoid macular edema . full - thickness mh is an anatomic defect in the fovea with interruption of all neural retinal layers . with the use of high - resolution oct , it has been shown that idiopathic mhs are initiated during perifoveal pvd as a consequence of the dynamic anteroposterior vmt process . this anteroposterior vmt may cause intraretinal cavitation with progression to dehiscence of the outer retinal layers and complete detachment of the cyst roof giving rise to a full - thickness defect . stages of the development of mh from focal vmt to complete aperture together with accompanying symptoms have been described by gass [ 18 , 19 ] . the introduction of enzymatic vitreolysis , which can result in the liberation of vmt , opens highly interesting new perspectives in this field . a few studies have been specifically addressed to the epidemiology of idiopathic vmt due to the overlapping of this condition with other ophthalmological diseases . a prevalence of isolated idiopathic vmt , without mh , has been estimated as approximately 22.5 cases per 100 000 of the general population , with an incidence of 0.6/100 000 persons - year . in different observational and intervention studies , the mean age of patients with vmt was around 6570 years ( range 4864 ) , with a predominance of females [ 4 , 15 ] . regarding the prevalence of mh , it has been reported around 0.1 to 0.8 in adults aged > 40 years , with an age - adjusted incidence of 7.8 cases per 100 000 of the general population per year . also , the risk of development of mh in the fellow eyes , without manifestations of pvd , has been estimated at around 712% after 5 years and 17% at 20 years . approximately two - thirds of patients with mh are women , and the disease is unilateral in 80% of cases . an increase in serum fibrinogen level has been reported as a risk factor for mh , whereas the use of estrogen replacement therapy in women decreases the risk . in subjects with myopia , now , nearly two decades since the introduction of oct , it is possible to assess and define the pathologic progression of disorders affecting vitreoretinal interface with a high level of accuracy and reproducibility . on the basis of oct - derived anatomic findings , a unified classification scheme for disease of the vitreomacular interface has been developed . with this purpose , a group of experts in diseases of the vitreoretinal interface ( international vitreomacular traction study group , ivts ) have proposed a classification system for diseases of the vitreomacular interface . this evidence - based classification is a clinically applicable system that is predictive of therapeutic outcomes and is useful for the execution and comparative analysis of clinical studies . vma represents a specific stage of partial vitreous detachment in the perifoveal area without retinal abnormalities . in previous classifications , vma is the equivalent of a stage 1 pvd [ 11 , 15 , 27 , 28 ] . vma is characterized by elevation of the cortical vitreous above the retinal surface , with the vitreous remaining attached within a 3 mm radius of the fovea ( as defined arbitrarily ) . the angle between the vitreous and the inner retinal surface is acute , and the retina displays no abnormalities in contour or morphological features of oct . vma is not accompanied by visual impairment and may be considered a normal finding in the natural course of pvd . also , vma may be subclassified by the size of the adhesion into focal ( 1500 m ) or broad ( > 1500 m ) . the cutoff of 1500 m corresponds to the area of increased vitreous adhesion to the fovea . vma usually resolves spontaneously as part of the normal process of pvd , although it may progress to vmt and , for this reason , periodic monitoring with oct is necessary . macular traction due to progression of pvd causes anatomic changes in contour of the foveal surface , intraretinal pseudocyst formation , and disappearance of foveolar depression , which typically results in reduced or distorted vision . the following anatomic criteria should be present at least in one oct image to classify an eye as having vmt : ( a ) evidence of perifoveal vitreous cortex detachment from the retinal surface , ( b ) attachment of the vitreous cortex to the macula within a 3 mm radius of the fovea , and ( c ) association of this attachment with distortion of the foveal surface , intraretinal structural changes , foveal detachment from the retinal pigment epithelium ( rpe ) , or a combination of these findings , without full - thickness interruption of all retinal layers . vmt can also be subclassified as focal or broad ( using the same cutoff of 1500 m ) depending on the width of the vitreous attachment . distortion of the foveal profile , formation of intraretinal cysts , intraretinal cavitation , subretinal fluid , and , even , rpe detachment can be observed . on the other hand , proliferation of residual of vitreous tissue provides the anatomic substrate to form an epiretinal membrane ( erm ) , which in turn may appear at any stage of vitreous separation . although spontaneous resolution of vmt may occur , traction on a large surface or the presence of erm is poor prognostic factor . in symptomatic patients , enzymatic vitreolysis or vitrectomy may be indicated . as stated above , full - thickness mh is an anatomic defect in the fovea featuring interruption of all neural retinal layers . the observation of the anatomic opening on several scans through the fovea is an unequivocal sign . according to the aperture size , mhs are considered small ( < 250 m ) , medium ( 250 to 400 m ) , and large ( diameter > 400 m ) . also , on the basis of oct findings , mh can be categorized according to the presence or absence of vmt . only patients with mh and concomitant vmt are candidates for pharmacologic vitreolysis . the correlations between mh stages commonly used in clinical practice and oct - based images proposed by the ivts group are shown in table 1 . primary mh results from vitreous traction on the fovea from anomalous pvd ( incomplete or inadequate separation of the vitreoretinal interface ) , whereas secondary mhs are caused by other pathologic conditions and do not have preexisting or concurrent vmt . secondary mhs have been reported in cases of blunt ocular trauma , lightning strike , high myopia [ 25 , 31 ] , macular schisis , macular telangiectasia type 2 , occlusion of the central retinal vein , diabetic macular edema , uveitis , and age - related macular degeneration . the availability of oct , particularly spectral domain oct ( sd - oct ) , has allowed a more accurate diagnosis and precise assessment of adhesion of the vitreous to the macula , differentiating vma from vmt . before the introduction of oct , only patients with advanced vma could have been diagnosed by biomicroscopy and , for this reason , the rates of spontaneous deterioration reported were high ( 64% ) . studies using sd - oct have shown that incomplete vitreous detachment with persistent vitreoretinal adhesions is more frequently observed than by clinical diagnosis . during the physiological process of pvd , the vitreous remains attached to the foveal region in the last stages ( figure 1 ) , so that , vma can be considered a normal stage in the natural history of pvd associated with vitreous aging [ 13 , 26 ] . only when symptoms are present or when foveal anatomic changes are observed , vma can be considered a pathological process . recently , john et al . investigated the spontaneous clinical course in 106 eyes of 81 patients identified as having vma by sd - oct and classified into three grades , with a mean follow - up of 18 months ( range 1 to 91 ) . the authors defined three grades to classify adherence : grade 1 ( 41% ) was incomplete cortical vitreous separation with attachment at the fovea , grade 2 ( 52% ) was the grade 1 findings and any intraretinal cysts , and grade 3 ( 7% ) was the grade 2 findings and the presence of subretinal fluid . by the last follow - up , spontaneous release of vma occurred in 32% of cases ( 34 eyes , in 30% , 30% , and 57% of grades 1 , 2 , and 3 , resp . ) . no changes were observed in 23 , 31 , and 2 eyes ( 52% of the total ) , and progression occurred in 7 , 8 , and 1 eye of grades 1 , 2 , and 3 , respectively ( 16% of the total ) . the authors conclude that the clinical course of patients with vma managed by initial observation was generally favourable in asymptomatic patients or with minimal symptoms of vmt . studying the retinal surface with sd - oct , it has been observed that pvd appears to begin in the perifoveal region , with a slow clinical course taking even years until complete separation of the vitreous from the papilla ( figure 2 ) . in most patients this process is asymptomatic but , in some cases , pvd may be complicated by macular pathology . in a oct study of eyes with macular edema secondary to vmt , published in 2012 , complete and spontaneous resolution of traction the clinical course of vma , particularly in asymptomatic patients , remains to be fully elucidated . systematic examination with sd - oct has been associated with an increase in diagnostic rates and has allowed assessing more accurately the course of this physiological process that may evolve into vmt , remain stable , or resolve spontaneously . therefore , in the presence of a vma syndrome , the first approach is to reexamine the patients using oct at a period of 3 months . even in cases of evolution to a vmt syndrome , observation still remains an option , given the possibility of spontaneous resolution of vmt . ocriplasmin is a truncated form of human plasmin that induces liquefaction of the vitreous and separation of the vitreous cortex from the retinal surface due to proteolytic activity against main components of the vitreomacular adhesion . the efficacy and safety of ocriplasmin have been evaluated in two pivotal , phase 3 clinical trials ( tg - mv-006 y tg - mv-007 ) carried out in the united states and europe . both studies were very similar except for the ratio of randomized assignments to ocriplasmin and placebo , which was 2 : 1 in the tg - mv-006 study and 3 : 1 in the tg - mv-007 . overall , 652 patients were randomized , 464 were assigned to treatment with a single intravitreal injection of ocriplasmin ( 125 g ) and 188 to a placebo intravitreal injection . the primary endpoint was the pharmacologic resolution of vma at day 28 , as determined by oct . secondary endpoints included the percentage of patients with complete pvd and nonsurgical closure of full - thickness mh at day 28 . eligible patients had symptomatic focal vma as seen on oct and a best - corrected visual acuity of 20/25 or less . exclusion criteria were high myopia ( more than 8 diopters or axial length > 26 mm ) , prior vitrectomy or prior laser photocoagulation of the macula , and other eye diseases that may affect visual acuity . patients with a mh > 400 m in diameter were also excluded . of note , the presence of an erm was not a criterion for exclusion . at day 28 , vma resolved in 26.5% of ocriplasmin - injected eyes and in 10.1% of placebo - injected eyes ( p < 0.001 ) . the between - group differences did not change substantially at 6 months ( 26.9% ocriplasmin versus 13.3% placebo , p = 0.001 ) . also , 72% of patients with resolution of vma showed the release during the first seven days . results of adhesion release were better in patients without erm ( 37.4% in the ocriplasmin group versus 14.3% in the placebo group , p < 0.001 ) . with regard to secondary variables ( day 28 ) , 13.4% of patients treated with ocriplasmin showed total pvd as compared to 3.7% of those treated with placebo ( p < 0.001 ) . also , nonsurgical closure of full - thickness mh was achieved in 40.6% of ocriplasmin - treated patients and in 10.6% of placebo - treated patients ( p < 0.001 ) . according to the investigator 's criteria , all patients could be treated with vitrectomy in the framework of the study if macular disease did not resolve . at 6 months , vitrectomy was performed in 17.7% of patients in the ocriplasmin group and in 26.6% of those in the placebo group ( p = 0.02 ) . at 6 months , there were statistically significant differences in favour of ocriplasmin in the gain of two or more lines ( 23.7% versus 11.2% , p < 0.001 ) or three or more lines ( 12.3% versus 6.4% , p = 0.02 ) . most adverse events were related to the development of pvd induced by ocriplasmin injection ( floaters and photopsia ) . there was a slightly higher incidence of retinal tears or detachments in the placebo group , which was attributed to the higher proportion of patients treated by means of vitrectomy in this group . the favourable results obtained in both clinical trials allowed approval of the use of intravitreal injection of ocriplasmin for the treatment of symptomatic vmt and mh by the food and drug administration ( fda ) in the united states , in november 2012 , and by the european medicines agency ( ema ) in may 2013 . outside the context of clinical trials , recent reports have provided data of the use of ocriplasmin in daily practice . in a retrospective study , 17 patients with symptomatic vmt were treated with a single intravitreal injection of ocriplasmin 0.125 mg . by day 28 , resolution of vmt was verified by sdoct in eight patients ( 47.1% ) , 7 of which ( 87.5% ) had already experienced release by day 7 . those who did not have traction release showed no statistically significant change in vma diameter . four of the five patients ( 80% ) with mh at baseline experienced resolution of their mh after injection . significant differences in visual acuity were not observed ( 20/49 at baseline and 20/46 at final follow - up ) . it should be noted that patients meeting the four positive predictor criteria ( younger than 65 years , no erm at baseline , traction < 1500 m , and phakic lens status ) showed a response rate of 75% ( three of four eyes ) . transient outer segment ellipsoid zone loss was documented in 7 cases ( 41.1% ) and subretinal fluid presence following injection was noted in 5 cases ( 29.4% ) . in another study of 19 patients with symptomatic vma treated with intravitreal ocriplasmin , resolution of vma results were significantly affected by lens status , with adhesion release in 53% of phakic patients , whereas no case of resolution of adhesions was observed in pseudophakic patients . also , closure of mhs after treatment was found in 3 of 6 patients ( 50% ) . visual acuity remains stable , with a slight tendency towards improvement in the majority of cases . only one patient showed an important loss of visual acuity ( from 20/70 to 20/200 ) due to progression of vmt to a full - thickness mh . the proportion of patients who had any ocular adverse event was 68.4% in the ocriplasmin group and 53.5% in the placebo group ( p < 0.001 ) . the most common complications included vitreous floaters ( ocriplasmin 16.8% versus placebo 7.5% , p = 0.002 ) , photopsia ( 11.8% versus 2.7% , p < 0.001 ) , blurred vision ( 8.6% versus 3.2% , p = 0.01 ) , and visual impairment ( 5.4% versus 1.6% , p = 0.02 ) . there were no differences between the groups in terms of severe ocular adverse events , including development of mh ( 5.2% versus 8.6% ) , retinal detachment ( 0% versus 1.6% ) , and reduced visual acuity ( 0.6% versus 0.5% ) . however , since the real - world use of the drug began , there have been some unfavourable reports of visual disturbances after ocriplasmin injection , including transient but profound visual decline , raising concerns regarding its safety . of 976 patients receiving ocriplasmin injection in clinical trials , 9 patients were reported to have experienced an acute decrease in vision , some to the hand motions level , within 24 hours of injection . in 8 of these 9 patients , in the clinical trials of ocriplasmin , dyschromatopsia , and electroretinographic ( erg ) changes occurred in a significantly greater number of eyes treated with ocriplasmin than in eyes receiving placebo [ 20 , 40 ] . freund et al . recently reported a single case report demonstrating changes seen in the outer photoreceptor segments by sd - oct . since the clinical trials used only time - domain oct with inferior resolution to sd - oct , it is possible that these cases may have been overlooked . in another study in which 17 patients were included , almost all the patients who responded to the treatment ( 7/8 ) had ellipsoid zone changes on the sd - oct ( figure 3 ) . these patients also had transient reduction of visual acuity and demonstrated subretinal fluid during the release process with almost the exact time course as the loss of the os ellipsoid zone . the loss of the os ellipsoid zone occurred after an average of 5 days after injection of ocriplasmin and the mean time of resolution on oct was 29.3 days . the occurrence and resolution of subretinal fluid occurred at an average of 4.8 days and 30 days after injection , respectively . however , in a retrospective review of 62 eyes with symptomatic vma treated with ocriplasmin , subretinal fluid appeared in 37% of cases , with persistence of fluid in 30% of cases after 5 months of follow - up . other studies have also shown resolution of the ellipsoid zone changes in most patients within weeks or months after ocriplasmin injection [ 43 , 44 ] . alteration of the ellipsoid zone on sd - oct and a significant decrease in erg amplitudes have been also reported in two patients with release of symptomatic vmt after ocriplasmin injection [ 45 , 46 ] . it is possible that this transient effect of the medication may be due to a diffuse enzymatic effect of the protease on the photoreceptors or the retinal pigment epithelium throughout the retina . the greater reduction in scotopic function compared with photopic function suggests that rod photoreceptors may be more susceptible than cone photoreceptors to the effects of ocriplasmin . if this transient affect occurs for both rods and cones , it may explain the dyschromatopsia , contrast sensitivity changes , dark adaptation issues , and erg changes reported in the ocriplasmin clinical trials . an ongoing phase 3b , 24-month randomized clinical trial which will evaluate erg and microperimetry in ocriplasmin - treated eyes compared to sham , will provide additional clarifications on the observed egr changes and dyschromatopsia events ( oasis study ; ntc01429441 ) already reported . surgery of idiopathic mh with ilm peeling is a very safe procedure , with good anatomic and functional results and scarce postoperative complications . data provided by clinical trials have shown that peeling of the ilm significantly increases mh closure rates and is also associated with significantly lower percentages of reoperation and reopening . therefore , ilm peeling is a cost - effective technique and the procedure of choice for all patients with idiopathic full - thickness mh susceptible to undergo surgical treatment [ 4853 ] . broad ilm peeling to the vascular arcades is recommended , so that tangential traction forces on the mh edges are removed facilitating approximation and closure . in cases of large mh ( > 400 m ) with increased risk of failure of primary surgery , alternative techniques have been proposed , such as the inverted ilm flap technique in which instead of completely removing the ilm , a remnant attached to the margins of the mh is left in place . this ilm remnant is then inverted upside down to cover the mh . with the use of this technique closure rates of 98% compared to 88% with the standard technique have been achieved . for refractory mh to the standard technique or for secondary mh after vitrectomy when peeling of the ilm has been already performed , an autologous transplantation of the ilm remnants introduced into the hole with subsequent gas tamponade contributes to the improvement of anatomic and visual outcomes . vital dyes have become effective and useful tools for identifying ocular tissues during vitrectomy , thereby facilitating ilm peeling and ensuring complete removal of this delicate membrane . the most frequently used vital dyes include triamcinolone acetonide suspension in balanced salt solution ( bss ) ( triesence ) , indocyanine green and infracyanine green , brilliant blue , and trypan blue with brilliant blue ( membrane blue - dual ) . triamcinolone suspension in bss is not a true dye but is very useful for the identification of vitreous remnants and the posterior hyaloid . deposition of crystals on the ilm surface helps the achievement of complete removal of the membrane , although it is less effective than vital dyes because triamcinolone does not increase the rigidity of ilm . indocyanine green and infracyanine green possess a great affinity for the matrix components of the ilm and produce intense staining of the ilm . besides the ability of indocyanine and infracyanine green to stain the ilm , they cause an increase in the biomechanical stiffness of the ilm , thereby facilitating its peeling . although in europe they are no longer used because of potential toxicity , they continue to be used in the united states [ 58 , 59 ] . brilliant blue has a remarkable affinity for the ilm and , although ilm staining is less intense than that achieved with indocyanine green , causes adequate staining of the ilm and may be used without fluid - air exchange . in europe the combination of trypan blue and brilliant blue allows staining of the erm , posterior hyaloid , and ilm simultaneously . this combination has a lower density than water and bss and circumvents the need for fluid - air exchange . there is controversy regarding posturing in mh surgery . although most authors recommend face - down posturing 90% of time for 10 days , different studies have reported successful hole closure in the absence of face - down positioning , given that isolation of the macula by gas tamponade maintaining the macula dried seems to be the most important factor for closure [ 6163 ] . in this respect , oct studies have shown that hole closure occurs during the first postoperative day independently of the types of gas tamponade and posturing , so that after vitrectomy with wide ilm peeling , gas tamponade would be sufficient ( preferably short - acting gases , such as sf6 ) at nonexpansible concentration , without the need of face - down posturing , avoiding the prone position during 3 to 5 days . this approach may be also indicated for phakic patients because it does not seem to increase the incidence of cataracts . combined phacovitrectomy or sequential vitrectomy and phacoemulsification are safe and effective for the treatment of mh , with equivalent anatomic and functional results . in most cases , idiopathic mh affects patients older than 50 years in which some degree of lens opacity is frequent . moreover , cataract develops in 75% to 95% of patients undergoing vitrectomy for mh within 3 years after surgery . for this reason , both cost and discomfort are lower with a single surgical procedure , and functional recovery is more rapid . combined phacovitrectomy may also decrease the risk of reopening after cataract extraction in the two - step surgical approach [ 66 , 67 ] . however , combined vitrectomy , phacoemulsification , and intraocular lens ( iol ) implantation may be associated with complications , including a high degree of postoperative anterior chamber inflammation and a higher risk of iol dislocation or papillary capture , generally as a result of excess gas tamponade and/or poor compliance to positioning . therefore , the decision of the combined versus the two - step procedure should be individualized according to the characteristics of each case and the patient 's and surgeon 's preferences . in the study of the moorfields macular hole ( mmhs ) group , an overall closure rate of 81% at 2 years was achieved in mhs stages 2 , 3 , and 4 as well as an improvement in visual acuity of 6/36 to 6/18 , which was clearly superior to results obtained in the observation group . in the vitrectomy for treatment of macular hole study ( vmhs ) , the rate of anatomic closure was 69% and the final visual acuity was higher in the operated than in nonoperated eyes ( 20/115 versus 20/166 ) . once peeling of the ilm has become popular , closure rates of 90% to 100% were reported [ 7175 ] . however , the use of indocyanine green was associated with potential toxicity in some cases and , for this reason , trypan blue and brilliant blue are in widespread use in some countries , with closure rates of 94% to 100% , without apparent severe side effects [ 7780 ] . despite its clear indication and safety in mh surgery , ilm peeling is a traumatic procedure that has acute effects on the underlying retinal nerve fiber layer . ilm peeling often results in temporary swelling of the arcuate nerve fiber layer ( sanfl ) which may be the earliest manifestation of dissociated nerve fiber layer ( donfl ) which occurs later in the postoperative period . however it is probably a transient feature that does not affect visual recovery . although peeling of the ilm has been widely adopted in mh surgery , the high percentages of hole closure obtained in the years prior to systematic ilm peeling add uncertainty about whether to use it in all cases . recently , spiteri cornish et al . [ 81 , 82 ] carried out a systematic review and meta - analysis to assess the success of hm surgery with ilm peeling compared with the nonpeeling technique . four randomized clinical trials comparing both techniques were identified [ 48 , 49 , 51 , 81 , 82 ] . there was no evidence of a difference in the primary outcome ( distance visual acuity at six months ) , nor in distance visual acuity at 12 months between randomized groups . overall , 66.2% achieved a visual acuity equal or greater than 69 letters on etdrs charts ( corresponding snellen visual acuity 20/40 ) and 77.9% gained more than three etdrs lines . improvement of visual acuity was higher in patients in which primary anatomic closure was achieved ( final visual acuity 72.8 7.6 letters and a mean improvement of 21.6 7.1 letters ) than in eyes in which further surgery was required ( 66.4 8.6 and 17.4 7.7 letters , resp . ) . however , visual improvement was obtained somewhat earlier in the ilm peeling group and , at 3 months , improvement was greater if ilm peeling was performed . in addition , the percentage of primary closure was higher in the ilm peeling as compared with no peeling ( 89.9% versus 50.3% , with an odds ratio ( or ) of 9.27 and 95% confidence interval [ ci ] of 4.9817.24 ) . when reoperations were excluded from the analysis , the ilm peeling group continued to have more favourable results ( or 3.99 , 95% ci 1.639.75 ) . also , in mh stage 2 , the efficacy rate was better for ilm peeling than no peeling ( 91.6% versus 61.3% , with an or of 6.19 ; 95% ci 1.6523.20 ) [ 48 , 49 , 83 ] . this higher success rate was not accompanied by an increase of perioperative complications , neither in the reports in which the ilm was stained with indocyanine green . in the meta - analysis , the rate of intraoperative complications was 19.32% for the ilm peeling group as compared with 21.1% for the nonpeeling group ( or 0.94 , 95% ci 0.471.87 ) . the most frequent intraoperative complications were small retinal hemorrhage ( 619% ) , retinal tears ( 5.432% ) , retinal detachment ( 26% ) , and choroidal hemorrhage ( 03% ) . according to these data , the authors conclude that ilm peeling offers more favourable cost - effectiveness compared with no peeling in mh surgery [ 81 , 82 ] . although anatomic closure in mh surgery is achieved in more than 90% of cases , sometimes it does not correlate well with improvement in visual acuity . multiple studies using oct have assessed hole configuration in an attempt to establish a correlation with postoperative visual acuity [ 8491 ] , emphasizing the importance of changes in the outer retina . defined a macular hole index ( mhi ) as a ratio of hole height to base diameter of hole , calculated from oct transverse images of the macular area , establishing that a mhi 0.5 was correlated with better postoperative visual acuity than mih < 0.5 . ruiz - moreno et al . described the diameter hole index ( dhi ) as a ratio between minimum hole diameter and base diameter , showing the minimum diameter was the best preoperative predictive prognostic factor . different studies have shown a direct correlation between integrity of the hyperreflective line as is / os junction of photoreceptors and postoperative improvement of visual acuity . in the study of kitaya et al . , postoperative vision 0.7 was correlated with good reconstitution of the is / os junction . however , sano et al . showed that a continuous is / os line was not a reliable prognostic factor in the early postoperative period given that abnormalities of the is / os line seen on sd - oct can be gradually repaired , with achievement of a continuous is / os line at 6 months . spaide and curcio assessed the correlation of the outer retina analyzed by means of sd - oct and histopathological findings , showing that the hyperreflective line identified as is / os junction of photoreceptors corresponded to the ellipsoid portion of the photoreceptor inner segment , containing mitochondria . wakabayashi et al . using sd - oct described that reconstitution of the external limiting membrane ( elm ) was more important to predict subsequent restoration of the foveal photoreceptor layer than the ellipsoid zone restoration . restoration of elm seems to be a necessary factor for reconstitution of the ellipsoid band , with subsequent migration of photoreceptors and complete closure of the full - thickness mh . ruiz - moreno et al . have analyzed 164 eyes with mh treated by vitrectomy and ilm peeling showing that restoration of the ellipsoid portion of the photoreceptor inner segment is an important prognostic factor for visual rehabilitation after mh surgery . reopening of the hole ( figure 4 ) is one of the best known complications after initially successful mh treatment with vitreous surgery [ 67 , 91101 ] . peeling of the ilm during primary mh surgery is one of the factors that has been mostly related to the incidence of reopening , varying between 0% and 8% in eyes with ilm peeling [ 67 , 9597 ] and between 2% and 16% in eyes with no peeling [ 67 , 9395 , 97 ] . the variable percentages reported in the studies are due in part to differences in the length of follow - up , with higher rates associated with prolonged follow - up periods . paques et al . reported a 9.5% incidence with a mean follow - up of 2 years , whereas scott et al . found a 12% incidence with a mean follow - up of 7 years . kumagai et al . analyzed the results of surgery in a series of 877 cases of mh , increasing the reopening percentage to 28.1% with no ilm peeling . the incidence of recurrence was 0.39% in eyes with peeling of the ilm , increasing to 7.2% with no peeling . besides no peeling , statistically significant risk factors for reopening were myopia of more than 6 diopters and intraoperative retinal tears . retinal tears treated with laser may be one of the factors that increase the development of erm , with subsequent tangential traction and reopening of the mh . no peeling of the ilm may be associated with a higher risk of erm formation [ 92 , 93 , 97 , 100 ] . yoshida and kishi observed the presence of erm in all cases of reopening of the hole . however , kumagai et al . did not report erm in none of the cases with hole reopening assessed by sd - oct . in relation to the incidence of reopening with bilateral mh , duker et al . reported bilateral reopening in 38% of cases , christmas et al . in 59% , scott et al . in 38% , and kumagai et al . in 14.9% . cataract surgery in the postoperative period of mh surgery has been involved in the reopening of mh . paques et al . observed that 73% of cases of hole reopening occurred after a secondary cataract surgery . bhatnagar et al . reported that in the presence of cystic macular edema after secondary cataract surgery , there was a sevenfold increase in the risk of reopened holes , and garca - arum et al and sheidow and gonder reported cystoid edema in combined surgical procedures and that the incidence of hole reopening did not increase in secondary cataracts . with regard to treatment of persisting mh , ilm peeling and erm removal should be performed in those cases in which these procedures were not performed at the initial macular surgery , together with long - acting gas tamponade ( c3f8 ) and strict face - down positioning during the first postoperative days . in these patients , when ilm peeling and removal of the erm have been performed in the first surgical procedure , the success of reoperation decreases . in a series of 30 patients reported by d'souza et al . with initial ilm peel who underwent repeat surgery involving vitrectomy , enlargement of ilm rhexis , and gas tamponade with c3f8 , more extensive ilm peeling causing tangential traction due to fibrosis of dissection margin may contribute to the anatomic closure . the use of growth factors , such as platelet - derived growth factors as a stimulus of glial progenitor cells , may be useful if the ilm has been adequately peeled ( figure 5 ) , as well as the use of heavy silicone oil in patients with positioning difficulties . based on the aforementioned data and as shown in the schematic representation in figure 6 , patients with vma can be observed without the need of any intervention . in cases of vmt if the patient is asymptomatic , a follow - up control at 3 months may be sufficient . during this interval , the patient should be advised to perform periodic self - examinations with the amsler grid or monocular reading tests . in case of symptoms , intensity and disability should be assessed . there is no consensus criterion regarding the degree of vision loss that should be considered significant and amenable to treatment . however , in the tg - mv-006 y tg - mv-007 clinical trials , patients with visual acuity equal or lower than 20/25 were eligible , so that this level of visual impairment can be already considered to be susceptible of treatment . also , other causes that may justify decreased visual acuity should be excluded . metamorphopsia clinically significant for the patient and visual loss progression are also key factors at the time of adopting a more interventional therapeutic attitude . despite these considerations , a period of observation may be an option for these patients , because spontaneous resolution is still possible . in case of deciding an active treatment , the presence of other associated macular diseases , such as erm , should be excluded . when traction is 1500 m , enzymatic vitreolysis with ocriplasmin is the treatment of choice . in the presence of > 1500 m traction or erm , surgical treatment with vitrectomy 400 m in size with mvt and in the absence of erm , enzymatic vitreolysis with ocriplasmin is again the most recommendable option . in cases of holes > 400 m , or in the absence of evident vmt , or in the presence of erm , vitrectomy is the first option . patients undergoing enzymatic vitreolysis with intravitreal injection of ocriplasmin should be evaluated at 7 and 30 days . most cases of vmt or mh resolve within the first week of treatment and also at this time the occurrence of potential treatment - related complications should be excluded . if resolution of vmt and/or hole closure had not occurred after a month of treatment , the likelihood of success is highly improbable and vitrectomy can be performed . patients with lamellar mh or pseudomacular holes in which traction is usually absent are also candidates for enzymatic vitreolysis . in cases of vmt associated with other retinal diseases , such as age - related macular degeneration , diabetic macular edema , or vitreomacular interface pathology in the myope , it is still too early to make a recommendation on the impact of enzymatic vitreolysis with ocriplasmin in the treatment of these conditions , and we should await for results of ongoing clinical trials on this topic . finally , in all cases , the final decision regarding treatment with enzymatic vitreolysis with ocriplasmin or vitrectomy should be consensuated with the patient . all cases in which the use of ocriplasmin is considered a first treatment option can be successfully treated by means of vitrectomy . also , it may be possible that patients who initially are not ideal candidates for enzymatic vitreolysis may have their pathologic condition solved by treatment with ocriplasmin . favourable prognostic factors for the choice of vitreolysis have been identified including young age and phakic status , but difficulties to maintain postoperative face - down posture or the waiting lists for vitrectomy are variables that should also be considered . enzymatic vitreolysis based on the intravitreal injection of ocriplasmin is a treatment option with proven efficacy and adequate safety profile in selected patients with vmt and mh . in cases of vmt , treatment with ocriplasmin is indicated when traction is 1500 m and in the absence of concurrent macular diseases ( erm ) . in the case of mh , the hole diameter should be 400 , traction has to be present , and erm should be absent . when resolution of the process after one month of the procedure is not achieved , vitrectomy with ilm peeling would be the surgical treatment of choice .

the paper presents a review of the sequence of events of posterior vitreous detachment ( pvd ) , vitreomacular adhesion ( vma ) , vitreomacular traction ( vmt ) , and macular hole ( mh ) from their pathophysiological aspects , clinical features , diagnostic implications , and current management strategies . a treatment algorithm to be used in clinical practice in patients with vma , vmt , and mh based on the presence of symptoms , visual acuity , associated epiretinal membrane , and width of the vitreous attachment is presented . observation , pharmacologic vitreolysis with ocriplasmin , and surgical treatment are positioned as treatment options in the different steps of the therapeutic algorithm , with clear indications of the paths to be followed according to the initial presenting manifestations and the patient 's clinical course .

1. Introduction 2. Diagnosis, Definition, and Classification of VMT and MH 3. Treatment Options 4. Practical Considerations: Therapeutic Algorithm 5. Conclusions

PMC3021274

laparoscopic pyelolithotomy was successfully performed in a pelvic kidney with an operative time of 310 minutes . the use of intraoperative fluoroscopy and a semi - automatic suturing device greatly facilitated the procedure . the patient 's operative pain was managed with 3 doses of ketorolac ; she resumed a regular diet the day after surgery , and was discharged on the first postoperative day . for patients with a large stone in the renal pelvis of an ectopic kidney , laparoscopic pyelolithotomy provides an effective approach . pelvic kidneys are typically incidental findings , but may pre - sent due to underlying obstructive or calculous disease . the anatomical characteristics of the ectopic kidney can pose a significant challenge to the treatment of calculous disease . treatment of larger renal calculi in these kidneys had been uniformly by open surgery until laparoscopically guided percutaneous nephrostolithotomy ( pcnl ) was described , initially , by esghi , et al . , and later by toth , et al . recently , 2 cases of laparoscopic pyelolithotomy have been reported in a pelvic kidney . in one , due to urine leakage , an indwelling stent and urethral catheter were maintained for 8 days . hospitalization lasted 6 days . in the second case report , intraoperative fluoroscopy and laparoscopic suturing were used to perform the procedure , but the pyelotomy closure was not tested for watertightness and the patient experienced peritoneal leakage of urine after the foley catheter was removed . this necessitated replacement of the foley catheter for an unspecified length of time and the length of hospitalization was not reported . herein we report the third case of a laparoscopic pyelolithotomy in a pelvic kidney ; in this case , use of the carterthomason needle - point suture passer ( inlet medical inc . , eden prairie , mn ) and laparoscopic suturing of the pyelotomy with an endostitch device ( auto suture , norwalk , ct ) resulted in a waterproof closure . a 63-year old woman was referred for endourological treatment of a symptomatic 2.5-cm - x-1.5 cm renal pelvis stone in her left pelvic kidney . intravenous urography ( ivu ) and abdominal / pelvic computed tomography defined the pelvic kidney 's location relative to other structures ; there was mild pyelocaliectasis noted ( figures 1 & 2 ) . ivu demonstrating ectopic right kidney and left pelvic kidney with laminated 2.5 cm stone at the ureteropelvic junction . the patient was turned to a 30-degree lateral , left side up position and a 15 mm hg co2 pneumoperitoneum was initiated with the veress needle using a lateral inflation technique . three 12 mm laparoscopic ports were placed : left lower quadrant in the midclavicular line , umbilicus , and right lower quadrant in the pararectus area . holding sutures were placed inferiorly and superiorly on the anterior surface of the renal pelvis using intracorporeal suturing ; suture delivery through the skin was accomplished via a carter - thomason needle - point suture passer ( inlet medical inc . , eden prairie , mn ) . a 3 cm long pyelotomy was made and a 10 mm biopsy forceps was used to grasp the stone , which was removed intact after the 12 mm cannula was withdrawn from the port site . a running closure of the renal pelvis was accomplished using the endostitch device with a 2 - 0 polysorb suture ( auto suture , norwalk , ct ) . the occlusion balloon catheter was changed to an indwelling 7 f double pigtail stent and a bladder catheter was left in place overnight . the total operative time was 310 minutes ( including pre- and postoperative stent placement ) . abdominal/ pelvic ct scan demonstrating left pelvic kidney with mild pyelocaliectasis and the stone at the ureteropelvic junction ( arrow at stone ) . after initiating general anesthesia , a 7 f/ 11.5 mm balloon occlusion catheter was then passed over the guidewire into the renal pelvis of the left pelvic kidney . the balloon was inflated with 1 cc of contrast material and was then snugged down at the ureteropelvic junction . postoperatively , the patient 's discomfort was managed by a total of 45 mg of intravenous ketorolac ( 3 doses ) followed by 2 doses of an oral narcotic . a follow - up ivu , one week later , revealed the left pelvic kidney to be functional , stone - free , and without extravasation . our experience ( table 1 ) is similar to the prior reports of successful laparoscopic pyelolithotomy for a large renal calculus in a pelvic kidney . placement of a retrograde ureteral catheter was instrumental in allowing expeditious localization of the renal pelvis at laparoscopy and for testing the closure of the pyelotomy . closure of the renal pelvis was facilitated by the use of holding sutures and the endostitch device . comparison of laparoscopic pyelolithotomy and pcnl for renal pelvis stones > 1.5 - 2 cm . mac= monitored anesthesia care , pcnu= percutaneous nephroureteral stent the patient experienced minimal pain and a brief hospitalization , findings consistent with previous reports of laparoscopic pyelolithotomy for a normally positioned kidney . in these reports , the operative time has averaged 2 - 5 hours using a 3- or 4-port approach . the renal pelvis was sutured closed in only one case and all patients had a surgical drain placed prior to fascial closure . while a stone - free rate of 100% was recorded among 9 patients , it is of note that 3 of those 9 patients required conversion to an open procedure . while these reports concluded that laparoscopic pyelolithotomy in a eutopic kidney was of value in a situation where shock wave lithotripsy ( swl ) or pcnl had failed or could not be done , we believe this represents a rare situation . indeed , despite a vast experience at washington university with laparoscopic renal surgery and a large surgical stone population ( e.g. an approximate annual urolithiasis case load of 500 to 600 swl , 100 to 150 ureteroscopies , and 50 to 100 pcnl ) this is the singular case of a laparoscopic pyelolithotomy at our institution . with regard to stone treatment in an ectopic or pelvic kidney , we believe the treatment strategy should be similar to that used for stones in a eutopic kidney . hence , swl or ureteroscopy remain first line therapy for smaller calculi ( less than 2 cm ) , while laparoscopic - assisted percutaneous , pure laparoscopic , or open procedures are reserved for larger calculi . from this standpoint , it is of note that 2 cases of laparoscopically guided pcnl have been reported in pelvic kidneys . the operative time was not reported for these cases and the length of hospital stay was 6 days in one report . we believe that given advances in laparoscopic equipment it may be just as simple to handle the stone removal entirely laparoscopically . the benefits of this approach would potentially include a shorter hospital stay and less postoperative morbidity as occurred in the present case . for urologists interested in laparoscopic pyelolithotomy in this situation , the use of intraoperative fluoroscopy to identify the renal pelvis , the placement of holding sutures , the use of the carter - thomason needle - point suture passer , and intracorporeal suturing with the endostitch device are recommended .

the anatomical characteristics of an ectopic kidney can pose significant challenges to the treatment of calculous disease . this report examines the feasibility of a laparoscopic approach for management of a large renal calculous in a pelvic kidney and reviews current literature regarding this subject .

Background and Objectives: Methods and Results: Conclusions: INTRODUCTION CASE REPORT DISCUSSION

PMC5376308

malaria , which is caused by a protozoan parasite of the plasmodium genus , represents a serious global health concern given that nearly half of the world population is at risk of infection ( who , 2014 ) . although several anti - malarial drugs for treating the symptomatic stage ( or blood stage ) of malarial infection are commercially available ( antony and parija , 2016 ) , their efficacy has declined appreciably in the last few decades owing to widespread drug resistance developed by the parasite ( breman et al . , chloroquine was the first - line malaria treatment for many decades until drug - resistant p. falciparum strains became common . the drug causes a dose - dependent decrease in hemozoin formation ( chou and fitch , 1992 , slater and cerami , 1992 ) and an associated increase in toxic free heme in the food vacuole of the parasite ( combrinck et al . , 2013 , loria et al . , 1999 ) . over the past few decades , researchers have proposed many different mechanisms for chloroquine action , including 1 ) dna intercalation ( meshnick , 1990 ) , 2 ) alteration of digestive food vacuole ph ( yayon et al . , 1985 ) , 3 ) inhibition of heme polymerase ( yayon et al . , 1985 ) , and 4 ) formation of a toxic chloroquine - ferriprotoporphyrin ix complex ( sugioka et al . , yet , there is no consensus on the exact mechanisms by which chloroquine kills the parasite and under what circumstances they operate . shedding light on the possible actions of chloroquine is critical for developing more potent drugs or combination drug treatments against the parasite and for understanding how the parasite can develop resistance against them . here we used systems biology model and transcriptional profiles of p. falciparum to obtain information about metabolic and biological precursors that determine the physiological or pathophysiological state of the parasite . specifically , we used the p. falciparum transcriptomic data obtained by hu and colleagues during the intraerythrocytic development cycle ( idc ) at different concentrations of chloroquine ( hu et al . , 2010 ) . our primary goal was to use these data to investigate the effect of chloroquine on p. falciparum dna replication . we chose to study this effect because it has been proposed as a mechanism of chloroquine action in different plasmodium species ( gutteridge et al . , 1972 , meshnick , 1990 , polet and barr , 1968a , polet and barr , 1968b ) , in bacteria ( ciak and hahn , 1966 ) , and in bioassays examining the effect of chloroquine on rna and dna polymerases ( cohen and yielding , 1965 , o'brien et al . we first developed a method that integrates the information embedded in the transcriptome data with the whole - genome metabolic network model for p. falciparum ( fang et al . , 2014 ) to predict the metabolic phenotype corresponding to those transcription data . we validated the developed method by using a data set from an independent study that jointly capture transcriptomic and metabolomic data from p. falciparum , to correctly predict the metabolic phenotype tied to genetic perturbation of two krebs cycle enzymes ( ke et al . , 2015 ) . we then focused on the inhibition of dna replication in p. falciparum ( as a consequence of chloroquine treatment ) a critical metabolic phenotype ( ciak and hahn , 1966 , cohen and yielding , 1965 , polet and barr , 1968a ) , and used our method to simulate the effect of chloroquine on the metabolic fluxes of p. falciparum during the idc . we identified genes that were substantially altered in response to chloroquine treatment and linked to the inhibition of p. falciparum dna replication . the cohort of identified genes suggests that dna replication is inhibited by the downstream effect of heme accumulation . specifically , our analysis suggests that continuous accumulation of heme inhibits redox metabolism , carbon fixation , and pyrimidine metabolism , which leads to inhibition of dna replication and facilitates death of the parasite . our results provide a mechanistic explanation for why parasites with an efficient redox metabolism may have a lower sensitivity to chloroquine ( kasozi et al . , 2013 , lehane et al . , 2012 , metabolic flux profiles of p. falciparum strains 3d7 ( pf3d7 ) and dd2 ( pfdd2 ) during the idc were estimated by using transcriptome data previously reported by llinas and colleagues ( llinas et al . , 2006 ) , who extracted cdna from parasites cultured in erythrocytes and hybridized them to dna microarrays to yield hourly levels of gene expression for the pf3d7 ( or pfdd2 ) strain during the idc . the temporal resolution of these hourly gene expression profiles allowed us to make high - resolution time - dependent metabolic flux predictions during the idc . we used previously reported gene expression data ( hu et al . , 2010 ) to simulate the effect of chloroquine on the time - dependent metabolic fluxes of pf3d7 and pfdd2 during the idc . briefly , hu and colleagues ( hu et al . , 2010 ) administered chloroquine 18 h after erythrocyte invasion at concentrations of 41 , 72 , and 144 nm for pf3d7 and at 43 nm for pfdd2 , and obtained gene expression profiles every hour for 8 h during the idc following the chloroquine treatment . the model was identical to a previously published model ( fang et al . , 2014 ) , with one minor modification : we added reactions explicitly carried out by pdx2 ( pf11_0169 ) and pdx1 ( mal6p1.215 ) . this was done by 1 ) modifying an old reaction that incorrectly assumed that pyridoxal 5-phosphate ( plp ) was synthesized from ribulose 5-phosphate and 2 ) adding a new ( and separate ) reaction for pdx1 because experiments suggest that it can synthesize plp independent of the ammonia generated by pdx2 ( hanes et al . , 2008 ) . in the updated model , pdx2 hydrolyzes l - glutamine to yield glutamate and ammonia while pdx1 incorporates the ammonia generated by pdx2 to yield plp from the substrates ribose 5-phosphate and glyceraldehyde 3-phosphate ( gengenbacher et al . , 2006 ) , with the isomerization of ribose 5-phosphate to ribulose 5-phosphate as a side reaction . we used gene - to - reaction mappings available in the metabolic network model to obtain reaction expressions rexp , which depend on the expression of a gene ( or genes ) catalyzing a particular metabolic reaction . for a reaction catalyzed by a single enzyme , rexp was equal to the expression of the gene catalyzing that reaction however , if a reaction was associated with more than one gene , then their gene - to - reaction mappings were defined by using the boolean operators and and or . we implemented these operations by taking the minimal ( for the and operator ) and maximal ( for the or operator ) values of the associated gene expression data ( song et al . , 2014 ) . , 2014 ) that integrates hourly gene expression data with a whole - genome metabolic network model to estimate the hourly metabolic flux profiles of p. falciparum during the 48-h long idc period . to take advantage of additional studies that collect gene expression data as a function of genetic or chemical perturbations , we modified the method because such studies usually do not provide the same granularity and density of gene expression data to fully cover the entire idc . therefore , we aimed to develop a method that could utilize a reduced transcriptomic data set to predict alterations in metabolic flux profiles during the idc . briefly , we used the following steps to estimate metabolic fluxes under no - stress conditions : step 1 : estimate nutrient fluxes sufficient for the nominal growth rate ( 40% of the maximum growth rate).step 2 : estimate reaction fluxes vin for metabolite i corresponding to the nominal growth rate constrained by the nutrient fluxes obtained in step 1.step 3 : estimate reaction fluxes vit for reaction i at any time point t during the idc by incorporating time - dependent transcriptomic profiles under stoichiometric and nutrient uptake constraints . step 1 : estimate nutrient fluxes sufficient for the nominal growth rate ( 40% of the maximum growth rate ) . step 2 : estimate reaction fluxes vin for metabolite i corresponding to the nominal growth rate constrained by the nutrient fluxes obtained in step 1 . step 3 : estimate reaction fluxes vit for reaction i at any time point t during the idc by incorporating time - dependent transcriptomic profiles under stoichiometric and nutrient uptake constraints . the last step was achieved by globally minimizing for all reactions and time points the value of t that minimized the weighted absolute difference , ig|vitritvin| , where g represents the set of unidirectional reactions with known gene - to - reaction mapping . the factor rit = rexp , itmin(rexp , it)mean(rexp , it)min(rexp , it ) ; where , rexp , it represents reaction expression of ith reaction at time t obtained from the gene - to - reaction mapping of the p. falciparum metabolic network , according to the approach outlined in section 2.2 . this factor , rit , ensured that the mean of the ith reaction during the idc was equal to vin . , we estimated the organism - level impact of a drug on metabolism by using transcriptomic data obtained after drug treatment . the original formulation outlined above ( steps 13 ) can not be directly translated to stress conditions unless the complete transcriptomic profile for each stress condition during the entire idc is available ( fang et al . , 2014 ) . therefore , we introduced a parameter i to account for up- or down - regulation of individual reactions in response to stress to simulate the effect of exogenous stress on metabolic flux profiles during the idc . we modified the factor rit to iritmin(irit)mean(rit)min(irit ) , where i denotes the median relative change in the reaction expression of the ith reaction following chloroquine treatment , as observed in previous experiments ( hu et al . , 2010 ) . note that the mean of each individual modifying factor rit , i.e. , 1nj=1tnrij , obtained by summing the values across all time points in the idc , tracks with i ; that is , when the mean is less than one , greater than one , or equal to one , i<1 , i>1 , or i=1 , respectively . in other words , i increases or decreases the nominal flux of the ith reaction during the entire idc depending on the relative increase or decrease in the ith reaction expression . in principle , a drug may differentially affect gene transcription at different time points during the idc . here , we assumed that the median change in gene transcription after a stress treatment was representative of the transcriptional response throughout the idc . we used a previously published data set ( ke et al . , 2015 ) to validate our methodology to simulate the effect of external stress on metabolic fluxes during the idc . ke and colleagues ( ke et al . , 2015 ) constructed a p. falciparum mutant with two tricarboxylic acid ( tca ) cycle enzymes deleted ( idh/kdh ) , cultured wild - type and mutant parasites in erythrocytes , and obtained matched transcriptomic and metabolomic data . we used this data set because it describes changes in gene expression ( transcriptomics ) and phenotype ( metabolomics ) under the same experimental stress conditions . if the reaction fluxes estimated from previously reported transcriptome data ( ke et al . , 2015 ) adequately represent the reaction fluxes in vitro , then the predicted changes in reaction fluxes should be sufficient to predict the changes observed in the corresponding metabolomic data . we used a computer model of mitochondrial oxidative phosphorylation and the tca cycle ( wu et al . , 2007 ) to predict changes in tca cycle metabolites in response to changes in these estimated reaction fluxes ( ke et al . the kinetic parameters of this computer model were determined and validated by using extensive experimental data . we simulated wild - type reactions by running this model , with its default parameters , for 2000 s ( time required to reach steady - state condition ) and stored the concentrations of tca cycle metabolites . we performed simulations for the mutant parasite in the same way , albeit after scaling the model parameters that govern tca cycle enzyme activities by the estimated increase or decrease in reaction fluxes of those enzymes . we used a freely available software package called bisen ( vanlier et al . , 2009 ) to implement the tca cycle computer model ( wu et al . , 2007 ) . p. falciparum takes up nutrients to synthesize dna , rna , proteins , and phospholipids . these processes are not constitutively activated , because the parasite will synthesize them as needed throughout the idc . we used a validated in - house developed method ( fang et al . , 2014 ) to simulate time - dependent dna synthesis during the idc . for comprehensiveness , we introduce the constraint governing the rate of dna synthesis in the metabolic network model : cdatpdatp+cdctpdctp+cdgtpdgtp+cdttpdttpdna+(cdatp+cdctp+cdgtp+cdttp)diphosphate . here , datp , dctp , dgtp , and dttp refer to deoxyadenosine , deoxycytidine , deoxyguanosine , and deoxythymidine triphosphates , respectively . we refer the reader to the original article ( fang et al . , 2014 ) for further details on this method for simulating time - dependent p. falciparum macro - molecules during the idc . the criticality of a gene is determined by optimizing the following objective function : idnacimi , subjected to : smxnvnx1=0 and vlbvvub . here , dna is the gene set containing the metabolites that contribute to dna synthesis ( introduced above ) and ci is the contribution of the ith metabolite ( mi ) . in addition , s is the stoichiometry matrix , v is a vector containing all metabolic reactions of the p. falciparum metabolic network model , m is the number of metabolites in the network , n is the number of reactions in the network , and vlb and vub are the upper and lower bounds , respectively , of metabolic reactions . we deemed a reaction as critical if there was no dna synthesis after knocking out the reaction in silico . chloroquine administration inhibits dna replication in malaria parasites ( cohen and yielding , 1965 , polet and barr , 1968a ) . 2010 ) also show that metabolic genes critical for p. falciparum dna synthesis are suppressed by chloroquine treatment . we sought to identify the genes that are substantially altered in response to chloroquine and cause inhibition of p. falciparum dna synthesis . obvious candidates are genes that are down - regulated and critical for p. falciparum dna synthesis . however , not all such genes will have a pronounced effect on dna synthesis , which depends on the extent to which an individual gene is down - regulated and/or the individual contribution of that gene to the dna synthesis of the parasite . this suggests that many combinations of such genes may underlie the metabolic response associated with chloroquine treatment . , 1990 ) , we aimed at identifying the minimal number of genes ( hereinafter referred to as the core genes ) that are sufficient to completely account for the effect of chloroquine on dna synthesis by the parasite . we identified the core genes based on the bayesian information criteria ( bic ) score ( schwarz , 1978 ) , which is given by , nln(ssen)+kln(n ) , where sse=i=1n(victlvicqn)2max(vctl)min(vctl ) and n denotes the number of time points , k the number of genes being tested , vctl the estimated rate of dna synthesis under control conditions , and vcqn the estimated rate of dna synthesis after chloroquine treatment ( with =1 for the k genes being tested ) . we implemented an algorithm that starts with a set of size k=1 and increases the size of the test set as long as a given combination of genes for the new set ( size k+1 ) has a bic score lower than the minimal bic score of the previous size . we exhaustively repeated this process at each chloroquine dose ( 41 , 72 , and 144 nm for pf3d7 , and 43 nm for pfdd2 ) . we modified our previous approach ( fang et al . , 2014 ) to simulate the effect of external stress , as depicted in fig . 1 and described in the materials and methods section . we validated the methodology by demonstrating that reaction fluxes obtained in response to stress were sufficient to predict metabolic phenotypes corresponding to that stress . 2a shows the biochemical pathways incorporated into the computer model used to predict the functional consequences of altered fluxes of tca cycle enzymes in response to previously studied genetic perturbations ( ke et al . , 2015 ) . 2b shows the predicted alterations in tca cycle metabolites in response to genetic perturbations of tca cycle enzymes ( fig . 2a , red circles ) . we calculated the altered metabolite concentrations by scaling the control parameters in the tca cycle model ( wu et al . , 2007 ) by the median of the predicted changes in tca cycle enzyme activities ( fig . 2c ) . 2b , filled bars ) qualitatively captured the trends of relative change in experimentally measured tca cycle metabolites ( fig . 2b , open bars ) . 2b , filled bars ) was not within the margin of error of the experimental measurements ( fig . 2b , open bars ) . we examined all possible enzymes / transporters ( e.g. , citrate synthase , pyruvate dehydrogenase , aspartate aminotransferase , citrate - malate antiporter ) whose perturbation could yield mitochondrial citrate levels higher than those in the wild - type system . in each scenario , either the predicted citrate levels did not increase or the predicted oxaloacetate levels increased . therefore , based on our model analysis , the source of this increase in the experimentally reported citrate level appears to be non - mitochondrial in nature . in agreement with previous experimental observations ( cohen and yielding , 1965 , polet and barr , 1968a ) , our simulations suggested that chloroquine treatment results in dose - dependent inhibition of dna replication ( fig . a , blue diamonds ) and 43 nm ( pfdd2 , panel b , blue diamonds ) , the rate of dna synthesis during the ring stage ( the first 18 h after infection ) was less inhibited than it was during the late trophozoite and early schizont phases ( beyond 30 h after infection ) . these results are in agreement with previous observations ( yayon et al . , 1983 ) . because many studies suggest that pfdd2 is less sensitive to chloroquine than pf3d7 ( moneriz et al . , 2011 ) , dna synthesis might have been expected to be more inhibited in pf3d7 than in pfdd2 . in our simulations , however , a similar concentration of chloroquine resulted in greater dna synthesis inhibition in pfdd2 ( fig . 3b ; blue diamonds versus black circles , paired - sample t - test ; p < 0.0001 ) than in pf3d7 ( fig . 3a ; blue diamonds versus black circles , paired - sample t - test ; p = 0.15 ) . these differences likely stem from differences in the relative gene expression ratios of pfdd2 and pf3d7 . notably , the transcriptional profile for pfdd2 was more down - regulated than that for pf3d7 at a similar concentration of chloroquine . in other words , the percentage of down - regulated genes critical for p. falciparum dna synthesis was greater in pfdd2 than in pf3d7 ( table s1 ) . together with the highly similar ic50 values reported previously ( hu et al . , 2010 ) for pf3d7 ( 41 nm ) and pfdd2 ( 43 nm ) , these results suggest that the pfdd2 strain used here may have diverged from its true ( chloroquine - resistant ) phenotype . we found that 81 metabolic reactions in our network model are critical for p. falciparum dna synthesis . we denoted these reactions as critical because their elimination resulted in abolished dna synthesis ( see section 2.6 ) . of these 81 reactions , 64 had a known gene association whereas 17 did not . these genes belonged to pathways involved in glycolysis , pentose phosphate synthesis , pyruvate metabolism , purine metabolism , pyrimidine metabolism , redox metabolism , and mitochondrial metabolism . in the online supplement ( see spreadsheet ) , we provide detailed information , including the reaction name , chemical reaction , gene association , and pathway , of all reactions critical for p. falciparum dna synthesis . experimental observations ( cohen and yielding , 1965 , polet and barr , 1968a ) and the present simulations ( fig . 3 ) indicate that chloroquine treatment inhibits replication of p. falciparum dna , which in turn suggests that pathways essential for dna synthesis are inhibited . 4 shows the relative change in the amount of dna synthesized by the parasite during the idc period in response to chloroquine treatment . table s2 lists the actual amounts of synthesized dna for both strains under each simulated condition . we observed a dose - dependent decrease in the amount of dna synthesized by the parasite during the idc ( fig . 4 , red markers ) . we used bic scores ( schwarz , 1978 ) and employed the algorithm described in section 2.7 to identify core genes that caused this net decrease of synthesized dna . preventing only the core genes from being down - regulated by chloroquine was sufficient to restore the amount of dna synthesized to control values ( fig . 4 , green markers ) ; for these simulations , the reaction expressions of the core genes were set to be equal for control and chloroquine - treated conditions . a list of the core genes identified for each strain and chloroquine dose ( table s3 ) suggests that thioredoxin reductase and ribonucleotide reductase substantially affect the amount of synthesized dna . for pfdd2 , only two genes ( thioredoxin reductase and ribonucleotide reductase ) caused the nearly 50% reduction in the net amount of synthesized dna ( table s3 and fig . 4 ) . these genes were not mentioned in the original transcriptome analysis of hu and colleagues , who used a fundamentally different approach to interpret their transcriptome data ( hu et al . , 2010 ) . these authors focused on genes that were either co - regulated or showed strongly differential expression ( 8 log2-fold change ) in response to a drug . in contrast , our methodology of coupling transcriptomic changes with a metabolic network model can predict physiological outcomes as they relate to parasite metabolism , regardless of the magnitude of transcriptional change for an individual gene . transcriptional changes are not proportional to changes in metabolic reaction fluxes ( moxley et al . , 2009 ) . our analysis suggests that low - amplitude alterations in these identified genes ( table s3 ) were sufficient to cause a significant reduction in parasite dna synthesis . several existing methods integrate whole - genome metabolic networks and transcriptome data ( colijn et al . , 2009 , song et al . , 2014 , recently , we developed a method that predicts metabolic flux profiles of p. falciparum during the idc ( fang et al . the transcriptome data of p. falciparum obtained under different stress conditions ( e.g. , drug exposure , genetic mutation , etc . ) contain information about its functional state and can be used to predict qualitative alterations , if any , in metabolic flux profiles during the idc . however , such data are often collected at a limited number of time points ; for instance , hu and colleagues ( hu et al . , 2010 ) obtained p. falciparum transcriptome data at only 8 selected time points during the idc . currently , there is no approach to integrate limited data , such as those reported previously ( hu et al . , 2010 ) , with a whole - genome network to predict metabolic flux profiles during the idc . this is mainly because such an approach requires gene expression data throughout the idc to predict the corresponding metabolic flux profiles . therefore , we developed an approach that uses gene expression data obtained under a stress condition to predict alterations of metabolic fluxes that correspond to that condition . we validated the developed method by integrating transcriptome data obtained under a given stress condition and predicting metabolomic data corresponding to that stress ( ke et al . , , we simulated the effect of chloroquine on the metabolic flux profiles of p. falciparum during the idc . chloroquine inhibits the formation of hemozoin and leads to heme accumulation , which is toxic to the parasite ( fitch , 1998 ) . however , the specific mechanism underlying this toxicity is not known ( ginsburg et al . , 1998 , some experiments suggest inhibition of dna replication as a potential mechanism of chloroquine action ( cohen and yielding , 1965 , kwakye - berko and meshnick , 1989 , polet and barr , 1968a ) . we developed a method that successfully predicted a reduction in the ability of the parasite to synthesize dna in response to chloroquine treatment ( see fig . we identified twelve distinct genes that were substantially affected in pf3d7 and pfdd2 after chloroquine treatment ; importantly , preventing their down - regulation in response to chloroquine treatment was sufficient for restoring the p. falciparum dna synthesis inhibited by chloroquine treatment ( see table 1 ) . these genes belonged to pathways involved in glycolysis , redox metabolism , and pyrimidine metabolism . a closer look at these genes reveals a potential mechanism of chloroquine action that unifies previous theories and observations involving heme ( fitch , 1998 ) , dna replication ( polet and barr , 1968a ) , and oxidative stress ( lehane et al . , 2012 , radfar et al . , 5 , suggests that administration of chloroquine has two major metabolic effects : i.it interferes with the function of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , which reduces the synthesis of phosphoenolpyruvate , whose carbon fixation activity is essential for anaplerotic tca metabolism and redox balance ( storm et al . , 2014).ii.its interference of thioredoxin reductase ( trxr ) reduces conversion of oxidized thioredoxin trx ( s2 ) to reduced thioredoxin trx ( sh2 ) , which is essential for donating electrons to reactions catalyzed by thioredoxin peroxidase ( tpx ) and ribonucleotide reductase ( rnr ) . tpx is essential for converting h2o2 ( reactive ) to h2o ( harmless ) ( pannala and dash , 2015 ) , and rnr for converting ribonucleoside 5-diphosphates species ( adp , udp , gdp and cdp ) , to deoxy - species ( dadp , dudp , dgdp and dcdp ) which are used for dna synthesis ( munro and silva , 2012 ) . it interferes with the function of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , which reduces the synthesis of phosphoenolpyruvate , whose carbon fixation activity is essential for anaplerotic tca metabolism and redox balance ( storm et al . , 2014 ) . its interference of thioredoxin reductase ( trxr ) reduces conversion of oxidized thioredoxin trx ( s2 ) to reduced thioredoxin trx ( sh2 ) , which is essential for donating electrons to reactions catalyzed by thioredoxin peroxidase ( tpx ) and ribonucleotide reductase ( rnr ) . tpx is essential for converting h2o2 ( reactive ) to h2o ( harmless ) ( pannala and dash , 2015 ) , and rnr for converting ribonucleoside 5-diphosphates species ( adp , udp , gdp and cdp ) , to deoxy - species ( dadp , dudp , dgdp and dcdp ) which are used for dna synthesis ( munro and silva , 2012 ) . for example , our analysis points to trxr and gapdh as candidate molecules that determine the efficacy of chloroquine . here , we focused on candidates susceptible to a low dose of chloroquine because several pathways will be inhibited at higher concentrations ( see table s3 ; chloroquine = 144 nm ) . in addition , proteins that transport chloroquine out of food vacuoles should decrease the effective concentration of chloroquine . targeting these pathways while administering chloroquine treatment should enhance the efficacy of chloroquine at a given effective concentration . the hypothesis that chloroquine kills the malarial parasite by dna intercalation is decades old ( meshnick , 1990 ) . although it was the prime candidate mechanism of chloroquine from the 1950s to the 1970s , it has now largely been abandoned for the past two decades . our model analyses do not identify a drug target or targets ; however , they suggest that the effect of chloroquine administration leads to inhibition of dna synthesis . according to our model , the parasite responds to chloroquine by reducing the expression of genes critical for dna synthesis . although chloroquine could specifically intercalate with the identified core gene set in accord with the dna intercalation theory of chloroquine ( meshnick , 1990 , o'brien et al . , 1966a heme is a more plausible molecular inhibitor , given that it accumulates in large amounts within the parasite 's food vacuole and is capable of causing dna damage ( ishikawa et al . , 2010 , rahman et al . , 1997 ) , protein oxidation ( kitatsuji et al . , 2016 ) , nadph oxidation ( bodaness , 1983 ) , and apoptosis ( chiabrando et al . , 2014 ) . however , there is no direct evidence suggesting that heme causes any of these damages in p. falciparum . chloroquine , which was once a highly effective drug , is now abandoned in many malaria endemic regions because of widespread resistance . knowledge of this mechanism allowed us to identify metabolic pathways that could be exploited to increase chloroquine efficacy . for example , our model analyses suggest that administration of h2o2 would potentiate parasite killing by chloroquine , consistent with in vitro experiments ( malhotra et al . , similarly , our analyses suggest the hypothesis that targeting parasite - specific pathways that synthesize phosphoenolpyruvate and reduce thioredoxin would potentiate parasite killing . targeting these pathways , while administering amodiaquine ( a chloroquine analogue ) , should result in higher efficacy than administering chloroquine , especially because amodiaquine accumulates more than chloroquine in the parasite s food vacuole and has a mechanism similar to that of chloroquine ( hawley et al . , 1996 ) . our aim was to identify and investigate enzymes that contribute to decreased dna replication in response to chloroquine treatment . using a transcriptomics - based approach , we can identify cellular responses that are specific to chloroquine by expression data taken from chloroquine - treated parasites . although other antimalarial drugs may activate similar stress pathways , the details of the response will be specific to the particular conditions . the approach makes no assumptions on the underlying molecular actions that cause or affect the response . the effect of chloroquine on dna replication is likely to be indirect , being mediated by heme accumulation , which evokes multiple toxic effects in the cell . we simulated the effect of chloroquine over the entire idc based on the available transcriptome data taken from an eight - hour window during the trophozoite stage . ideally , for any specific inhibitor , it is desirable to have hourly transcriptional readouts to predict the effect of a systemic perturbation throughout the entire idc . in the case presented here , the largest effect of chloroquine occurs during the trophozoite stage and the protocol for collecting experimental transcriptomic data was designed to characterize transcriptional changes during this stage . our use of these data to identify changes in metabolite fluxes throughout the idc relative to untreated controls rests on the assumption that altering the expression of genes not associated with any biological function specific to a particular stage will have minimal physiological effects at other stages . this is an assumption that is specific to p. falciparum , because malaria relies on the execution of a highly regimented transcriptional program during the idc , which initiates specific stage - dependent cellular functions . these assumptions were ultimately corroborated because the observed metabolite phenotype changes are mainly associated with the trophozoite stage and metabolic alterations during previous time points were non - existent or immaterial to dna synthesis . herein , we investigated the potential mechanism by which chloroquine inhibits dna synthesis in p. falciparum and kills the parasite . we first developed an approach ( that requires limited transcriptome data ) to simulate the effect of exogenous stress on p. falciparum metabolic fluxes during the idc . we then developed an algorithm employing bayesian information theory to identify potential pathways that inhibit dna synthesis in response to chloroquine treatment . our analysis suggests that 1 ) chloroquine inhibits dna synthesis via inhibition of redox metabolism , carbon fixation , and anaplerotic tca cycle metabolism ; 2 ) p. falciparum may maintain dna synthesis after chloroquine treatment by preserving a reduced pool of thioredoxin and by invoking alternate mechanisms for synthesizing phosphoenolpyruvate , and 3 ) a drug or combination of drugs that target redox metabolism , carbon fixation , and heme accumulation could be an effective way to target the parasite . the method developed here used limited transcriptome data to identify pathways that underlie the inhibition of dna synthesis of p. falciparum in response to chloroquine treatment . this suggests that the developed method could also be suitable to simulate and capture the effect of other drugs ( or stress conditions ) on complex metabolic and physiological responses of p. falciparum , based solely on transcriptome data .

chloroquine , long the default first - line treatment against malaria , is now abandoned in large parts of the world because of widespread drug - resistance in plasmodium falciparum . in spite of its importance as a cost - effective and efficient drug , a coherent understanding of the cellular mechanisms affected by chloroquine and how they influence the fitness and survival of the parasite remains elusive . here , we used a systems biology approach to integrate genome - scale transcriptomics to map out the effects of chloroquine , identify targeted metabolic pathways , and translate these findings into mechanistic insights . specifically , we first developed a method that integrates transcriptomic and metabolomic data , which we independently validated against a recently published set of such data for krebs - cycle mutants of p. falciparum . we then used the method to calculate the effect of chloroquine treatment on the metabolic flux profiles of p. falciparum during the intraerythrocytic developmental cycle . the model predicted dose - dependent inhibition of dna replication , in agreement with earlier experimental results for both drug - sensitive and drug - resistant p. falciparum strains . our simulations also corroborated experimental findings that suggest differences in chloroquine sensitivity between ring- and schizont - stage p. falciparum . our analysis also suggests that metabolic fluxes that govern reduced thioredoxin and phosphoenolpyruvate synthesis are significantly decreased and are pivotal to chloroquine - based inhibition of p. falciparum dna replication . the consequences of impaired phosphoenolpyruvate synthesis and redox metabolism are reduced carbon fixation and increased oxidative stress , respectively , both of which eventually facilitate killing of the parasite . our analysis suggests that a combination of chloroquine ( or an analogue ) and another drug , which inhibits carbon fixation and/or increases oxidative stress , should increase the clearance of p. falciparum from the host system .

Introduction Materials and methods Results Discussion Limitations of the current study Conclusion

PMC3401724

situs inversus totalis , a congenital anomaly present in approximately 0.01% of the population , results in complete mirror - image reversal of all the thoracic and abdominal organs . the laparoscopic literature has several reports of successful bariatric procedures in patients with situs inversus despite the extremely low prevalence of this condition.[13 ] while several equivalent alternatives are available in the bariatric algorithm , more recently the laparoscopic sleeve gastrectomy ( sg ) has been gaining traction as an effective means of weight loss in patients with morbid obesity.[47 ] a 39-year - old woman who had previously undergone an open gastric banding procedure 20 months earlier for morbid obesity presented with increased weight gain . her weight at presentation was 106 kg ( body mass index 42 kg / m ) , a percentage excess weight loss ( ewl ) of only 1.9% . her medical history was significant for hypertension , noninsulin - dependent diabetes mellitus , hypothyroidism , obstructive sleep apnea , and situs inversus totalis . the presence of situs inversus was initially discovered during her first weight loss operation and confirmed by computed tomography of the abdomen [ figure 1 ] . the operating surgeon began the case laparoscopically , but upon recognizing the altered anatomy made the decision to convert to an open gastric band placement . given the failure of this first procedure to produce meaningful weight loss , the patient was scheduled for laparoscopic removal of the gastric band with concurrent sg . coronal computed tomographic scan of the abdomen and pelvis with oral and intravenous contrast demonstrating the mirror - image anatomy . panel a displays a cross - section of the band in its position on the right side of the abdomen . panel b further demonstrates the altered anatomy with left - sided liver and right - sided stomach the patient was positioned supine on the operating room table . secondary to the presence of situs inversus , the veress needle was placed in the right upper quadrant to avoid the liver . a 12-mm trocar was placed five fingerbreadths above the umbilicus , just to the right of midline . next , a 5-mm trocar was placed in the left lower quadrant approximately two fingerbreadths below the umbilicus in the midclavicular line . a 12-mm port was then placed under direct vision in the left midclavicular line approximately five fingerbreadths above the umbilicus . a 12-mm port was placed in the right upper quadrant in the midclavicular line , and a 5-mm port was placed in the right upper quadrant one fingerbreadth below the costal margin in the midaxillary line [ figure 2 ] . exploration revealed extensive adhesions as a result of her previous open procedures . at this point , the stomach was identified and noted to be severely adhered to the liver , which was , in turn , adherent to the abdominal wall . the stomach was mobilized , the band was identified , and the band buckle was cut and removed . the previous fundoplication was taken down using a 3.5-mm depth 45-mm length stapler , and the greater curvature and short gastric vessels were divided using a harmonic scalpel . after performing the dissection all the way to the angle of his and exposing the left crux of the diaphragm , a 34-french bougie was placed into the stomach with the tip of the bougie sitting in the proximal duodenum . the formation of the gastric sleeve with a 4.8-mm depth 45-mm length stapler was started from a point 5 cm proximal to the pylorus muscle and directed toward the angle of his [ figure 3 ] . eight total firings were used , thus dividing the stomach completely and forming a gastric tube over the 34-french bougie . at the most proximal aspect of the stomach , several dense adhesions to the diaphragm were encountered and eventually taken down . following the formation of the gastric tube , the staple line was oversewn with 2 - 0 absorbable suture using an endo stitch ( covidien inc . , after submerging the gastric tube underwater , air was pumped into the scope , and subsequently no leak was appreciated . before removal of the last trocar , the detached stomach was placed into a specimen retrieval bag and removed from the peritoneal cavity via the 12-mm port in the right upper quadrant . the same trocar site was slightly extended to allow for removal of the port and band . finally , a jackson pratt drain was placed by the sleeve for surveillance purposes . novel port placement ( copyright sages 2010 , soper nj , swanstrom ll , eubanks ws , eds . mastery of endoscopic and laparoscopic surgery ) intraoperative image ( white arrow indicates the angle of his ; blue arrows indicate several gastrohepatic adhesions , and black arrows indicate the greater curvature of the stomach ) the postoperative course was complicated by a leak resulting in a 3.9 2.8 cm collection in the gastrosplenic ligament adjacent to the surgical suture line [ figure 4 ] . a jackson pratt drain was left alongside the sg at the time of surgery as this is the surgeons protocol for revisional surgery . the patient underwent an upper endoscopy and placement of a 12-cm removable polyflex stent ( boston scientific inc . , natick , ma ) for management of the leak [ figure 5 ] . over the next few weeks further imaging demonstrated interval migration of the stent into the stomach with no change in the size or location of the perigastric collection [ figure 6 ] . the patient was managed conservatively despite the stent movement and had eventual resolution of her symptoms . the stent was subsequently removed without any complications . currently , the patient is 2 years out from the laparoscopic sg . postoperative upper gi series demonstrating extravasation of contrast at the most proximal aspect of the staple line completion radiograph following endoscopic placement of a removable stent across the anastamotic leak site coronal computed tomographic scan of the abdomen and pelvis with oral and intravenous contrast demonstrating complete migration of the stent into the body of the stomach secondary to the presence of situs inversus , the veress needle was placed in the right upper quadrant to avoid the liver . a 12-mm trocar was placed five fingerbreadths above the umbilicus , just to the right of midline . next , a 5-mm trocar was placed in the left lower quadrant approximately two fingerbreadths below the umbilicus in the midclavicular line . a 12-mm port was then placed under direct vision in the left midclavicular line approximately five fingerbreadths above the umbilicus . a 12-mm port was placed in the right upper quadrant in the midclavicular line , and a 5-mm port was placed in the right upper quadrant one fingerbreadth below the costal margin in the midaxillary line [ figure 2 ] . exploration revealed extensive adhesions as a result of her previous open procedures . at this point , the stomach was identified and noted to be severely adhered to the liver , which was , in turn , adherent to the abdominal wall . the stomach was mobilized , the band was identified , and the band buckle was cut and removed . the previous fundoplication was taken down using a 3.5-mm depth 45-mm length stapler , and the greater curvature and short gastric vessels were divided using a harmonic scalpel . after performing the dissection all the way to the angle of his and exposing the left crux of the diaphragm , a 34-french bougie was placed into the stomach with the tip of the bougie sitting in the proximal duodenum . the formation of the gastric sleeve with a 4.8-mm depth 45-mm length stapler was started from a point 5 cm proximal to the pylorus muscle and directed toward the angle of his [ figure 3 ] . eight total firings were used , thus dividing the stomach completely and forming a gastric tube over the 34-french bougie . at the most proximal aspect of the stomach , several dense adhesions to the diaphragm were encountered and eventually taken down . following the formation of the gastric tube , the staple line was oversewn with 2 - 0 absorbable suture using an endo stitch ( covidien inc . , after submerging the gastric tube underwater , air was pumped into the scope , and subsequently no leak was appreciated . before removal of the last trocar , the detached stomach was placed into a specimen retrieval bag and removed from the peritoneal cavity via the 12-mm port in the right upper quadrant . the same trocar site was slightly extended to allow for removal of the port and band . finally , a jackson pratt drain was placed by the sleeve for surveillance purposes . novel port placement ( copyright sages 2010 , soper nj , swanstrom ll , eubanks ws , eds . mastery of endoscopic and laparoscopic surgery ) intraoperative image ( white arrow indicates the angle of his ; blue arrows indicate several gastrohepatic adhesions , and black arrows indicate the greater curvature of the stomach ) the postoperative course was complicated by a leak resulting in a 3.9 2.8 cm collection in the gastrosplenic ligament adjacent to the surgical suture line [ figure 4 ] . a jackson pratt drain was left alongside the sg at the time of surgery as this is the surgeons protocol for revisional surgery . the patient underwent an upper endoscopy and placement of a 12-cm removable polyflex stent ( boston scientific inc . , natick , ma ) for management of the leak [ figure 5 ] . over the next few weeks further imaging demonstrated interval migration of the stent into the stomach with no change in the size or location of the perigastric collection [ figure 6 ] . the patient was managed conservatively despite the stent movement and had eventual resolution of her symptoms . postoperative upper gi series demonstrating extravasation of contrast at the most proximal aspect of the staple line completion radiograph following endoscopic placement of a removable stent across the anastamotic leak site coronal computed tomographic scan of the abdomen and pelvis with oral and intravenous contrast demonstrating complete migration of the stent into the body of the stomach to our knowledge , this is the first published report of a laparoscopic sg after open gastric banding in a patient with situs inversus totalis . however , modifications in the standard trocar placement can help make the procedure more technically feasible . awareness of the inherited condition before undertaking the operation allows for advanced planning and preparation . in this case , the history of prior open gastric banding made the operation especially tricky , primarily due to the numerous adhesions encountered . revisional surgery is always difficult , but when performed in a patient with situs inversus may be even more challenging . after encountering the initial disorientation , experienced we believe the postoperative leak occurred secondary to the difficult dissection around the gastric fundus , independent of the altered anatomy . these anastamotic leaks can be classified as early or late , depending on how soon postoperatively they occur . in the early period ( <3 days ) , many advocate primary repair . washout and drainage is also an excellent alternative in patients with severe inflammation , signs of sepsis , or hemodynamic instability . the endoscopic placement of polyester or nitinol covered stents is a fairly recent innovation , allowing patients to be maintained on oral enteral nutrition . there have been several reports in the literature supporting their use in both early and late leaks.[911 ] the accepted leak rate for sg is 1.6% at the esophagogastric junction and 0.8% at lower points along the staple line . laparoscopic vertical sg after open gastric banding in a patient with situs inversus can be performed safely by experienced laparoscopic surgeons . while our case yielded a good result , other similar cases are needed to establish the reproducibility and long - term efficacy of the procedure .

while several equivalent alternatives are available in the bariatric algorithm , more recently the laparoscopic sleeve gastrectomy ( sg ) has been gaining traction as an effective means of weight loss in patients with morbid obesity . we present the case of a 39-year - old woman with situs inversus totalis , who was taken to the operating room for laparoscopic sg . the patient had previously undergone a failed open gastric banding procedure 20 months earlier . awareness of the inherited condition before performing the operation allows for advanced planning and preparation . subsequent modifications to the standard trocar placement help make the procedure more technically feasible . to our knowledge , this is the first published report of a laparoscopic sg after open gastric banding in a patient with situs inversus totalis . after encountering the initial disorientation , we believe experienced laparoscopic surgeons can perform this procedure successfully and safely .

INTRODUCTION CASE REPORT Operation Postoperative DISCUSSION CONCLUSION

PMC3085755

current techniques in acl surgery have been associated with satisfactory long - term results in the majority of patients . however , there remains a considerable subset , up to 30% of patients , with unsatisfactory outcomes [ 2 , 7 ] . specifically , patients report problems relating to rotational instability and return to previous level of activity [ 3 , 10 ] . it has been suggested that a more anatomical approach to restore the original acl anatomy may benefit these patients [ 4 , 17 ] . some authors have advocated placing a single graft in a position closer to the oblique femoral attachment of the acl [ 14 , 15 ] . however , it is not possible to fully restore normal knee kinematics with a single graft , regardless of the position [ 13 , 16 ] . the double bundle reconstruction technique ( dbt ) for acl reconstruction aims at restoring the acl anatomy with its two bundles and is gaining popularity [ 11 , 17 ] . anatomic studies have demonstrated the presence of two functional bundles within the acl , the anteromedial ( am ) and the posterolateral ( pl ) bundle [ 1 , 8 ] . although it is somewhat of a simplification , the double bundle description of the acl is generally accepted as an anatomic model for understanding the complex structure and function of the ligament [ 6 , 9 ] . the am bundle often obscures the pl bundle , and it may appear that only one bundle is present without careful inspection . the goal of this study was to describe the presence of the double bundle structure from an arthroscopic point of view , and to evaluate the value of different portals in knee arthroscopy . we prospectively examined 60 knees during standard arthroscopy . in each knee , the double bundle acl structure was evaluated , along with the usefulnes of different portals for visualization . all knees that were included in the study had a previous x - ray and mri in order to rule out any significant changes to the bone , and to ensure that the acl was intact . all patients were less than 60 years and had no history of acl injury . surgical indications for the 60 total subjects examined included 31 cases treated for meniscal findings alone , 21 treated for articular cartilage findings alone , and 8 cases of a combined repair of meniscus and cartilage . there were 25 female and 35 male knees , 29 right knees , and 31 left knees included in the study . age distribution ranged between 16 and 60 , with an average age of 40.2 years . arthroscopy started with an anterolateral portal ( alp ) located just lateral to the patellar tendon using the inferior pole of the patella as a vertical landmark , and an anteromedial portal ( amp ) approximately 0.5 cm medial to the edge of the patella tendon , 1 cm superior to the joint line , and 1 cm inferior to the tip of the patella . for each knee , the acl anatomy and the visibility of the pl bundle through the alp and amp were evaluated with and without retraction of the am bundle according to the description of the two bundles by girgis et al . and arnoczky . gross biomechanics of the two bundles using a probe were also assessed . for statistical analysis with the arthroscope in the alp , we were able to distinguish the am and pl bundle in 17 cases ( 28.3% ) ( fig . 1 ) . in the remaining cases ( n = 43 , 71.7% ) , the pl bundle was obscured by the am bundle , and visualization was only possible with retraction of the am bundle with a probe ( fig . 2 ) . switching the arthroscope to the amp , differentiation of the am and pl bundle without using a probe to retract am was possible in 40 cases ( 66.7% ) ( fig . 3 ) . in the remaining cases ( n = 20 , 33.3% ) , visualization of the pl bundle was possible only after retraction of the am bundle ( table 1 ) . there was a statistically significant better visibility of the pl bundle using the amp ( p < 0.05).fig . 2anterolateral portal : pl bundle with retraction of am bundle , pl bundle loose with knee flexionfig . bundlepl bundleanterolateral portaln = 17 ( 28.3%)anteromedial portaln = 40 ( 66.7% ) * * p < 0.05 anterolateral portal : pl bundle without retraction of am bundle anterolateral portal : pl bundle with retraction of am bundle , pl bundle loose with knee flexion anteromedial portal : pl bundle without retraction of am bundle visualization of the pl bundle the femoral insertion site of the acl was semilunar at the inner surface of the lateral condyle . the centre of the pl bundle visualized more shallow than the centre of the am bundle with the knee held in 90 flexion , while both insertion sites were oriented horizontally ( fig . 4 ) . the pl bundle insertion was located at the posterolateral aspect of the tibial attachment with a close approximation of the pl bundle to the posterior root of the lateral meniscus . gross assessment using a probe while applying a flexion - extension motion pattern to the knee demonstrated a taut am bundle throughout the range of motion and a tightening of the pl bundle with knee - extension ( figs . 2 , 5 ) . pl bundle tight with knee extension anteromedial portal : pl bundle without retraction of am bundle anteromedial portal : pl bundle tight with knee extension the principal finding of the present study was that it is possible to distinguish the double bundle structure in acl anatomy as described by girgis et al . and arnoczky by applying a two portal knee arthroscopy technique . the visualization of the acl anatomy seems to be improved through the use of a medial portal . the anteromedial portal not only helps in visualization of the double bundle structure but also in distinguishing the am and pl portions of the femoral attachment sites . consistent with the literature , we found the pl bundle tightening when the knee is extended [ 6 , 12 ] . this study is limited by the fact that we observed only acl intact knees and did not consider cases with a torn acl , where the anatomic position of the attachment sites is sometimes obscured by the knee injury and bony changes that occur between injury and reconstruction . therefore , we stress the need for further arthroscopic evaluation of different portals in acl reconstruction . the clinical relevance of this study is that it can be beneficial to establish an additional medial working portal . the amp can be used for the arthroscope and a better visualization of the femoral insertion site of the acl at the inner surface of the lateral condyle and a precise femoral tunnel placement can be achieved . reported the use of an additional medial portal without any increase in morbidity to patients for acl surgery .

in order to describe the arthroscopic presence of the double bundle structure and to evaluate the value of different portals in knee arthroscopy , we assessed the am and pl bundle anatomy . we prospectively examined the knees of 60 patients undergoing arthroscopic surgery for pathology unrelated to the acl . arthroscopy was performed in a two portal technique using an anterolateral ( alp ) and an anteromedial ( amp ) portal . with the arthroscope in the alp , we could distinguish an am and pl bundle in 28% . switching the arthroscope to the amp , differentiation of the bundles was possible in 67% . in all remaining cases visualization of the pl bundle was possible after retraction of the am bundle . use of amp increased visualization of the pl bundle . it seems reasonable to perform arthroscopy for acl reconstruction with the arthroscope in the amp and to establish an additional medial working portal to increase the visualization of the femoral acl insertion sites for optimal femoral tunnel placement .

Introduction Materials and methods Results Discussion Conclusion

PMC4499988

abnormal knee kinematics are considered to be an important factor influencing the long - term outcomes after cruciate ligament injuries1,2,3 . in order to improve long - term outcomes of treatments for the patients with cruciate ligament injuries , therefore , it is important to precisely evaluate the kinematics of knees with cruciate ligament injuries . the motion of a knee joint with ligament injury is commonly evaluated by static methods such as the lachman test , posterior drawer test , valgus stress test , and arthrometric measurements , e.g. kt-1000 . however , patients with knee ligament deficiencies usually complain about their symptoms under dynamic conditions . therefore , there is a possibility that the instability of the knee joint evaluated in static conditions does not reflect the functional disabilities of its ligament injury1 , 3,4,5 . three - dimensional motion analysis using optical skin marker systems has been widely used as an evaluation method of body kinematics in dynamic conditions with muscular activations . this approach provides six - degrees - of - freedom motion of the kinematics of the knee during activities of daily living without restricted conditions in vivo . andriacchi et al . developed a point cluster technique ( pct ) , which employs an overabundance of markers ( a cluster ) placed on each segment to minimize the effects of skin movement artifacts6 . the pct can be extended to minimize skin movement artifact by optimal weighting of the markers according to their degree of deformation . the criteria of the normal range of three - dimensional knee kinematics during walking remain uncertain . side - to - side difference in the range of knee motion is widely used as a criterion of the normal range of knee motion1 . to detect an abnormal knee motion during walking , it is useful to determine the normal range of the side - to - side difference in knee kinematics during walking based on the data using the pct . to the best of our knowledge , however , no study has examined this ; therefore , the purpose of the present study was to determine the normal range of the side - to - side difference in knee kinematics using pct during walking based on three dimensional kinematics of the knee of healthy subjects . the study was approved by the research ethics committee of hakodate national college of technology and informed consent was obtained from each subject before the test . twenty - one healthy normal volunteers without knee pain or an episode of injury to the legs were recruited for the study . the subjects were 11 males and 10 females with an average age of 27.0 ( sd 7.2 ) years , and ranging from 21 to 47 years , and an average body mass index of 20.6 ( sd 2.4 ) kg / m , ranging from 16.8 to 27.7 kg / m . the subjects were tested bilaterally at a self - selected normal walking speed during their walking for 8 m after the first four strides . the subjects walked barefoot on a hard surface at an average walking speed of 1.25 m / s . the subjects took a rest before each trial until they had recovered from fatigue . the kinematic data were collected by a three - dimensional motion analysis system using four infrared light cameras ( rrorflex , qualisys ab inc . , ground reaction forces during the tests were also measured using two multi - component force plates ( or6 , advanced mechanical technology inc . , three - dimensional motion data were processed using qualisys track manager ( qualisys track manager 3d , qualisys ab inc . , force data were used to identify the time of heel strike in each gait cycle . a gait cycle was defined as the interval between heel strike to heel strike on the ipsilateral side . light - reflective markers were placed by physical therapists well - trained in the pct and body surface anatomy6 . kinematic measurements were made of 21 light - reflective markers arranged on two limb segments , creating separate cluster groups of 11 markers on the thigh and 10 markers on the shank . the pct employs an overabundance of markers ( a cluster ) placed on each segment to minimize the skin motion artifact due to segmental form change by muscle contraction and marker - oscillation6 . the pct is based on a cluster of points uniformly distributed on the limb segment . each point is assigned an arbitrary mass , which can be varied at each time step . the center of mass and the inertia tensor of the cluster of points are calculated as described below . the eigenvalues and the eigenvectors of the inertia tensor are the principal moments of inertia and the principle axes of the point cluster . the eigenvalues are invariant to movement if the segment is behaving as a rigid body . if the eigenvalues change from their value in the rest position , then the segment where the markers are placed has deviated from rigid body movement . the individual weighting is adjusted by redistributing the weight factors at each time step to minimize the eigenvalue changes . examined the pct using simulation trials with systematic and random components of deformation error introduced into marker position vectors7 . they also tested the pct in vivo with a subject fitted with an external fixation device ( ilizarov ) . the average location and orientation measurement errors of the pct were 0.6 mm ( sd 0.6 mm , maximum approximately 3 mm ) and 0.5 degrees ( sd 0.5 degrees , maximum approximately 4 degrees ) respectively7 . another study has also reported an accuracy of 0.79 degrees ( sd 0.48 degrees ) for flexion - extension , 1.79 degrees ( sd 0.93 degrees ) for internal - external rotation , and 0.40 degrees ( sd 0.12 degrees ) for abduction - adduction8 . intra - class correlations coefficients ( 1 , 5 ) and standard errors of measurement of the pct have been calculated using the data of a previous study and indicate substantial or almost perfect reliability9 . in the present study , the position coordinate data were converted to flexion - extension ( fe ) , abduction - adduction ( aa ) , internal - external ( ie ) rotation , and the anterior - posterior ( ap ) , medial - lateral ( ml ) , superior - inferior ( si ) translation of the tibia with respect to a gait cycle6 . data from three trials were averaged and the average was used for the analysis of each limb . the value of the right leg subtracted from that of the left leg was defined as the side - to - side difference in rotation / translation in each stage of the gait cycle . we then determined the average side - to - side differences and their 95% confidence intervals ( cis ) . for statistical analysis , one - way analysis of variance and post - hoc bonferroni tests were performed to compare the average side - to - side difference of each motion among 0% , 20% , 40% , 60% and 80% of the time of a gait cycle . differences were considered statistically significant at p < 0.05 with bonferroni adjustment for post - hoc multiple comparisons . based on the ground reaction forces of the force plates , the stance and swing phases during walking were determined . as a result , 0% to mean 61.6% ( sd 1.3% ) of the gait cycle was regarded as the stance phase . therefore , 0% , 20% , 40% , 60% and 80% of the gait cycle were considered as the early stance phase , the middle stance phase , the late stance phase , the early swing phase and the late middle swing phase , respectively . the average knee kinematics pattern of the right and left legs during a gait cycle are shown in fig . 1.the average knee kinematics pattern of the right and left legs during a gait cyclea ) flexion - extension ; b ) adduction - abduction ; c ) internal - external rotation of the tibia . regarding fe of the knee during walking , the average values ( 95% cis ) of side - to - side differences at each time frame of normal walking were 4.3 degrees ( 3.0 to 5.7 degrees ) , 4.9 degrees ( 3.5 to 6.3 degrees ) , 4.4 degrees ( 2.9 to 5.9 degrees ) , 7.4 degrees ( 5.2 to 9.6 degrees ) and 5.4 degrees ( 3.4 to 7.3 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . 2.the 95% confidence intervals ( cis ) of side - to - side differences of the normal subjectsa ) flexion - extension ( fe ) ; b ) abduction - adduction ( aa ) ; c ) internal - external ( ie ) rotation ; d ) anterior - posterior ( ap ) translation ; e ) medial - lateral ( ml ) translation ; f ) superior - inferior ( si ) translation , table 1table 1.the average of side - to - side differences and their 95% confidence intervals in each stage of the gait cyclewalking cycle0%20%40%60%80%rotation ( degrees)flexion - extension4.3 ( 3.05.7)4.9 ( 3.56.3)4.4 ( 2.95.9)7.4 ( 5.29.6)5.4 ( 3.47.3)abduction - adduction3.8 ( 2.65.1)4.5 ( 2.76.4)3.2 ( 2.14.3)5.4 ( 3.67.2)8.9 ( 5.911.9)internal - external7.4 ( 5.59.4)5.7 ( 3.77.8)4.7 ( 3.36.1)6.2 ( 4.08.4)7.4 ( 4.710.2)translation ( mm)anterior - posterior9.7 ( 6.712.7)7.7 ( 4.510.9)6.8 ( 4.19.6)9.2 ( 5.013.4)12.8 ( 6.818.8)medial - lateral6.3 ( 3.79.0)5.3 ( 3.47.1)5.5 ( 3.77.3)6.7 ( 4.98.5)5.9 ( 3.78.1)superior - inferior7.7 ( 4.510.8)8.1 ( 4.211.9)8.2 ( 4.112.4)8.9 ( 4.413.5)9.8 ( 9.818.8)(mean , 95% ci ) . a : post - hoc bonferroni tests ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . b : post - hoc bonferroni test ( vs. 40% p<0.001 ) ) . there were no significant differences in the side - to - side differences of fe among 0% , 20% , 40% , 60% , and 80% of the gait cycle ( p=0.086 ) . the average side - to - side differences ( 95%cis ) of aa were 3.8 degrees ( 2.6 to 5.1 degrees ) , 4.5 degrees ( 2.7 to 6.4 degrees ) , 3.2 degrees ( 2.1 to 4.3 degrees ) , 5.4 degrees ( 3.6 to 7.2 degrees ) and 8.9 degrees ( 5.9 to 11.9 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . the side - to - side difference of aa at 80% of the walking cycle was significantly greater than those at 0% , 20% , 40% and 60% of the gait cycle ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . the average side - to - side differences ( 95%cis ) of ie rotation were 7.4 degrees ( 5.5 to 9.4 degrees ) , 5.7 degrees ( 3.7 to 7.8 degrees ) , 4.7 degrees ( 3.3 to 6.1 degrees ) , 6.2 degrees ( 4.0 to 8.4 degrees ) and 7.4 degrees ( 4.7 to 10.2 degrees ) at 0% , 20% , 40% , 60% , and 80% of the gait cycle , respectively ( fig . there were no significant differences in the side - to - side differences of ie rotation among 0% , 20% , 40% , 60% , and 80% of the gait cycle ( p=0.332 ) . at 0% , 20% , 40% , 60% , and 80% of the gait cycle , the average values ( 95% cis ) of side - to - side differences of ap translation were 9.7 mm ( 6.7 to 12.7 mm ) , 7.7 mm ( 4.5 to 10.9 mm ) , 6.8 mm ( 4.1 to 9.6 mm ) , 9.2 mm ( 5.0 to 13.4 mm ) and 12.8 mm ( 6.8 to 18.8 mm ) , respectively ( fig . the side - to - side difference of ap translation at 80% of the gait cycle was significantly greater than that at 40% of the gait cycle ( p=0.001 ) . the average side - to - side differences ( 95%cis ) of ml translation were 6.3 mm ( 3.7 to 9.0 mm ) , 5.3 mm ( 3.4 to 7.1 mm ) , 5.5 mm ( 3.7 to 7.3 mm ) , 6.7 mm ( 4.9 to 8.5 mm ) and 5.9 mm ( 3.7 to 8.1 mm ) , respectively ( fig . 2-e , table 1 ) . there were no significant differences in the side - to - side differences of ml translation among 0% , 20% , 40% , 60% , and 80% of the walking cycle ( p=0.875 ) . similarly the average side - to - side differences ( 95%cis ) of si translation were 7.7 mm ( 4.5 to 10.8 mm ) , 8.1 mm ( 4.2 to 11.9 mm ) , 8.2 mm ( 4.1 to 12.4 mm ) , 8.9 mm ( 4.4 to 13.5 mm ) and 9.8 mm ( 9.8 to 18.8 mm ) , respectively ( fig . there were no significant differences in the side - to - side differences of sl translation among 0% , 20% , 40% , 60% , and 80% of the walking cycle ( p=0.947 ) . the average knee kinematics pattern of the right and left legs during a gait cycle a ) flexion - extension ; b ) adduction - abduction ; c ) internal - external rotation of the tibia the 95% confidence intervals ( cis ) of side - to - side differences of the normal subjects a ) flexion - extension ( fe ) ; b ) abduction - adduction ( aa ) ; c ) internal - external ( ie ) rotation ; d ) anterior - posterior ( ap ) translation ; e ) medial - lateral ( ml ) translation ; f ) superior - inferior ( si ) translation ( mean , 95% ci ) . a : post - hoc bonferroni tests ( vs. 0% : p<0.001 , vs. 20% : p<0.001 , vs. 40% : p<0.001 , vs. 60% : p<0.001 ) . b : post - hoc bonferroni test ( vs. 40% p<0.001 ) for clinical application , the knee kinematics of a case with the posterior cruciate ligament injury ( pcl ) were also evaluated . a 37 year - old male suffered a knee injury while playing rugby football . the case showed grade-3 positive in the posterior drawer test , and posteromedial rotatory instability11 . the mri images indicated a pcl injury . at two months after injury , evaluation of 3-d kinematics during walking based on the side - to - side differences of kinematic data during each stage of gait , the tibia of the injured leg was shifted posteriorly in ap translation during 20% to 60% walking cycle , and shifted internally in ie rotation during 0% to 60% gait cycle ( fig . 3fig . 3the side - to - side difference of the femur relative to the tibia in the case with pcl and pms injuries with the 95% confidence intervals ( cis ) of the normal subjects . a ) anterior - posterior ( ap ) translation ; b ) internal - external ( ie ) rotation ) . the side - to - side difference of the femur relative to the tibia in the case with pcl and pms injuries with the 95% confidence intervals ( cis ) of the normal subjects . a ) anterior - posterior ( ap ) translation ; b ) internal - external ( ie ) rotation the present study investigated the side - to - side differences in kinematics of the knee joint during walking by healthy subjects . the side - to - side differences in adduction - abduction rotation and anterior - posterior translation of the tibia were found to be highly dependent on the stage of gait , and they were significantly larger in the swing phase than in the stance phase . therefore , our findings suggest that it is important to know the normal ranges of the side - to - side differences in knee kinematics in each stage of the gait cycle , in particular those of adduction - abduction and anterior - posterior translation of the knee , in order to evaluate the knee kinematics of subjects during walking . the reason why the side - to - side differences of adduction - abduction rotation and anterior - posterior translation of the tibia during walking are larger in the swing phase than in the stance phase remain unknown . one possible reason is mechanical restraint of the motion of the tibia during walking . the leg is not subject to weight bearing in the swing phase , and the tibia can move with less mechanical restraint in the swing phase than in the stance phase . without weight bearing , the knee is relaxed and the joint kinematics are likely to be influenced by ligament balance , rather than joint morphology . the knee kinematics during walking of a pcl injury case are also presented . for this case , an abnormal side - to - side difference in internal rotation of the tibia during walking as well as an abnormal side - to - side difference in posterior translation of the tibia were found . li et al . reported that pcl deficiency significantly increases posterior translation of the tibia during knee flexion under a weight - bearing condition using a dual - orthogonal fluoroscopic system12 . however , there was no significant difference in internal - external rotation between pcl - injury and normal knees throughout the range of flexion . in our present study , the case might have had associated injuries of posteromedial structures of the knee . previous cadaver studies have shown that additional injuries of posteromedial structures significantly increase internal rotation of the tibia as well as posterior translation of the tibia in pcl deficient knees13,14,15 . regarding clinical relevance , our findings indicate that the evaluation of the side - to - side difference of knee - joint kinematics provides useful information for clinical practice . however , caution is required in the clinical application of the evaluation of the side - to - side difference of knee - joint kinematics during walking because , there is a possibility that the contralateral leg would functionally adapt to the condition after trauma or disease . some investigators have reported that the contralateral leg as well as the injured leg functionally adapt to the condition after the injury of the acl and show the so - called quadriceps - avoidance gait16,17,18,19 . therefore , the evaluation of the side - to - side difference of knee joint kinematics may underestimate the knee kinematics of an injured leg . while this approach can measure in vivo kinematics of the knee in a dynamic condition , namely in a more physiological condition , it has been reported to have low accuracy due to skin movement artifacts20 , 21 . the recently described pct employs an overabundance of markers ( a cluster ) placed on each segment to minimize the effects of skin movement artifact6 . the pct can be extended to minimize skin movement artifacts by optimal weighting of the markers according to their degree of deformation6 . furthermore pct permits direct in vivo measurement of the complete six - degrees - of - freedom motion of the kinematics of the knee during activities of daily living . pct has , therefore , recently attracted attention , since the data of three dimensional kinematics of the knee during activities of daily living including walking are considered to reflect function of the knee joint . walking speed , surface condition , and shoes presumably influence the results of gait analysis . the knee kinematics measured in this study during walking and gait speed were similar to those reported by other investigators22,23,24,25,26 . first , since the point cluster technique is based on skin makers , the accuracy of the relative positions of the femur with respect to the tibia depends on the marker attachment technique of the examiner . second , the sample size of the present study , 21 , was not large enough to determine the normal range of the side - to - side - difference of the knee kinematics during walking . third , the effect of age or gender differences was not investigated in the present study . however , no significant differences have been reported between age groups and gender in previous knee kinematics studies27,28,29 , suggesting their effects in this study would have been small . despite the above - noted limitations , the mean side - to - side differences and their 95% cis as objective criteria of normal knee kinematics are useful information for the functional evaluation of subjects with disorders of the knee joint . in conclusion , the knee kinematics of healthy subjects during walking were measured using the pct , and the normal range of side - to - side differences in knee kinematics were determined for five stages of the gait cycle . the side - to - side differences in aa rotation and ap translation were highly dependent on the stage of gait ; therefore , the normal ranges of the side - to - side differences in knee kinematics of each stage of gait , in particular aa rotation and ap translation of the tibia , are useful information for the evaluation of the normal and pathological knee kinematics during walking .

[ purpose ] the purpose of this study was to determine the normal range of the side - to - side difference in three dimensional knee kinematics measured by the point cluster technique ( pct ) . [ subjects ] the subjects were twenty - one healthy normal volunteers without knee pain or an episode of injury to the legs . [ methods ] the subjects were tested bilaterally at a self - selected normal walking speed and six degrees of freedom knee kinematics were measured using the pct , and the 95% confidence intervals of the average side - to - side differences in flexion - extension ( fe ) , adduction - abduction ( aa ) , internal - external ( ie ) rotation , and anterior - posterior ( ap ) , medial - lateral ( ml ) , superior - inferior ( si ) translation in each stage of the gait cycle were determined . [ results ] the average side - to - side differences and their 95% confidence intervals in rotation / translation in each stage of the gait cycle were determined . the side - to - side differences in aa rotation and ap translation of the tibia were significantly larger in the swing phase than in the stance phase . [ conclusion ] the side - to - side differences in aa rotation and ap translation were highly dependent on the stage of the gait cycle . therefore , the normal ranges of the side - to - side differences in knee kinematics in each stage of the gait cycle , in particular aa rotation and ap translation of the tibia , is useful information for evaluating knee kinematics during walking .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4378297

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

multi - disciplinary research has revealed that electrically conductive contact of the human body with the surface of the earth ( grounding or earthing ) produces intriguing effects on physiology and health . such effects relate to inflammation , immune responses , wound healing , and prevention and treatment of chronic inflammatory and autoimmune diseases . the purpose of this report is two - fold : to 1 ) inform researchers about what appears to be a new perspective to the study of inflammation , and 2 ) alert researchers that the length of time and degree ( resistance to ground ) of grounding of experimental animals is an important but usually overlooked factor that can influence outcomes of studies of inflammation , wound healing , and tumorigenesis . specifically , grounding an organism produces measurable differences in the concentrations of white blood cells , cytokines , and other molecules involved in the inflammatory response . we present several hypotheses to explain observed effects , based on current research results and our understanding of the electronic aspects of cell and tissue physiology , cell biology , biophysics , and biochemistry . an experimental injury to muscles , known as delayed onset muscle soreness , has been used to monitor the immune response under grounded versus ungrounded conditions . grounding reduces pain and alters the numbers of circulating neutrophils and lymphocytes , and also affects various circulating chemical factors related to inflammation .

Introduction Summary of findings to date Effects on sleep Effects on pain and the immune response Anatomical and biophysical aspects Discussion Conclusion

PMC3687268

this study combines four coordinated protocols sharing the same dias artificial pancreas technology conducted at the universities of padova ( italy ) and montpellier ( france ) , the university of virginia ( uva ) , and the sansum diabetes research institute , santa barbara , california . to test whether a smart phone is capable of running outpatient closed - loop control , we have configured a system comprising available components , which were linked as follows : cgm idex dias ( running all closed - loop computations , user interface , and communications to peripheral devices ) idex pump . the idex is an experimental device manufactured by insulet ( bedford , ma ) , which combines a dexcom seven plus receiver and omnipod insulin pump . in addition , dias transferred data in real time to a central location allowing remote monitoring of patient state and system functions . the primary engineering end point was the percent time with all system communications working properly ; the protocol criterion for success in this early feasibility study was this time reaching > 80% of the total time of investigation . secondary end points included the estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and control algorithm performance . the clinical goal was to assess patients and clinicians subjective impressions of the system , i.e. , the feasibility of its ambulatory use , including patient usability and wearability . a total of 20 adults ( age 2165 years ) with type 1 diabetes were studied ( 5 subjects at each site ) . before the tests , a pilot subject was performed in italy , france , and in the united states . all participants were experienced insulin pump users and were required to have the following : prestudy hba1c of 69% ; predefined insulin pump parameters for basal rates , carbohydrate ratios , and insulin sensitivity factors ; and proper mental status / cognition . the exclusion criteria were directed toward safety and included recent history of diabetic ketoacidosis or severe hypoglycemia , pregnancy , breastfeeding , or intention of becoming pregnant ( females ) , uncontrolled arterial hypertension , and conditions that may increase the risk of hypoglycemia or infections . in addition , the united states based studies received food and drug administration approval ( ide # g120032 ) and the european studies received appropriate national - level certifications . all studies were registered with clinicaltrials.gov ( nct01578980 for uva / sansum , nct01447992 for padova , and nct01447979 for montpellier ) . after consent and screening , subjects were trained to use the omnipod insulin pump ( insulet ) and participated in a 3- to 7-day pump initiation if needed . two dexcom seven plus sensors ( dexcom , san diego , ca ) were inserted 2472 h before admission ; throughout the trials , the sensors were calibrated per manufacturer s instructions using commercial glucometers . a calibration was performed before dinner at 7:00 p.m. , thereby allowing for further system - required calibrations to be performed during the timeframes before dinner and before breakfast . per food and drug administration recommendation , an additional ( one - time ) calibration was entered by the study staff if there was a discrepancy in the two sensor readings of 20% or if the cgm was reading < 70 mg / dl and the hemocue value was > 85 mg / dl . participants in italy , france , and virginia stayed at hotels , and participants in california resided at a guest house like outpatient research unit of the sansum consisting of a living room , kitchen , four bedrooms , and bathrooms . the participants in the european studies were admitted individually , one subject at a time ; uva had both single and double admissions ; at sansum , all five subjects were admitted concurrently . subjects checked in by 5:00 p.m. and met with the study team , which confirmed that the subjects had brought their insulin , pump supplies , and regular medications . the subject s pump was removed and the study pump containing the subject s insulin was started . connections were established between dias and one sensor designated as primary ( via the idex ) , the insulin pump ( via the idex ) , and the remote monitoring site . the subject was then introduced to dias operation ; the orientation took 1520 min to complete . the dias user manual ( supplementary data ) and advice from the study team were available to the subjects at all times . after this introduction , the subjects were in charge of their interactions with dias , controlling the system via its graphical user interface . the protocol continued for 42 h. during the first evening / night of study , dias was used in open - loop mode with the subject s home insulin parameters . at 7:00 a.m. on day 2 , the system was switched into closed - loop mode and remained in closed - loop control for 29 h until the subject was discharged at 12:00 p.m. on day 3 . meals were delivered to the patient s room from local restaurants or consumed at local dining facilities ( e.g. , dining out at a restaurant in padova or a hotel buffet breakfast at uva ) . the carbohydrate content of the meals was estimated by the subject and proper entry of the desired carbohydrate amount into dias was confirmed by the study physician , but there were no dietary restrictions . when the subjects were outside of their room , they were accompanied by a member of the study team and dias were remotely monitored continually . figure 1 describes the timeline of the studies in europe and in the united states . protocol design in european ( a ) and united states ( b ) investigation centers . 1b ) were as follows : in padova and montpellier , the patient was moved to the hospital at 7:00 a.m. on day 2 of the study for initiation of closed - loop control and remained in the hospital for 10 h before returning to the hotel for the rest of the study ; in the united states studies the control algorithm was switched into safety - only mode for the night ( 11:00 p.m. to 7:00 a.m. ) , as requested by the food and drug administration . a study physician , a nurse , and a technician were located in nearby rooms to provide assistance if needed . reference blood glucose readings were measured simultaneously by finger stick with a hemocue ( hemocue ab , ngelholm , sweden ) and a commercial glucometer beginning at 7:00 p.m. on the evening of admission and continuing every 2 h during the day . overnight , there were no scheduled finger sticks ; reference blood glucose measurements were taken only if dias or the secondary sensor alarm indicated hypoglycemia or hyperglycemia , or if the two sensors had readings diverging by > 20% . nursing staff checked dias and secondary cgm readings hourly overnight and system alarms were monitored remotely for the dias and with a baby monitor to capture alarms from the secondary cgm . any dias hypoglycemia red - light warning triggered treatment with 15 g fast - acting carbohydrate ( e.g. , juice ) , whereas hyperglycemia red - light warnings prompted checking the insulin pump for occlusion or malfunction . mmol / l ( 250 mg / dl ) was followed by a -hydroxybutyrate test ( finger stick precision xtra -ketone measurement ) ; confirmed -hydroxybutyrate level > 0.6 mmol / l was a criterion for discontinuation of the trial . any hemocue reading < 80 mg / dl was followed - up with additional finger sticks at least every 15 min and any hemocue blood glucose < 70 mg / dl was treated with fast - acting glucose . the hub of the dias system was an off - the - shelf smart phone running the android operating system . to ensure the operation of the smart phone as a medical device , its operating system was modified to disable processes not related to closed - loop control operation and to include self - checks of system integrity . the communications between dias , the idex , and the pump and the sensor were wireless , giving the patient the freedom to be fully detached from the dias controller . the system components worn by the patient included an insulet omnipod insulin pod and a dexcom seven plus sensor / transmitter . the patient additionally wore a pouch containing a communication box ( either viliv s5 tablet or galaxy nexus phone ) attached to the idex . the idex and the communication box were only needed for automated data transfer and pump control at this early feasibility stage . these devices did not have any computing or patient interaction functions and were abandoned in subsequent studies . the subject controlled dias using graphical user interface , which allowed the following : initializing the system with the average daily insulin dose , basal rate , carbohydrate ratio , and correction factor ; displaying cgm traces and insulin delivery graphs ; and real - time interaction , such as entries of sensor calibrations , meal carbohydrate content , premeal capillary glucose level , and other information the subject wished to provide ( e.g. , exercise or hypoglycemia treatment ) . two traffic - light signals presented the degree of risks for hypoglycemia or hyperglycemia as follows : green light , no risks detected ; yellow light , the system is working actively to mitigate the risks by either attenuating insulin delivery if hypoglycemia is anticipated or administering correction insulin if hyperglycemia is predicted ; and red light , which signifies that risks can not be eliminated by adjustment of insulin alone and intervention is required to either consume carbohydrate or ensure that insulin is delivered properly . dias operated in two modes , open - loop ( first 13 h of each study ) controlling the pump per each patient s preset basal / bolus delivery instructions and displaying cgm and insulin delivery information or closed - loop ( hours 1442 of each study ) running a closed - loop control algorithm . both modes of operation included fully automated transfer of data from the sensor to dias and commands from dias to the insulin pump . user input was required only before meals and whenever the system signaled imminent risk for hypoglycemia or hyperglycemia . the closed - loop control algorithm included two modules : 1 ) safety supervision responsible for prediction of hypoglycemia , attenuation , or discontinuation of insulin delivery if hypoglycemia is anticipated and warnings if hypoglycemia is imminent and can not be prevented by insulin discontinuation alone ( 19 ) and 2 ) range - correction module responsible for injecting correction boluses . the clinical use of this algorithm is described in detail in a recent publication as standard control to range ( 12 ) ; details on its engineering architecture also have been published ( 20 ) . occasional cgm data loss ( up to 20 min ) did not stop the operation of the controller ; during loss of pump communication , insulin was not delivered . in addition , dias transmitted data in real time through either 3 g ( telephone network ) or wifi to two servers ( uva and montpellier ) , which allowed team members to log - in for remote observation from their locations . the server connections were one - directional : dias transmitted data out but could not be controlled from a remote location for safety reasons . the transmitted data contained glucose traces , insulin infusion by the pump , and technical information about the functioning of the control algorithm but did not contain any subject identifiers ; the monitoring web sites were password - protected . achieving statistical significance was not an objective of this early - feasibility investigation . the data analysis corresponded to the goals of the study and included estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and percent time of active system operation . post hoc analyses included t test and nonparametric comparisons of open versus closed - loop parameters of glucose control observed during the study ; however , the study was not powered for this outcome . before inclusion in the analyses , cgm data were sent through retrospective recalibration using reference blood glucose readings as discussed in a recent editorial ( 21 ) . a total of 20 adults ( age 2165 years ) with type 1 diabetes were studied ( 5 subjects at each site ) . before the tests , a pilot subject was performed in italy , france , and in the united states . all participants were experienced insulin pump users and were required to have the following : prestudy hba1c of 69% ; predefined insulin pump parameters for basal rates , carbohydrate ratios , and insulin sensitivity factors ; and proper mental status / cognition . the exclusion criteria were directed toward safety and included recent history of diabetic ketoacidosis or severe hypoglycemia , pregnancy , breastfeeding , or intention of becoming pregnant ( females ) , uncontrolled arterial hypertension , and conditions that may increase the risk of hypoglycemia or infections . all protocols were approved by the review boards of the participating institutions . in addition , the united states based studies received food and drug administration approval ( ide # g120032 ) and the european studies received appropriate national - level certifications . all studies were registered with clinicaltrials.gov ( nct01578980 for uva / sansum , nct01447992 for padova , and nct01447979 for montpellier ) . after consent and screening , subjects were trained to use the omnipod insulin pump ( insulet ) and participated in a 3- to 7-day pump initiation if needed . two dexcom seven plus sensors ( dexcom , san diego , ca ) were inserted 2472 h before admission ; throughout the trials , the sensors were calibrated per manufacturer s instructions using commercial glucometers . a calibration was performed before dinner at 7:00 p.m. , thereby allowing for further system - required calibrations to be performed during the timeframes before dinner and before breakfast . per food and drug administration recommendation , an additional ( one - time ) calibration was entered by the study staff if there was a discrepancy in the two sensor readings of 20% or if the cgm was reading < 70 mg / dl and the hemocue value was > 85 mg / dl . participants in italy , france , and virginia stayed at hotels , and participants in california resided at a guest house like outpatient research unit of the sansum consisting of a living room , kitchen , four bedrooms , and bathrooms . the participants in the european studies were admitted individually , one subject at a time ; uva had both single and double admissions ; at sansum , all five subjects were admitted concurrently . subjects checked in by 5:00 p.m. and met with the study team , which confirmed that the subjects had brought their insulin , pump supplies , and regular medications . the subject s pump was removed and the study pump containing the subject s insulin was started . connections were established between dias and one sensor designated as primary ( via the idex ) , the insulin pump ( via the idex ) , and the remote monitoring site . the subject was then introduced to dias operation ; the orientation took 1520 min to complete . the dias user manual ( supplementary data ) and advice from the study team were available to the subjects at all times . after this introduction , the subjects were in charge of their interactions with dias , controlling the system via its graphical user interface . the protocol continued for 42 h. during the first evening / night of study , dias was used in open - loop mode with the subject s home insulin parameters . at 7:00 a.m. on day 2 , the system was switched into closed - loop mode and remained in closed - loop control for 29 h until the subject was discharged at 12:00 p.m. on day 3 . meals were delivered to the patient s room from local restaurants or consumed at local dining facilities ( e.g. , dining out at a restaurant in padova or a hotel buffet breakfast at uva ) . the carbohydrate content of the meals was estimated by the subject and proper entry of the desired carbohydrate amount into dias was confirmed by the study physician , but there were no dietary restrictions . when the subjects were outside of their room , they were accompanied by a member of the study team and dias were remotely monitored continually . figure 1 describes the timeline of the studies in europe and in the united states . protocol design in european ( a ) and united states ( b ) investigation centers . 1b ) were as follows : in padova and montpellier , the patient was moved to the hospital at 7:00 a.m. on day 2 of the study for initiation of closed - loop control and remained in the hospital for 10 h before returning to the hotel for the rest of the study ; in the united states studies the control algorithm was switched into safety - only mode for the night ( 11:00 p.m. to 7:00 a.m. ) , as requested by the food and drug administration . a study physician , a nurse , and a technician were located in nearby rooms to provide assistance if needed . reference blood glucose readings were measured simultaneously by finger stick with a hemocue ( hemocue ab , ngelholm , sweden ) and a commercial glucometer beginning at 7:00 p.m. on the evening of admission and continuing every 2 h during the day . overnight , there were no scheduled finger sticks ; reference blood glucose measurements were taken only if dias or the secondary sensor alarm indicated hypoglycemia or hyperglycemia , or if the two sensors had readings diverging by > 20% . nursing staff checked dias and secondary cgm readings hourly overnight and system alarms were monitored remotely for the dias and with a baby monitor to capture alarms from the secondary cgm . any dias hypoglycemia red - light warning triggered treatment with 15 g fast - acting carbohydrate ( e.g. , juice ) , whereas hyperglycemia red - light warnings prompted checking the insulin pump for occlusion or malfunction . mmol / l ( 250 mg / dl ) was followed by a -hydroxybutyrate test ( finger stick precision xtra -ketone measurement ) ; confirmed -hydroxybutyrate level > 0.6 mmol / l was a criterion for discontinuation of the trial . any hemocue reading < 80 mg / dl was followed - up with additional finger sticks at least every 15 min and any hemocue blood glucose < 70 mg / dl was treated with fast - acting glucose . the hub of the dias system was an off - the - shelf smart phone running the android operating system . to ensure the operation of the smart phone as a medical device , its operating system was modified to disable processes not related to closed - loop control operation and to include self - checks of system integrity . the communications between dias , the idex , and the pump and the sensor were wireless , giving the patient the freedom to be fully detached from the dias controller . the system components worn by the patient included an insulet omnipod insulin pod and a dexcom seven plus sensor / transmitter . the patient additionally wore a pouch containing a communication box ( either viliv s5 tablet or galaxy nexus phone ) attached to the idex . the idex and the communication box were only needed for automated data transfer and pump control at this early feasibility stage . these devices did not have any computing or patient interaction functions and were abandoned in subsequent studies . the subject controlled dias using graphical user interface , which allowed the following : initializing the system with the average daily insulin dose , basal rate , carbohydrate ratio , and correction factor ; displaying cgm traces and insulin delivery graphs ; and real - time interaction , such as entries of sensor calibrations , meal carbohydrate content , premeal capillary glucose level , and other information the subject wished to provide ( e.g. , exercise or hypoglycemia treatment ) . two traffic - light signals presented the degree of risks for hypoglycemia or hyperglycemia as follows : green light , no risks detected ; yellow light , the system is working actively to mitigate the risks by either attenuating insulin delivery if hypoglycemia is anticipated or administering correction insulin if hyperglycemia is predicted ; and red light , which signifies that risks can not be eliminated by adjustment of insulin alone and intervention is required to either consume carbohydrate or ensure that insulin is delivered properly . dias operated in two modes , open - loop ( first 13 h of each study ) controlling the pump per each patient s preset basal / bolus delivery instructions and displaying cgm and insulin delivery information or closed - loop ( hours 1442 of each study ) running a closed - loop control algorithm . both modes of operation included fully automated transfer of data from the sensor to dias and commands from dias to the insulin pump . user input was required only before meals and whenever the system signaled imminent risk for hypoglycemia or hyperglycemia . the closed - loop control algorithm included two modules : 1 ) safety supervision responsible for prediction of hypoglycemia , attenuation , or discontinuation of insulin delivery if hypoglycemia is anticipated and warnings if hypoglycemia is imminent and can not be prevented by insulin discontinuation alone ( 19 ) and 2 ) range - correction module responsible for injecting correction boluses . the clinical use of this algorithm is described in detail in a recent publication as standard control to range ( 12 ) ; details on its engineering architecture also have been published ( 20 ) . occasional cgm data loss ( up to 20 min ) did not stop the operation of the controller ; during loss of pump communication , insulin was not delivered . in addition , dias transmitted data in real time through either 3 g ( telephone network ) or wifi to two servers ( uva and montpellier ) , which allowed team members to log - in for remote observation from their locations . the server connections were one - directional : dias transmitted data out but could not be controlled from a remote location for safety reasons . the transmitted data contained glucose traces , insulin infusion by the pump , and technical information about the functioning of the control algorithm but did not contain any subject identifiers ; the monitoring web sites were password - protected . the data analysis corresponded to the goals of the study and included estimation of the failure rates of system components , frequency analysis of lost or inaccurate cgm records , and percent time of active system operation . post hoc analyses included t test and nonparametric comparisons of open versus closed - loop parameters of glucose control observed during the study ; however , the study was not powered for this outcome . before inclusion in the analyses , cgm data were sent through retrospective recalibration using reference blood glucose readings as discussed in a recent editorial ( 21 ) . the focus of this investigation was on the concept of using dias as a smart phone based control algorithm and user interface host . we assessed dias in terms of human factors and usability , system and component performance , performance of the control algorithm , utility of remote monitoring , and clinical events . before this study , a formative evaluation of the dias user interface heuristic evaluation ( expert review ) was followed by three focus groups with type 1 diabetic patients with varying exposure to diabetes technology ( n = 13 ) . feedback was gathered on various system components addressing user interaction , system features , and capabilities . change recommendations were prioritized , and users were asked to rate the system on several criteria . users indicated the importance of maintaining all existing insulin pump and cgm device functionalities ( 22 ) . the dias graphical user interface ( supplementary data ) proved to be reliable and well - understood by the subjects . all were able to easily navigate through the graphical user interface commands on their own in both open - loop and closed - loop modes of operation , view cgm and insulin information , and administer meal or correction boluses as needed . one subject used a hotel treadmill , one subject in italy rode a bike , one subject in france walked to nearby museums , and five subjects took a shower with the pouch hanging just outside of the shower . system wearability was evaluated in relative terms , comparing dias to previous laptop - based systems . with the transition to a smart phone as a system hub and to wireless data transmission , the weight of a closed - loop control system was reduced several - fold . figure 2a presents photos of dias displaying cgm and insulin delivery traces and the entire system worn by a study subject . dias communicated wirelessly with the idex / communication box ; these devices are placed in a pouch on the patient s belt . the idex communicated wirelessly to an omnipod insulin pump and to a dexcom sensor visible as attached on the subject . the communication range of the idex with the insulin pod and dexcom sensor was 5 inches , which necessitated the use of a belt pouch . with this set - up , the subjects were able to maintain activities of daily living , a necessary first step toward routine outpatient use . a : photos of the dias smart phone displaying cgm and insulin delivery traces ( left ) and the entire system worn by a study subject ( right ) . each of the five subjects participating simultaneously in these trials is represented by an icon on the computer screen . table 1 presents metrics of the technical performance of the artificial pancreas system overall and during the open - loop and closed - loop portions of the study . two subjects described developed hyperglycemia with ketones because of pump site or pod failures in the initial open - loop portion of the study and were rescheduled . only the completed second study data for the rescheduled subjects are included in this analysis . additionally , the three pilot subjects for each country were not included in the analysis ; the data of the first two from italy and france were recently published ( 18 ) . overall , the artificial pancreas system was functional 98% of the time , which exceeded the initially set primary end point goal of 80% . performance metrics for the functioning of the artificial pancreas system used in these studies and of its primary components one element of system connectivity should be noted . in the european studies and in the first united states based studies we used a viliv s5 tablet to communicate with the idex , which was then replaced by a more reliable samsung galaxy nexus smart phone . as evident from table 1 , this replacement had a substantial effect on system reliability , reducing almost five - fold the number of unplanned system restarts because of loss of signal transmission . because the communication box was dedicated solely to data transmission , its replacement did not affect the conceptual or the computing outcomes of the study . further , table 1 presents data on the performance of the principal system components : the cgm , dias , and the insulin pump . of particular importance for fully integrated closed - loop control is the stability of interdevice connections ( sensor idex smart phone idex insulin pump ) . table 1 presents the availability of cgm and insulin pump communications with dias during the study . although the study was not designed to test algorithm performance or to compare open - loop versus closed - loop , table 2 presents a set of glycemic control metrics and certain post hoc comparisons of open - loop versus closed - loop nights using retrospectively recalibrated cgm data ( 21 ) . the outpatient performance of the controller was similar to its inpatient performance of this same control algorithm observed in a previous study ( standard control to range , 12 ) ; thus , first indications are that a different platform ( e.g. , a smart phone ) under different outpatient conditions may achieve similar performance as a laptop - based system in the hospital . on open - loop versus closed - loop control , we observed 80 vs.72% time within target range ( p = 0.22 ) and 0.53 vs. 0.27 hypoglycemic episodes 3.9 mmol / l ( 70 mg / dl ) per 24 h ( p = 0.16 ) ; in other words , there were no significant differences between open - loop and closed - loop control overnight , which is an expected result for standard control to range ( 12 ) . performance of the control algorithm figure 2b presents a screenshot of the remote monitoring system operation during the study at sansum . each of the five subjects participating simultaneously in this study is represented by an icon on the computer screen . the icon summarizes real - time information , including patient identification number , current cgm reading and direction of change , the state of the hypoglycemia and hyperglycemia alerts , and a message informing the technician of possible risks or system malfunction . safety supervision module was active for three patients ( identification numbers 211 , 212 , and 214 ) as indicated by yellow hypoglycemia lights . each icon can be clicked during a monitoring session , which will display more detailed information for this subject , including detailed records of insulin delivery , glucose data , and the algorithm functions . throughout the study , observation of the participants was performed mainly through the remote monitoring system , which proved to be a useful tool . on six occasions during the study ( two during open - loop and four during closed - loop control ) , carbohydrate treatment was administered for blood glucose levels < 3.3 mmol / l ( 60 mg / dl ) , for a total of 0.17 events per 24 h of system operation . there were no instances of patient - initiated system shutdown , but the trials were discontinued by study staff on three occasions . the first two events occurred early , before initiation of closed - loop control , and the subjects were rescheduled for subsequent admissions , which concluded successfully . subject 1 experienced hyperglycemia of 260 mg / dl with -hydroxybutyrate level of 0.7 mmol / l 2 h after the insulin pump was initiated ( consistent with pod compared with insertion site failure ) . after the connection was reestablished with a new tablet , the insulin pod alarmed , prompting a pod change . the new pod occluded ( blood noted in pod tubing ) , resulting in -hydroxybutyrate of 1.3 mmol / l . 1 ) with the range controller switched off , delivering only basal rate ( 4.1 units in the previous 6 h ) . the subject s cgm glucose was noted to increase from 180 mg / dl to 295 mg / dl over the final 2 h , suggesting that a pod occlusion ( unconfirmed ) may have contributed to this event . these three patients were treated with subcutaneous insulin , resulting in prompt resolution of the mild ketosis . before this study , a formative evaluation of the dias user interface was conducted to evaluate the feasibility of the design for patient use . heuristic evaluation ( expert review ) was followed by three focus groups with type 1 diabetic patients with varying exposure to diabetes technology ( n = 13 ) . feedback was gathered on various system components addressing user interaction , system features , and capabilities . change recommendations were prioritized , and users were asked to rate the system on several criteria . users indicated the importance of maintaining all existing insulin pump and cgm device functionalities ( 22 ) . the dias graphical user interface ( supplementary data ) proved to be reliable and well - understood by the subjects . all were able to easily navigate through the graphical user interface commands on their own in both open - loop and closed - loop modes of operation , view cgm and insulin information , and administer meal or correction boluses as needed . one subject used a hotel treadmill , one subject in italy rode a bike , one subject in france walked to nearby museums , and five subjects took a shower with the pouch hanging just outside of the shower . system wearability was evaluated in relative terms , comparing dias to previous laptop - based systems . with the transition to a smart phone as a system hub and to wireless data transmission , figure 2a presents photos of dias displaying cgm and insulin delivery traces and the entire system worn by a study subject . dias communicated wirelessly with the idex / communication box ; these devices are placed in a pouch on the patient s belt . the idex communicated wirelessly to an omnipod insulin pump and to a dexcom sensor visible as attached on the subject . the communication range of the idex with the insulin pod and dexcom sensor was 5 inches , which necessitated the use of a belt pouch . with this set - up , the subjects were able to maintain activities of daily living , a necessary first step toward routine outpatient use . a : photos of the dias smart phone displaying cgm and insulin delivery traces ( left ) and the entire system worn by a study subject ( right ) . each of the five subjects participating simultaneously in these trials is represented by an icon on the computer screen . table 1 presents metrics of the technical performance of the artificial pancreas system overall and during the open - loop and closed - loop portions of the study . two subjects described developed hyperglycemia with ketones because of pump site or pod failures in the initial open - loop portion of the study and were rescheduled . only the completed second study data for the rescheduled subjects are included in this analysis . additionally , the three pilot subjects for each country were not included in the analysis ; the data of the first two from italy and france were recently published ( 18 ) . overall , the artificial pancreas system was functional 98% of the time , which exceeded the initially set primary end point goal of 80% . performance metrics for the functioning of the artificial pancreas system used in these studies and of its primary components one element of system connectivity should be noted . in the european studies and in the first united states based studies we used a viliv s5 tablet to communicate with the idex , which was then replaced by a more reliable samsung galaxy nexus smart phone . as evident from table 1 , this replacement had a substantial effect on system reliability , reducing almost five - fold the number of unplanned system restarts because of loss of signal transmission . because the communication box was dedicated solely to data transmission , its replacement did not affect the conceptual or the computing outcomes of the study . further , table 1 presents data on the performance of the principal system components : the cgm , dias , and the insulin pump . of particular importance for fully integrated closed - loop control is the stability of interdevice connections ( sensor idex smart phone idex insulin pump ) . table 1 presents the availability of cgm and insulin pump communications with dias during the study . although the study was not designed to test algorithm performance or to compare open - loop versus closed - loop , table 2 presents a set of glycemic control metrics and certain post hoc comparisons of open - loop versus closed - loop nights using retrospectively recalibrated cgm data ( 21 ) . the outpatient performance of the controller was similar to its inpatient performance of this same control algorithm observed in a previous study ( standard control to range , 12 ) ; thus , first indications are that a different platform ( e.g. , a smart phone ) under different outpatient conditions may achieve similar performance as a laptop - based system in the hospital . on open - loop versus closed - loop control , we observed 80 vs.72% time within target range ( p = 0.22 ) and 0.53 vs. 0.27 hypoglycemic episodes 3.9 mmol / l ( 70 mg / dl ) per 24 h ( p = 0.16 ) ; in other words , there were no significant differences between open - loop and closed - loop control overnight , which is an expected result for standard control to range ( 12 ) . figure 2b presents a screenshot of the remote monitoring system operation during the study at sansum . each of the five subjects participating simultaneously in this study is represented by an icon on the computer screen . the icon summarizes real - time information , including patient identification number , current cgm reading and direction of change , the state of the hypoglycemia and hyperglycemia alerts , and a message informing the technician of possible risks or system malfunction . safety supervision module was active for three patients ( identification numbers 211 , 212 , and 214 ) as indicated by yellow hypoglycemia lights . each icon can be clicked during a monitoring session , which will display more detailed information for this subject , including detailed records of insulin delivery , glucose data , and the algorithm functions . throughout the study , observation of the participants was performed mainly through the remote monitoring system , which proved to be a useful tool . on six occasions during the study ( two during open - loop and four during closed - loop control ) , carbohydrate treatment was administered for blood glucose levels < 3.3 mmol / l ( 60 mg / dl ) , for a total of 0.17 events per 24 h of system operation . there were no instances of patient - initiated system shutdown , but the trials were discontinued by study staff on three occasions . the first two events occurred early , before initiation of closed - loop control , and the subjects were rescheduled for subsequent admissions , which concluded successfully . subject 1 experienced hyperglycemia of 260 mg / dl with -hydroxybutyrate level of 0.7 mmol / l 2 h after the insulin pump was initiated ( consistent with pod compared with insertion site failure ) . after the connection was reestablished with a new tablet , the insulin pod alarmed , prompting a pod change . the new pod occluded ( blood noted in pod tubing ) , resulting in -hydroxybutyrate of 1.3 mmol / l . 1 ) with the range controller switched off , delivering only basal rate ( 4.1 units in the previous 6 h ) . the subject s cgm glucose was noted to increase from 180 mg / dl to 295 mg / dl over the final 2 h , suggesting that a pod occlusion ( unconfirmed ) may have contributed to this event . these three patients were treated with subcutaneous insulin , resulting in prompt resolution of the mild ketosis . technology advancements in the past year made possible the development of dias , wearable ambulatory artificial pancreas platforms using an off - the - shelf smart phone as a computational hub . besides more user - friendly touch - screen interface and wireless connectivity , one easily quantifiable result of the transition from a laptop - based to a phone - based closed - loop control is a significant reduction in the system weight , which brings the system one important step closer to ambulatory use . ultimately , this would lead to closing the loop with a portable minimally invasive system suitable for home use . industry is currently transitioning cgms and pumps to include wireless connectivity ; thus , dias is only the first of many portable devices that will be capable of wireless data exchange and fully integrated closed - loop control . at the time of this outpatient trial , the dexcom seven plus and the omnipod insulet pump had short - range wireless capability to communicate with an idex research platform . also , for the idex to establish wireless communication with dias , an intermediary tablet ( or a cell phone ) needed to be connected to the idex . hence , there was short - range wireless communication from the patient ( wearing a pod and sensor / transmitter ) to a pouch containing the idex and tablet ( or cell phone ) , and long - range wireless communication between the pouch and the dias artificial pancreas platform . these intermediate devices are now being phased out ; communication boxes are no longer necessary . such a technology improvement was anticipated in our study ; thus , we focused on the smart phone computing and user - interface capabilities of the dias , assuming that this would be the device that is here to stay . special emphasis should be placed on the fact that the subjects were operating the system by themselves most of the time . to the best of our knowledge , this is the first trial in which the subjects were responsible for the oversight of their closed - loop systems , a step that is critical for outpatient deployment of closed - loop control . based on this feedback , we conclude that the form factor of dias as an artificial pancreas platform is appropriate for outpatient use . however , before long - term efficacy studies comparing outpatient artificial pancreas with sensor - augmented pump therapy can proceed , system wearability during daily living and the reliability of device communications must be ensured . testing of the system at four different sites in three countries and in a variety of hotel and restaurant settings using one , two , or five systems concurrently provided an opportunity to challenge dias with multiple scenarios that are likely to be encountered in nonhospital and , ultimately , home settings . in general , the technical performance of the dias system with overall operational time of 98% exceeded the set goal of 80% . in retrospect , this goal may have been conservative , but before this study it was generally unclear whether a smart phone can run closed - loop control , and there was no experience to guide the choice of this goal . the principal system components sensor , dias , and insulin pump were reasonably reliable , with 0.03 , 0.09 , and 0.12 malfunction events necessitating device replacement per 24 h , respectively . occasional cgm data points were lost ( 8.1% ) , but this did not result in skipping control cycles or discontinuation of the study ; by design , control to range would function during transient absence of cgm data because the controller intervenes only if risks for hypoglycemia or hyperglycemia are detected ( 20 ) . finally , we must emphasize the utility of remote monitoring , which was available on site and at remote locations ( i.e. , studies in europe or in california were observed from virginia and vice versa in real time ) . this was a critical aspect for patient safety that allowed close supervision so that intervention could occur quickly if needed . our system allowed monitoring concurrently multiple patients , a feature that was tested at sansum with five patients simultaneously . this feature alone will allow acceleration of the number of subjects who could be studied at the same time , reducing staffing costs and making artificial pancreas research more efficient . in summary , a wearable inexpensive closed - loop control platform ( dias ) combined with real - time remote monitoring , this system opens the possibility for large pivotal trials that will establish the artificial pancreas as a viable mainstream treatment strategy in type 1 diabetes .

objectiveto evaluate the feasibility of a wearable artificial pancreas system , the diabetes assistant ( dias ) , which uses a smart phone as a closed - loop control platform.research design and methodstwenty patients with type 1 diabetes were enrolled at the universities of padova , montpellier , and virginia and at sansum diabetes research institute . each trial continued for 42 h. the united states studies were conducted entirely in outpatient setting ( e.g. , hotel or guest house ) ; studies in italy and france were hybrid hospital hotel admissions . a continuous glucose monitoring / pump system ( dexcom seven plus / omnipod ) was placed on the subject and was connected to dias . the patient operated the system via the dias user interface in open - loop mode ( first 14 h of study ) , switching to closed - loop for the remaining 28 h. study personnel monitored remotely via 3 g or wifi connection to dias and were available on site for assistance.resultsthe total duration of proper system communication functioning was 807.5 h ( 274 h in open - loop and 533.5 h in closed - loop ) , which represented 97.7% of the total possible time from admission to discharge . this exceeded the predetermined primary end point of 80% system functionality.conclusionsthis study demonstrated that a contemporary smart phone is capable of running outpatient closed - loop control and introduced a prototype system ( dias ) for further investigation . following this proof of concept , future steps should include equipping insulin pumps and sensors with wireless capabilities , as well as studies focusing on control efficacy and patient - oriented clinical outcomes .

RESEARCH DESIGN AND METHODS Subjects Procedure Safety Technology User interface Control strategy Remote monitoring Statistical analysis RESULTS Human factors and usability System and component performance Performance of the control algorithm Utility of remote monitoring Clinical events CONCLUSIONS

PMC3024374

participants for this study were recruited from two urban areas ( yaound , the capital city of cameroon , and bamenda , the capital city of the north western region ) and two rural areas ( mbankomo and bafut ) of cameroon . the inhabitants of the urban sites are mainly middle - class public or private sector workers or businessmen . residential houses in the urban areas are grouped closely together and arranged in blocks with paved or graded road access . in the rural areas , the rural habitation pattern is sparse , with most of the homes and farms accessed only via foot paths . as a result of the absence of complete population registers , a sampling frame was established following enumeration of eligible adults in houses in delimited areas of the study sites . eligible participants were adults aged between 25 and 55 years without previously diagnosed diabetes or any known cardiovascular disease . all 3,854 eligible participants ( rural : n = 2,238 , mean age 35.3 7.7 years ; urban : n = 1,616 , mean age 35.4 8.3 years ) in the sampling frame were approached through door - to - door recruitment and invited to participate in the study . a total of 651 volunteers ( rural : n = 303 , mean age 38.5 8.3 years ; urban : n = 348 , mean age 37.9 9.1 years ) took part in this study . volunteers were provided detailed information about the study verbally and in writing , and invited to attend a testing session at their local hospital . volunteers were asked to refrain from eating , drinking or smoking starting at 10:00 p.m. the evening prior to testing . personal and sociodemographic data , as well as data on smoking , alcohol consumption , and fruit and vegetable intake were collected by self - report using an adapted world health organization ( who ) stepwise approach to sureveillance questionnaire . ethical approval for the study was obtained from the cameroon national ethics committee , and all participants provided signed informed consent . pregnant women and other volunteers who could not take part in exercise testing according to study protocol were excluded from the study . an exercise screening questionnaire , adapted from the who rose angina questionnaire was used to screen for preexisting cardiovascular disease which would contraindicate exercise testing . free - living physical activity was measured using a combined heart rate ( hr ) and movement sensor ( actiheart ; cambridge neurotechnology , cambridge , u.k . ) which has been validated in this population in free - living adults with doubly labeled water method as criterion ( 14 ) . the individual hr response to the mechanical work during an 8-min incremental step test was used for individual calibration of hr data ( 15 ) . during the test , hr was monitored in real time and the test was aborted if hr reached 90% of the age - predicted maximum hr ( 16 ) , if the subject could not keep up with the stepping protocol , or if they requested to stop . free - living physical activity was measured in 1-min epochs using the combined hr and movement sensor over 7 continuous days . the participants were requested to carry on with their normal habitual lifestyle and wear the monitor at all times except while showering , bathing , or swimming . participants with less than 2 full days of free - living data ( n = 51 ) were not included in these analyses . hr data were preprocessed ( 17 ) and converted to energy expenditure using the individual calibration derived from the step test , and the accelerometer data were converted to energy expenditure using group acceleration equations corresponding to level walking or running ( 15 ) . minute - by - minute hr and movement - derived physical activity intensity was combined in a branched equation model ( 18 ) to calculate free - living paee . basal metabolic rate ( ~resting energy expenditure [ ree ] ) was estimated using the oxford equations ( 19 ) , and total energy expenditure ( tee ) was calculated by adding ree and a component for the thermic effect of food ( equivalent to 10% of tee ) to the derived paee . energy expenditure variables are expressed per kilogram body weight to adjust for differences in body size . the distribution of intensity of activity was expressed as average daily time ( min / day ) spent at 1 met equal to time spent asleep or sedentary , between 1 and 3 mets equal to light physical activity , above 3 mets equal to moderate - to - vigorous physical activity . an estimate of cardiorespiratory fitness ( vo2max in ml of o2/kg / min ) was calculated as the intensity of work at the age - predicted maximum heart rate ( 15 ) by extrapolation of the individual heart rate response to mechanical work derived from the step test calibration . body weight was measured using electronic clinical scales , and body composition was measured using bio - impedance ( tanita tbf-531 scales ; tanita uk , uxbridge , middlesex , u.k . ) . bmi was calculated as the ratio of body weight ( kg ) to the square of height ( m ) and categorized as normal : < 25 ; overweight : 25 to < 30 ; and obese : 30 waist circumference was measured at the level of the midpoint between the lower costal margin and the anterior superior iliac crests . measurement was made to the nearest 0.1 cm using a nonstretch fiberglass tape measure with participants wearing light indoor clothing . central obesity was defined as waist circumference 80 cm in women or 94 cm in men , or using the cut points of the national cholesterol education program - adult treatment panel iii ( ncep - atp iii ) ( 20 ) when included to define the metabolic syndrome . measurements were made on the dominant arm after 5 min of rest with the subject seated . three measurements were taken at 1-min intervals , and the average of the three constituted the final blood pressure value . capillary blood glucose readings were measured after an overnight fast of at least 8 h , and then 2 h after ingestion of 75 g of glucose dissolved in 300 ml of water . blood glucose measurements were done on whole fresh capillary blood using a hemocue b - glucose analyzer ( hemocue ab , ngelholm , sweden ) . the metabolic syndrome was defined using the ncep - atp iii criteria ( 20 ) . to demonstrate the population distribution of global metabolic health , we computed a continuous summary metabolic risk score ( z - score ) largely on the basis of the components of the metabolic syndrome . this variable was derived by standardizing and then summing waist circumference , blood pressure ( average of systolic and diastolic ) , blood glucose ( average of fasting , 30-min , and 2-h glucose ) , triglycerides , body fat , fasting insulin , and inverse hdl cholesterol . the standardization of these continuous variables was computed by subtracting the sample mean from the individual value and then dividing by the sd of the sample mean . sex - specific calculations were made for waist circumference , body fat , fasting insulin , and hdl cholesterol . fasting blood samples were collected in the mornings between 7:30 and 9:30 a.m. plasma and serum samples were separated upon collection in a refrigerated centrifuge at 4c . samples were transported on dry ice by air to cambridge ( u.k . ) and stored at 80c until further analyses . assays were performed by the national institute for health research cambridge biomedical research centre , core biochemical assay laboratory . hdl cholesterol and triglycerides were measured using automated assays on the dade behring dimension rxl analyzer ( siemens healthcare , camberley , u.k . ) . hdl was measured using a homogenous accelerator selective detergent assay and triglycerides were measured using an enzymatic assay according to manufacturers specifications . ldl was calculated by the friedwald formula ( ldl = cholesterol hdl [ triglycerides/2.2 ] ) . fasting insulin was assayed in singleton on a 1235 autodelfia automatic immunoassay system using a two - step time resolved fluorometric assay kit manufactured by perkin elmer life sciences ( wallac oy , turku , finland ) . pmol / l , 3233 split proinsulin 1% at 2,800 pmol / l , and c - peptide < 0.1% at 5,280 analyses were carried out using stata version 10.1 ( statacorp , college station , tx ) . descriptive characteristics of the study sample are presented as means with sd , adjusted means with se , or as numbers with proportions , stratified by sex - specific paee quartiles . for dichotomous grouping variables , difference in means was assessed using the student t test and difference in proportion using the test or fisher exact test . independent associations between paee and the metabolic syndrome were examined in multivariate logistic regression models . there was no paee interaction by residential site or sex on its association with the metabolic syndrome . models were either unadjusted or adjusted for age , sex , residential site , smoking , alcohol consumption , fruit and vegetable consumption , and educational level ( as a surrogate for socioeconomic status ) . to illustrate the population impact of physical inactivity on metabolic health , we calculated the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee ( q1-paee ) . the population attributable fraction was computed as = ( prevalence of q1-paee in case subjects prevalence of q1-paee in control subjects)/(1 prevalence of q1-paee in control subjects ) . free - living physical activity was measured using a combined heart rate ( hr ) and movement sensor ( actiheart ; cambridge neurotechnology , cambridge , u.k . ) which has been validated in this population in free - living adults with doubly labeled water method as criterion ( 14 ) . the individual hr response to the mechanical work during an 8-min incremental step test was used for individual calibration of hr data ( 15 ) . during the test , hr was monitored in real time and the test was aborted if hr reached 90% of the age - predicted maximum hr ( 16 ) , if the subject could not keep up with the stepping protocol , or if they requested to stop . free - living physical activity was measured in 1-min epochs using the combined hr and movement sensor over 7 continuous days . the participants were requested to carry on with their normal habitual lifestyle and wear the monitor at all times except while showering , bathing , or swimming . participants with less than 2 full days of free - living data ( n = 51 ) were not included in these analyses . hr data were preprocessed ( 17 ) and converted to energy expenditure using the individual calibration derived from the step test , and the accelerometer data were converted to energy expenditure using group acceleration equations corresponding to level walking or running ( 15 ) . minute - by - minute hr and movement - derived physical activity intensity was combined in a branched equation model ( 18 ) to calculate free - living paee . basal metabolic rate ( ~resting energy expenditure [ ree ] ) was estimated using the oxford equations ( 19 ) , and total energy expenditure ( tee ) was calculated by adding ree and a component for the thermic effect of food ( equivalent to 10% of tee ) to the derived paee . energy expenditure variables are expressed per kilogram body weight to adjust for differences in body size . the distribution of intensity of activity was expressed as average daily time ( min / day ) spent at 1 met equal to time spent asleep or sedentary , between 1 and 3 mets equal to light physical activity , above 3 mets equal to moderate - to - vigorous physical activity . an estimate of cardiorespiratory fitness ( vo2max in ml of o2/kg / min ) was calculated as the intensity of work at the age - predicted maximum heart rate ( 15 ) by extrapolation of the individual heart rate response to mechanical work derived from the step test calibration . body weight was measured using electronic clinical scales , and body composition was measured using bio - impedance ( tanita tbf-531 scales ; tanita uk , uxbridge , middlesex , u.k . ) . bmi was calculated as the ratio of body weight ( kg ) to the square of height ( m ) and categorized as normal : < 25 ; overweight : 25 to < 30 ; and obese : 30 waist circumference was measured at the level of the midpoint between the lower costal margin and the anterior superior iliac crests . measurement was made to the nearest 0.1 cm using a nonstretch fiberglass tape measure with participants wearing light indoor clothing . central obesity was defined as waist circumference 80 cm in women or 94 cm in men , or using the cut points of the national cholesterol education program - adult treatment panel iii ( ncep - atp iii ) ( 20 ) when included to define the metabolic syndrome . measurements were made on the dominant arm after 5 min of rest with the subject seated . three measurements were taken at 1-min intervals , and the average of the three constituted the final blood pressure value . capillary blood glucose readings were measured after an overnight fast of at least 8 h , and then 2 h after ingestion of 75 g of glucose dissolved in 300 ml of water . blood glucose measurements were done on whole fresh capillary blood using a hemocue b - glucose analyzer ( hemocue ab , ngelholm , sweden ) . the metabolic syndrome was defined using the ncep - atp iii criteria ( 20 ) . to demonstrate the population distribution of global metabolic health , we computed a continuous summary metabolic risk score ( z - score ) largely on the basis of the components of the metabolic syndrome . this variable was derived by standardizing and then summing waist circumference , blood pressure ( average of systolic and diastolic ) , blood glucose ( average of fasting , 30-min , and 2-h glucose ) , triglycerides , body fat , fasting insulin , and inverse hdl cholesterol . the standardization of these continuous variables was computed by subtracting the sample mean from the individual value and then dividing by the sd of the sample mean . sex - specific calculations were made for waist circumference , body fat , fasting insulin , and hdl cholesterol . fasting blood samples were collected in the mornings between 7:30 and 9:30 a.m. plasma and serum samples were separated upon collection in a refrigerated centrifuge at 4c . samples were transported on dry ice by air to cambridge ( u.k . ) and stored at 80c until further analyses . assays were performed by the national institute for health research cambridge biomedical research centre , core biochemical assay laboratory . hdl cholesterol and triglycerides were measured using automated assays on the dade behring dimension rxl analyzer ( siemens healthcare , camberley , u.k . ) . hdl was measured using a homogenous accelerator selective detergent assay and triglycerides were measured using an enzymatic assay according to manufacturers specifications . ldl was calculated by the friedwald formula ( ldl = cholesterol hdl [ triglycerides/2.2 ] ) . fasting insulin was assayed in singleton on a 1235 autodelfia automatic immunoassay system using a two - step time resolved fluorometric assay kit manufactured by perkin elmer life sciences ( wallac oy , turku , finland ) . pmol / l , 3233 split proinsulin 1% at 2,800 pmol / l , and c - peptide < 0.1% at 5,280 analyses were carried out using stata version 10.1 ( statacorp , college station , tx ) . descriptive characteristics of the study sample are presented as means with sd , adjusted means with se , or as numbers with proportions , stratified by sex - specific paee quartiles . for dichotomous grouping variables , difference in means was assessed using the student t test and difference in proportion using the test or fisher exact test . independent associations between paee and the metabolic syndrome were examined in multivariate logistic regression models . there was no paee interaction by residential site or sex on its association with the metabolic syndrome . models were either unadjusted or adjusted for age , sex , residential site , smoking , alcohol consumption , fruit and vegetable consumption , and educational level ( as a surrogate for socioeconomic status ) . to illustrate the population impact of physical inactivity on metabolic health , we calculated the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee ( q1-paee ) . the population attributable fraction was computed as = ( prevalence of q1-paee in case subjects prevalence of q1-paee in control subjects)/(1 prevalence of q1-paee in control subjects ) . descriptive characteristics of the study sample stratified by sex and rural or urban residence are presented in supplementary table 1 . clinical and metabolic characteristics of the study sample stratified by sex - specific quartiles of paee are presented in table 1 . most of the individual metabolic and clinical parameters , as well as the clustered metabolic risk score or prevalence of the metabolic syndrome showed a significant linear trend across quartiles of paee . blood lipids were generally low in this sample with no sex or rural - urban differences . prevalence of metabolic syndrome was almost fourfold lower in the highest compared with the lowest quartile of paee . clinical and metabolic characteristics by quartiles of paee in adult cameroonians ( n = 552 ) data are age - adjusted means se or n ( % ) . bg , blood glucose ; bp , blood pressure ; ms , metabolic syndrome ; wc , waist circumference ; zms , clustered metabolic risk score . urban dwellers had a significantly lower paee than rural dwellers ( 44.2 21.0 vs. 59.6 23.7 kj / kg / day , p < 0.001 ) and a higher prevalence of the metabolic syndrome ( 17.7 vs. 3.5% , p < 0.001 ) ( supplementary tables 1 and 2 ) . there was no significant difference between the sexes in metabolic risk expressed as a continuous clustered metabolic score , but urban dwellers had a significantly higher mean score than rural dwellers ( 0.80 3.67 vs. 1.11 2.90 , p < 0.001 ) . this difference was observed to correspond to a rural - to - urban right shift in the population density distribution of the clustered metabolic z - score when stratified by residential area ( fig . a similar but reverse difference was observed with the population distribution of paee , with a rural - to - urban left shift in the distribution . population distribution of paee and clustered metabolic z - score in adults in rural and urban cameroon . table 2 presents independent associations between paee quartiles and prevalent metabolic syndrome . with the lowest quartile of paee as reference , there was a strong inverse association between paee and prevalent metabolic syndrome . compared with the lowest quartile of paee , being in the 2nd quartile of paee was associated with a reduction in the risk of prevalent metabolic syndrome , which was not statistically significant ( odds ratio 0.63 [ 95% ci 0.321.25 ] ) . the risk of prevalent metabolic syndrome was significantly lower for the 3rd and 4th quartiles of paee compared with the 1st quartile ( 0.32 [ 0.14 0.73 ] and 0.19 [ 0.08 0.49 ] , respectively ) . multivariate adjustments for age , sex , residential area , smoking , alcohol drinking , fruit and vegetable consumption , and educational level as potential confounders attenuated but did not change these associations . independent associations between paee and metabolic syndrome in adult cameroonians ( n = 552 ) data are odds ratio or odds ratio ( 95% ci ) . adjusted for age , sex , residential area , smoking , alcohol consumption , fruit and vegetable consumption , and number of years of education . defining the lowest quartile of paee to represent sedentary or inactive lifestyle , the population attributable fraction of prevalent metabolic syndrome due to a sedentary or inactive lifestyle was 26.3% ( 25.3% in women and 35.7% in men ) . expressing paee as a continuous variable , and assuming a linear relationship with the metabolic syndrome , each kj / kg / day of paee was associated with a 2.1% lower risk of prevalent metabolic syndrome ( odds ratio 0.98 , p = 0.03 ) , after adjustment for potential confounders as in table 2 , model 3 . this implies that an increase of 6.5 kj / kg / day of paee would correspond to approximately 13.7% risk reduction in prevalent metabolic syndrome . this amount of paee can be achieved by 30 min / day of brisk walking . our results suggest that free - living paee was significantly and independently associated with prevalent metabolic syndrome . this association persisted after multivariate adjustments for possible confounders and was similar in both urban and rural dwellers , even though rural dwellers were more physically active and had a more favorable metabolic profile than urban dwellers . low levels of physical activity may be contributing significantly to the metabolic syndrome in this population . theoretically , these data suggest a 26.3% population attributable fraction of prevalent metabolic syndrome as a result of being in the lowest quartile of paee . however , independent of several confounders , it is likely that small changes in paee may confer significant benefits in terms of lower risk of metabolic syndrome . because of the cross - sectional design of our study , we can not infer causality . the study sample included only adults aged 2555 years without known metabolic syndrome or cvd . therefore , these results may not be directly generalizable to younger or older individuals , as well as individuals with diagnosed prevalent metabolic or cardiovascular disease . the lack of complete population registers as well as the absence of fixed house addresses or telephone numbers limited our ability to use simple random sampling of participants . our use of volunteers from a study - established sampling frame may introduce sampling bias , which would limit the generalizability of these results even to our target population . however , our sample was similar to other larger population - based surveys with respect to bmi , blood pressure , and educational level ( 21,22 ) . separate from prevalence of risk factors , one could argue that educational level may have influenced volunteering for this study . however , the proportion of volunteers in this sample having at least a secondary education ( 44% of men and 35% of women ) was comparable to data from the 2004 demographic and health survey in cameroon ( 48% of men and 32% of women ) ( 21 ) . the observed inverse association between total volume of physical activity ( paee ) with the metabolic syndrome is comparable to results from previous studies which used objective measures of physical activity ( 12,13,23 ) ( 23 ) reported a significant inverse association between paee scaled for fat - free body mass and a clustered metabolic syndrome score . however , we scaled paee for body weight , not fat - free mass . although researchers agree that paee should be expressed taking into account differences in body size , the exact scaling factor applicable to body size is still a contentious issue . during light intensity locomotion activities , which are the main component of total activity in many people , the expression of paee per kilogram body weight may be more appropriate for the habitual free - living activity pattern , and is easy to interpret for clinical or public health usage . the importance of the distribution of free - living physical activity intensities , beyond total volume of physical activity , on metabolic health is still uncertain . many studies that have examined the effect of intensity distribution of physical activity on global metabolic risk have used self - report of physical activity ( 24 ) . studies that have used objective assessment of physical activity intensity distribution are less common ( 12,13 ) . most studies confirm the deleterious effect of increasing sedentary time or decreasing moderate - to - vigorous physical activity on the metabolic syndrome ( 12,24 ) . however , the benefits of increasing overall activity compared with shifting the intensity distribution toward more vigorous activity is still inconclusive . in this study we show that objectively measured total volume of physical activity had a significant independent association with the metabolic syndrome . our use of total volume of activity may be more applicable to health promotion initiatives in regions with low literacy . it may be more feasible to encourage overall physical activity than to recommend specific subdimensions of activity to less educated people . rapid urbanization of lifestyle in sub - saharan africa is characterized by increasing prevalence and clustering of metabolic risk factors such as diabetes , high blood pressure , and obesity ( 4,25 ) . our data show an evident rural - urban gradient of paee , which mirrors progression toward western levels of habitual physical activity . this transition is not only explained by increasing frequency of high risk individuals , but by a shift in the underlying population distribution of risk factors . our use of an aggregate variable for metabolic health ( the metabolic syndrome ) is relevant for assessing the effects of physical activity level on multiple risk factors in the whole population . further , the metabolic syndrome is associated with a higher morbidity and mortality ( 3 ) . we did not collect data on energy intake in this study because of the difficulties of collecting objective dietary data in a population - based study . however , we adjusted for self - reported fruit and vegetable intake in the analyses , which would capture part of the variance associated with healthy or unhealthy dietary habits . these data suggest a significant population burden of prevalent metabolic syndrome associated with being in the lowest quartile of paee , or a mean paee of 26 7 this level of physical activity corresponds to an average of 45 met - hours / week . typically , these individuals would be spending more than 80% of their time in sedentary activities with very little physical effort . encouraging people to increase their physical activity , even through reduction of sedentary time , may confer considerable health benefits especially in the least active individuals . in conclusion , reduction in physically active lifestyles as a result of urbanization may be critical in driving the current transition toward a higher burden of metabolic diseases in this region . public health interventions to increase physical activity may be expected to significantly curb the burden of metabolic syndrome in sub - saharan africa .

objectivewe examined the independent associations between objectively measured free - living physical activity energy expenditure ( paee ) and the metabolic syndrome in adults in rural and urban cameroon.research design and methodspaee was measured in 552 rural and urban dwellers using combined heart rate and movement sensing over 7 continuous days . the metabolic syndrome was defined using the national cholesterol education program - adult treatment panel iii criteria.resultsurban dwellers had a significantly lower paee than rural dwellers ( 44.2 21.0 vs. 59.6 23.7 kj / kg / day , p < 0.001 ) and a higher prevalence of the metabolic syndrome ( 17.7 vs. 3.5% , p < 0.001 ) . in multivariate regression models adjusted for possible confounders , each kj / kg / day of paee was associated with a 2.1% lower risk of prevalent metabolic syndrome ( odds ratio 0.98 , p = 0.03 ) . this implies a 6.5 kj / kg / day difference in paee , equivalent to 30 min / day of brisk walking , corresponds to a 13.7% lower risk of prevalent metabolic syndrome . the population attributable fraction of prevalent metabolic syndrome due to being in the lowest quartile of paee was 26.3% ( 25.3% in women and 35.7% in men).conclusionsurban compared with rural residence is associated with lower paee and higher prevalence of metabolic syndrome . paee is strongly independently associated with prevalent metabolic syndrome in adult cameroonians . modest population - wide changes in paee may have significant benefits in terms of reducing the emerging burden of metabolic diseases in sub - saharan africa .

RESEARCH DESIGN AND METHODS Physical activity measurement Anthropometry and clinical measures Metabolic syndrome and clustered metabolic risk score Biochemical assays Calculations and statistical analyses RESULTS CONCLUSIONS

PMC3317592

cyclic organic structures comprise a wide variety of interesting examples in strained , distorted , bent , and sterically hindered molecular systems in nanoscience . these structures have fascinated chemists for several decades not only because of their unique chemical bonding but also because of the potential to tailor their unique physical properties through variations in molecular size , shape , and crystallinity . cyclic nanostructures composed entirely of conjugated carbon subunits are especially fascinating because they exhibit a myriad of interesting electronic properties . in particular , because cycloparaphenylene nanostructures are composed of aromatic subunits with radially oriented p orbitals , these materials can support mobile charge carriers on the delocalized valence and conduction orbitals along the backbone chain . as a result , cycloparaphenylenes and other well - ordered macrocyles may find applications as conducting materials in molecular electronics , organic field - effect transistors ( ofets ) , and nonlinear optics ( nlos ) due to their unique optoelectronic properties . in general , the ability to control and understand the electronic response of organic nanostructures at the molecular level would have a tremendous impact in nanoscale optoelectronic technologies . carbon nanorings large enough to encapsulate fullerenes were first synthesized by kawase et al . and are composed of a conjugated carbon skeleton in a smooth belt - shaped structure . many of the nanorings synthesized by the kawase group are actually part of a larger family of molecules known as phenylacetylene macrocycles ( pams ) , which are composed of alternating phenyl and ethynyl subunits sequentially bonded to form a single ring . the phenyl subunits in pams are usually linked in the meta or para positions and can be composed of four to nine phenyleneethynylene units . in a very recent communication , jasti et al . synthesized and characterized a set of - , - , and -cycloparaphenylenes composed solely of phenyl rings sequentially connected to form a single nanohoop ( figure 1 ) . their synthesis was especially noteworthy because cycloparaphenylenes form the fundamental annular segments of armchair carbon nanotubes and may serve as templates toward the rational synthesis of carbon nanotubes with specific chiralities . [ n]-cycloparaphenylenes with n = 9 , 12 , and 18 . each cycloparaphenylene is composed solely of phenyl rings sequentially connected in the para position to form a single nanoring . a peculiar feature of these cycloparaphenylenes is their unusual optoelectronic properties as a function of molecular size . specifically , as the carbon nanoring size is increased , the lowest computed absorption energy becomes larger . this result is surprising and counterintuitive to our usual expectations ! for example , our chemical intuition from organic chemistry tells us that , as the number of repeat units in a macromolecule increases , the distance between molecular energy levels diminishes , effectively decreasing the optical absorption gap . this trend ( which actually arises from quantum confinement effects ) qualitatively describes the variation of absorbance properties in -conjugated systems as the number of monomer units increase in a polymer . however , in the case of cyclic nanorings , the excitation energies as a function of size seem to contradict this quantum - confinement - effect argument . to rationalize these trends , with a view of understanding the nature of electronic excitations in these nanorings , i carried out ab initio calculations on both cyclic and linear paraphenylenes ( figure 2 ) composed of 5 to 18 phenyl repeat units each . results obtained from time - dependent density functional theory ( tddft ) give electronic band gaps , optical absorption energies , and oscillator strengths that can be used to estimate exciton binding energies as a function of paraphenylene size . the tddft calculations also provide a two - dimensional real - space analysis of transition densities that represent coherent electronic transitions between ground and electronically excited states . these transition densities give a panoramic view of electronhole coherence and exciton delocalization in each of the paraphenylene systems . on the basis of overall trends in exciton binding energies and their spatial delocalization , i find that excitonic effects play a significant role in understanding the unique photoinduced dynamics of cycloparaphenylene nanoring systems . molecular structures and atom labels for ( a ) cyclic paraphenylenes and ( b ) linear paraphenylenes . the specific atom numbers depicted in these figures define an ordered basis for generating the density matrices discussed in section . the cyclic and linear paraphenylenes ( n = 518 ) analyzed in this work are shown in figure 2 . all quantum chemical calculations were carried out with the parameter - free pbe0 hybrid density functional that incorporates a fixed combination of 25% hartreefock exchange and perdew s gga corrections in the correlation contribution . have shown that the use of pure local and gradient - corrected functionals ( i.e. , lda and pbe ) results in unphysical unbound exciton states , whereas hybrid functionals partially overcome this problem by mixing in a fraction of nonlocal hartreefock exchange . on the basis of their studies , i have chosen the pbe0 functional for this work because the pbe0 kernel provides a balanced description of neutral excitons ( both singlets and triplets ) in conjugated polymers and carbon nanotubes . ground - state geometries of all cyclic and linear paraphenylenes were optimized at the pbe0/6 - 31g(d , p ) level of theory . geometry optimizations were calculated without symmetry constraints , and root - mean - squared forces converged to within 0.00003 au . at the optimized ground - state geometries , tddft calculations were performed with a larger , diffuse 6 - 31+g(d , p ) basis set to obtain the lowest four singlet vertical excitations . for both the ground - state and the tddft calculations , i used a high - accuracy lebedev grid consisting of 96 radial and 302 angular points . all ab initio calculations were performed with a locally modified version of gamess . within the tddft formalism , one obtains the excited - state electron density , (r ) |(r)| = i=1n|i(r)| composed of n - occupied molecular orbitals i(r ) as solutions from the time - dependent kohnsham equations . in the same way that one can calculate transition density matrices from time - dependent hartreefock orbitals , one can use orbitals from the noninteracting kohnsham system to form transition densities that include many - body correlation effects from the tddft formalism . , one can construct coordinate qv and momentum pv matrices with elements given by 12 where g and v are ground and excited states , respectively . the fermi operators ci and ci represent the creation and annihilation of an electron in the ith basis set orbital in . for the cyclic and linear paraphenylenes analyzed in this work , the qv and pv matrices each form a two - dimensional xy grid running over all the carbon sites along the x and y axes . the specific ordering of the carbon sites used in this work is shown in figure 2 . the ( pv)mn momentum matrix represents the probability amplitude of an electronhole pair oscillation between carbon sites m and n , and the ( qv)mn coordinate matrix gives a measure of exciton delocalization between sites m and n. each of these matrices provides a global view of electronhole coherence and exciton delocalization for optical transitions within the paraphenylene systems . in the ground - state optimizations for the linear paraphenylenes , the dihedral angle between adjacent benzene rings was calculated to be 37. this is close to the dihedral angle in isolated biphenyl and is consistent in all of the linear paraphenylenes regardless of length . in contrast , for the smallest and highly strained n = 5 cyclic paraphenylene , the benzene rings adopt a wide variety of smaller dihedral angles between 9 and 27. figure 3 shows the average dihedral angle between adjacent benzene rings as a function of paraphenylene size . for a given number of benzene rings , the average dihedral angle in the cyclic paraphenylenes is always smaller than their acyclic counterparts . however , as the size of the cyclic paraphenylene increases , the strain energy becomes smaller , and the dihedral angles between benzene rings increase to 36. the optimized cartesian coordinates for both the cyclic and the linear paraphenylenes can be found in the supporting information . average dihedral angle between adjacent benzene rings as a function of paraphenylene size . as the number of benzene rings increases , the average dihedral angle for the cyclic paraphenylenes asymptotically approaches the acyclic dihedral angle of 37. to investigate optoelectronic trends as a function of size and shape , optical absorption energies , eopt , were computed for all the cyclic and linear ground - state geometries . table 1 compares the lowest excitation energies and oscillator strengths between the cyclic and linear paraphenylenes , and figure 4 displays eopt as a function of size . the other higher - lying singlet excitations ( up to s4 ) can be found in the supporting information . as the number of benzene rings increases , the lowest excitation energies for both the cyclic and the linear paraphenylenes asymptotically approach a value of 3.48 ev . however , as figure 4 illustrates , the manner in which they approach this asymptotic value is considerably different : eopt for the cyclic systems increases with size , whereas eopt for the acyclic systems decreases . optical excitation energies ( eopt ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . eopt for the cyclic systems increases with size , whereas eopt for the acyclic systems decreases . all excitation energies were obtained from pbe0/6 - 31+g(d , p ) tddft calculations at pbe0/6 - 31g(d , p)-optimized geometries . another significant difference between the two systems is the variation of oscillator strengths as a function of size . for the linear paraphenylenes , the oscillator strengths increase linearly as a function of chain length , whereas the oscillator strengths for most of the even - membered cyclic systems are zero ( due to molecular symmetry ) . although the dft geometry optimizations were performed without symmetry constraints , most of the calculations for the smaller ( n 12 ) even - membered nanorings converged to a c2v - like symmetric structure . for nanorings containing an odd number of benzenes , the optimized structures have a reduced symmetry , and the oscillator strengths are nonzero . however , as the number of benzene rings increases , the strain energy becomes smaller , and several conformers with different dihedral angles can exist with similar energies . i found that these different conformers have various oscillator strengths ( due to reduced symmetry ) but nearly indentical energies . a complete analysis of the conformational landscape available to the nanorings is beyond the scope of this work and would have a negligible effect on the excitation energies in the larger systems . the small s1 s0 oscillator strengths obtained for the nanorings also have a close analogy with the very recent study by kilina et al . that calculated optoelectronic properties of finite - length carbon nanotubes . in their study , it was found that hybrid functionals determine the lowest singlet - excited state to be an optically inactive ( dark ) state with the optically active ( bright ) state lying higher in energy . similarly , for the large ( n 12 ) nanorings in this work , the second and third singlet excitations are bright states with strongly allowed transitions ( table si-1 in the supporting information ) . these trends in oscillator strengths for the nanorings can also be explained qualitatively in terms of a simple exciton model where one transition dipole moment is assigned to each benzene ring ( figure 5 ) . for the lowest s1 s0 excitation , the transition moments in both the linear and the cyclic paraphenylenes are aligned in a head - to - tail arrangement . however , because of their circular geometries , the s1 s0 transition moments in the cyclic paraphenylenes effectively cancel , while the total transition moment increases as a function of length in the linear systems ( for the larger , more flexible nanorings , some of the transition dipole moments have a small component perpendicular to the ring , and the vectorial sum is slightly nonzero ) . in contrast , for the other higher - lying singlet excitations , the transition dipoles are aligned in one direction for half of the nanoring and in the opposite direction for the other half of the ring , producing a net transition dipole and a nonzero oscillator strength . as a result , the structural difference between cyclic and linear geometries imposes additional symmetry constraints that determine the oscillator strengths in these conjugated systems . arrangements of individual transition dipole moments for ( a ) cyclic and ( b ) linear paraphenylenes during the s1 s0 excitation . the transition moments in the cyclic geometry effectively cancel , but the total transition moment increases proportionally with length in the linear systems . to provide further insight into these optoelectronic trends , i carried out a two - dimensional real - space analysis of density matrices for both the cyclic and the linear systems . figure 6 displays the absolute values of the coordinate density matrix elements , |(q1)mn| , for the lowest excitation energy ( s1 s0 ) in the n = 5 , 9 , 14 , and 18 paraphenylenes . the coordinate and momentum , |(p1)mn| , density matrices for all of the other paraphenylenes the x and y axes represent the benzene repeat units in the molecule , and the individual matrix elements are depicted by the various colors . contour plots of coordinate density matrices ( q1 ) for the lowest excitation energy ( s1 s0 ) in the cyclic and linear paraphenylenes . the x- and y - axis labels represent the number of benzene repeat units in the molecule . the elements of the coordinate matrix , qmn , give a measure of exciton delocalization between sites m ( x axis ) and n ( y axis ) . although the cyclic and linear paraphenylenes are composed of similar benzene repeat units , the density - matrix delocalization patterns in each system are considerably different . for the linear systems , the electronhole pair created upon optical excitation becomes primarily localized in the middle of the molecule and away from the edges . in contrast , the density matrices for the cyclic systems have significant off - diagonal elements that persist even for the largest n = 18 nanoring . the magnitude of the off - diagonal elements represents electronic coherence between different atoms , and figure 6 shows that the electronhole states are delocalized over the entire circumference in the cyclic systems . the coherence size , which characterizes the distance between an electron and a hole , is given by the width of the momentum density matrix along the coordinate axes in figure si-2 ( supporting information ) . in this work , i arbitrarily define the coherence width as the distance where the momentum decreases to 10% of its maximum value . these figures show that , for a given number of benzene rings , the coherence size in the linear paraphenylenes is slightly larger than their cyclic counterparts . for the smaller paraphenylenes ( n < 9 ) , the coherence size in the linear systems is larger by approximately one repeat unit in comparison with the cyclic systems . as the number of benzene units increases to 18 , the coherence size for both the cyclic and the linear systems approach the same value of 9 repeat units . both |(q1)mn| and |(p1)mn| also show that the linear systems only have strong optical coherences induced at their center , whereas the optical coherences in the cyclic geometries are nearly equally distributed throughout the entire molecule . these different density - matrix delocalization patterns and coherence sizes result in distinct excitonic properties in the cyclic and linear systems , an effect which i quantify in the next section . to understand electronhole interactions on a more quantitative level , i calculated exciton binding energies for the cyclic and linear paraphenylenes as a function of size . as illustrated in figure 7 , the exciton binding energy , eexc , is given by the difference between the quasiparticle energy gap [ ionization potential ( ip ) electron affinity ( ea ) ] and the optical absorption gap ( eopt ) . both the ip and the ea were obtained from pbe0/6 - 31+g(d , p ) electronic energy calculations on the n 1 , n , and n + 1 electron systems at the neutral pbe0/6 - 31g(d , p ) optimized geometry . in this work , the vertical ip is defined by 3 and the magnitude of the vertical ea is given by 4 with these definitions , the magnitude of the exciton binding energy is then 5 table 2 gives ionization potentials , electron affinities , and exciton binding energies for the cyclic and linear paraphenylenes , and figure 8 shows the quasiparticle energy gap ( ip ea ) as a function of size . schematic representation of energy levels defining the exciton binding energy ( eexc ) , optical absorption gap ( eopt ) , ionization potential ( ip ) , and electron affinity ( ea ) . quasiparticle energy gaps ( ip ea ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . the quasiparticle energy gap for the acyclic paraphenylenes decreases much more rapidly than the energy gap for the corresponding cyclic systems . as figure 8 shows , with the exception of some small fluctuations , the quasiparticle energy gap in both systems decreases with size , as expected from quantum - confinement effects . furthermore , the quasiparticle energy gap for the linear paraphenylenes decreases much more rapidly than the energy gap for the corresponding cyclic systems . the question therefore arises : why does the quasiparticle energy gap behave so differently in the linear and cyclic systems ? to address this question , we must take a closer look at the variations in aromatic character within the linear and cyclic geometries . first , in each of these organic systems ( regardless of molecular geometry ) , an electronic competition exists between maintaining the aromaticity of the individual benzene rings versus delocalization along the backbone chain . in the unstrained linear paraphenylenes , there is relatively little conjugation along the backbone of the system because the electrons are localized in each individual phenyl unit to maintain aromaticity . in contrast , the cyclic paraphenylenes have highly strained geometries that distort the electronic structure of the individual phenyl units . the strong deformation within the phenyl ring diminishes the overlap of orbitals , resulting in quinoidal character ( antibonding interactions within the phenyl ring and double - bond character connecting adjacent phenyl rings ) . as a result , the electronic states in the cyclic paraphenylenes are more delocalized in comparison with their acyclic counterparts , in agreement with the transition density matrix analysis discussed in section . more importantly , the quinoid form is energetically less stable than the aromatic form : the quinoid structure has a smaller quasiparticle gap because it involves destruction of aromaticity and a loss in stabilization energy . in other words , by increasing the quinoid character of the system , the highest - occupied orbital in the cyclic paraphenylenes becomes destabilized ( i.e. , raised in energy ) , and the quasiparticle energy gap is significantly reduced . to give a quantitative measure of aromaticity in these systems , i calculated the nucleus - independent chemical shift ( nics(1 ) ) at the pbe0/6 - 31g(d , p ) level of theory for each of the phenyl rings in the cyclic and linear geometries . in the nics(1 ) procedure suggested by schleyer et al . , the absolute magnetic shielding is computed at 1 above and 1 below the phenyl ring center . the resulting average nics(1 ) values give a measure of -orbital aromaticity , with more negative nics(1 ) values denoting aromaticity and more positive values corresponding to quinoidal character . as a reference point in this work , the nics(1 ) value for benzene at the pbe0/6 - 31g(d , p ) table 2 gives average nics(1 ) values of phenyl rings in the cyclic and linear paraphenylenes , and figure 9 shows the nics(1 ) values as a function of size . as anticipated from our qualitative discussion on aromaticity , the nics(1 ) values for the cyclic paraphenylenes in particular , for the smaller nanorings ( n < 8) , figure 9 shows that these structures have significant quinoidal character , resulting in unusually small quasiparticle energy gaps ( cf . however , as the size of the cyclic paraphenylene increases , the strain energy becomes smaller , and both the cyclic and the linear nics(1 ) values approach the same limit . it is also interesting to note that the fluctuations in nanoring nics(1 ) values shown in figure 9 are correlated with the deviations seen in figures 4 and 8 . specifically , the discontinuous change in nics(1 ) values at n = 8 , 10 , 13 , and 17 can also be found as discontinuities in optical excitation energies ( figure 4 ) and abrupt changes in quasiparticle energy gaps in figure 8 . even more intriguing is the direct correlation between nics(1 ) values of individual phenyl rings and the intensity of the transition density matrix elements discussed in section . returning to figure 6 , the excitonic density along the diagonal for n = 18 is not uniform ; in other words , there are four areas along the diagonal with maximal density at repeat units of 3 , 8 , 12 , and 17 . figure si-3a , b ( supporting information ) displays the nics(1 ) values of individual phenyl rings and the dihedral angle between adjacent phenyl rings for the n = 18 cyclic geometry . figure si-3a ( supporting information ) shows four distinct maxima ( i.e. , four phenyl units that have quinoidal , or delocalized , character ) at the same positions corresponding to maxima in the n = 18 transition density matrix . in contrast , figure si-3b ( supporting information ) shows that the dihedral angles are nearly identical ( all within 0.3 ) throughout the n = 18 nanoring . taken together , these figures show that the distribution of excitonic density in figure 6 is purely an electronic effect and not a result of conformational variations . average nics(1 ) values of phenyl rings in the cyclic and acyclic paraphenylenes as a function of size . all nics(1 ) calculations were obtained at the pbe0/6 - 31g(d , p ) level of theory . finally , figure 10 displays the exciton binding energies of both systems calculated from eq 5 . in contrast to figures 4 and 8 , the exciton binding energies in both systems show a smooth monotonic variation as a function of size . as expected , for a given number of benzene rings , the exciton binding energies in the cyclic paraphenylenes are always larger ( and decrease at a faster rate ) than their acyclic counterparts . this trend is in agreement with the coherence sizes of the momentum density matrices discussed in section . compared with the linear paraphenylenes , the average electronhole distance in the cyclic systems is smaller , leading to an increase in the binding energy . however , as the number of benzene units increases , the coherence sizes in both systems become nearly equal , and the binding energies asymptotically approach the same value . these results , in combination with the quasiparticle energy gaps and electronhole delocalization patterns , explain the anomalous absorption energy trends in the cyclic paraphenylenes . in the linear systems , the quasiparticle energy gap ( ip ea ) decreases much more rapidly than the exciton binding energy , eexc . as a result , the behavior of the ip ea term dominates the right - hand side of eq 5 , and the optical absorption gap ( eopt = ip ea eexc ) in the linear paraphenylenes decreases as a function of size . in contrast , ip ea in the cyclic systems decreases at a significantly slower rate than eexc ( cf . consequently , eopt in the cyclic paraphenylenes will be largely determined by the behavior of eexc . because eexc decreases faster than ip ea in the carbon nanorings , the resulting optical absorption gap in the expression eopt = ip ea eexcincreases as a function of size , in agreement with the tddft calculations . exciton binding energies ( ip ea eopt ) for the cyclic and acyclic paraphenylenes as a function of the number of benzene rings . for a given number of benzene rings , in this study , i investigated the optoelectronic properties in a series of cycloparaphenylenes that form the molecular structure of carbon nanorings . to understand their unusual electronic properties as a function of molecular size , i analyzed tddft excited - state energies , examined electronhole transition density matrices , and calculated quasiparticle and exciton binding energies for both the cyclic and the linear paraphenylenes systems . the tddft calculations and transition density analyses show that the cyclic and linear paraphenylenes exhibit dramatically different size - dependent effects that can be understood in the framework of a simple exciton theory . specifically , the main differences between the absorption spectra of the cyclic and linear paraphenylenes are caused by the subtle interplay of contributions to the electronhole interaction energy . in the linear systems , the exciton binding energy decreases slower than the quasiparticle energy gap , and the resulting optical absorption gap decreases with molecular size , as expected . in contrast , the effective coulomb attraction between an electronhole pair is larger in the cyclic geometry and decreases at a faster rate than the quasiparticle band gap . as a result , the overall optical absorption gap in the cyclic paraphenylenes increases as a function of size . in conclusion , i have shown that the different excitonic effects in the cyclic and linear paraphenylenes play a vital role in determining their distinct photophysical properties . the unique spatial distributions of electronhole states , as depicted by their transition density delocalization patterns , result in different electronhole interaction energies that ultimately control the electronic excitations in these systems . looking forward , it would be very interesting to see if the unusual optical trends resulting from the lowest , weakly allowed transition in these nanorings can be observed spectroscopically . the most direct way to observe these lowest transitions would be via low - temperature spectroscopic studies with an applied magnetic field to brighten the dark excitons . a magnetic flux through the open center of the nanorings would distort the circular symmetry of the electronic wave function and produce bright excitons due to the aharonovbohm effect . it is also possible that one can already observe these lowest transitions spectroscopically without the use of an applied magnetic field . because these nanorings are significantly more flexible than carbon nanotubes , the lowest dark exciton may be strongly allowed through symmetry breaking . vibronic coupling , which allows a borrowing of intensity from an energetically close bright state , may also play a significant role in observing these weakly allowed transitions . for the larger carbon nanorings in particular , the first bright state ( i.e. , the s2 state ) is energetically close to the lowest dark state . as a result , the anomalous size scaling of optical excitations should be more easily observed in the larger carbon nanorings . from a theoretical point of view , it would also be interesting to see how these effects change ( or if they change at all ) when the paraphenylenes are functionalized with both electron donor and acceptor functional groups to facilitate charge transfer . calculations of this type would require the use of recent long - range - corrected density functional methods , which are currently underway in my group . with these goals in mind , i anticipate that a critical understanding of excitonic effects in molecular nanostructures will provide a step toward their implementation in optoelectronic applications .

the electronic structure and size - scaling of optoelectronic properties in cycloparaphenylene carbon nanorings are investigated using time - dependent density functional theory ( tddft ) . the tddft calculations on these molecular nanostructures indicate that the lowest excitation energy surprisingly becomes larger as the carbon nanoring size is increased , in contradiction with typical quantum confinement effects . in order to understand their unusual electronic properties , i performed an extensive investigation of excitonic effects by analyzing electron - hole transition density matrices and exciton binding energies as a function of size in these nanoring systems . the transition density matrices allow a global view of electronic coherence during an electronic excitation , and the exciton binding energies give a quantitative measure of electron - hole interaction energies in the nanorings . based on overall trends in exciton binding energies and their spatial delocalization , i find that excitonic effects play a vital role in understanding the unique photoinduced dynamics in these carbon nanoring systems .

Introduction Theory and Methodology Results and Discussion Conclusion

PMC5213806

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

abstractthis article is a historiographical exploration of the special forms of knowledge generation and knowledge transmission that occur along local cultural boundaries in the modern neurosciences . following the inauguration of the so - called law on the re - establishment of a professional civil service in nazi germany on april 7 , 1933 , hundreds of jewish and oppositional neurologists , neuropathologists , and psychiatrists were forced out of their academic positions , having to leave their home countries and local knowledge economies and traditions for canada and the united states . a closer analysis of their living and working conditions will create an understanding of some of the elements and factors that determined the international forced migration waves of physicians and clinical neuroscientists in the twentieth century from a historiographical perspective . while i am particularly looking here at new case examples regarding the forced migration during the national socialist period in germany , the analysis follows german - speaking migr neurologists and psychiatrists who found refuge and settled in canada . these individuals form an understudied group of refugee medical professionals , despite the fact that the subsegments of refugee neurologists and clinical psychoanalysts in the united states , for example , have been a fairly well - investigated population , as the works of grob ( 1983 ) , lunbeck ( 1995 ) , or ash and soellner ( 1996 ) have shown . this article is primarily an exploration of the adjustment and acculturation processes of several highly versatile and well - rounded german - speaking physicians , who had received their prior education in neurology , psychiatry , and basic brain research . they were forced out of their academic home institutions and had to leave their clinical research fields as well as their disciplinary self - understanding behind on the other side of the atlantic .

Introduction Situating a cultural view in the historiography of forced migration in the neurosciences From neuropathology to psychoanalytic psychiatry and medical education: The case example of Karl Stern From clinical neurology and psychiatry to public mental health: The case example of Robert Weil From brain psychiatry to clinical neurochemistry: The case example of Heinz Lehmann Discussion

PMC1637882

epidemiologic studies of the link between particulate matter ( pm ) concentrations and mortality rates have yielded a range of estimates , leading to disagreement about the magnitude of the relationship and the strength of the causal connection . previous meta - analyses of this literature have provided pooled effect estimates , but have not addressed between - study variability that may be associated with analytical models , pollution patterns , and exposed populations . to determine whether study - specific factors can explain some of the variability in the time - series studies on mortality from particulate matter [ less than / equal to ] 10 microm in aerodynamic diameter ( pm(10 ) ) , we applied an empirical bayes meta - analysis . we estimate that mortality rates increase on average by 0.7% per 10 microg / m(3 ) increase in pm(10 ) concentrations , with greater effects at sites with higher ratios of particulate matter [ less than / equal to ] 2.5 microm in aerodynamic diameter ( pm(2.5))/pm(10 ) . this finding did not change with the inclusion of a number of potential confounders and effect modifiers , although there is some evidence that pm effects are influenced by climate , housing characteristics , demographics , and the presence of sulfur dioxide and ozone . although further analysis would be needed to determine which factors causally influence the relationship between pm(10 ) and mortality , these findings can help guide future epidemiologic investigations and policy decisions.imagesfigure 1

Images

PMC5260590

hurling is a stick and ball invasion field sport native to ireland ( reilly and collins , 2008 ) . the movement profile of players is stochastic in nature , where the timing of high speed running follows the ebb and flow of competitive play ( reilly and collins , 2008 ) . despite its spectator popularity very few research investigations have been conducted on hurling cohorts ( reilly and collins , 2008 ) . the training - recovery relationship is an important consideration in the training prescription of players and a clear understanding of training loads can help inform practice . recent reports have linked the training load and inadequate fitness to the high number of injuries observed within the gaelic sports ( blake et al . , 2014 ) . ( 2014 ) reported that the injury incidence rate for match play ( 61.75/1000 hr ) is 21 times that of training ( 2.99/1000 hr ) . this suggests that training does not adequately prepare players for the competitive demands of match - play . the training load ( tl ) in field sports can be divided into internal ( heart rate , rpe ) or external ( distance covered ) responses . an understanding of the tl allows for a more individualised understanding of the dose - response relationship of training and match play ( impellizzeri et al . , 2005 ; stagano et al . , 2007 ) . recent advancement in technology has improved the ease to which locomotion data collection can take place . there has been an increase in monitoring of the external training load in match and training situations through the use of global positioning satellite systems ( gps ) ( casamichana et al . the advent of this technology has resulted in total distance covered and distance covered at arbitrary demarcated thresholds of high speed locomotion being utilised to quantify the tl . the poor sensitivity and variability of these performance metrics within match play and training environments has been previously highlighted ( gregson et al . , 2010 ; kempton et al . , 2014 ; mclaren et al . , 2015 ; stevens et al . , 2016 ) . as a consequence these metrics may be unreliable to gain full appreciation of the tl . the understanding of the dose - response relationship has been advocated as an important consideration for coaches within the training process . monitoring the tl in team sports is difficult due to different exercise designs resulting in different physiological and mechanical demands . additional complications are presented in the form of inter - individual responses to the prescribed exercise ( akubat et al . , 2012 ) . o2 consumption relationship appears to be valid even during intermittent exercise ( esposito et al . , 2004 ) , the itrimp methodology has potential for use for monitoring the dose - response of players to specific training programs within a team sport environment ( manzi et al . , 2013 ) . indeed , measures of the internal tl have been validated with a dose - response relationship in team sports ( akubat et al . , 2012 ; both studies demonstrated that the fully individualized training impulse ( itrimp ) showed relationships with changes in aerobic - fitness measures of soccer players . previous studies ( manzi et al . , 2013 ) have shown that itrimp measures over a training period were correlated to the percentage improvement in vo2max ( r = 0.77 , p = 0.002 ) and yo - yoir1 performance ( r = 0.69 , p = 0.009 ) . ( 2014 ) through the integration of external ( total distance [ td ] ; high speed distance [ hsd ] ) and internal loading parameters ( itrimp ) . interestingly , these measures have shown a relationship with aerobic fitness measures in soccer players ( akubat et al . , 2014 ) . the investigation reported that both td : itrimp showed moderate to large correlations with vobla ( r = 0.65 ; p = 004 ; large ) and vobla ( r = 0.69 ; p = 0.03 ; large ) , with td not significantly related to vlt ( r = 0.28 ; p = 0.43 ; small ) and vobla ( r = 0.14 ; p = 0.69 ; small ) . this highlights the potential merits of using tl ratios to better quantify the dose - response of the tl . furthermore , studies within the rugby league ( weaving et al . , 2014 ) have shown that the integration of tl measures better explain a greater proportion of training component variance that internal or external measures of load alone fail to fully explain . at elite levels of hurling a disproportionate number of injuries occur during match play ( murphy et al . , 2007 ) with most occurring in the last quarter of games due to increased fatigue states ( blake et al . , this is in line with previous investigations ( watson , 1996 ) that have reported injury occurrences in hurling populations which are attributable to poor conditioning of players . given such a scenario , it would appear that a situation where an analysis and assessment of aerobic fitness training through data collected routinely at elite levels could prove useful . considering the potential ease of quantifying the hr ( internal load ) and the gps ( external load ) , both the internal and external output of exercise can be measured . if external : internal measures relate to measures of fitness that predetermine a given performance , more frequent analysis of training status and match performance could be made by practitioners . this would allow for more informed and proactive decisions related to player s fitness and fatigue status , therefore , providing a more thorough understanding of the training load paradigm in intermittent team sports . hence , the current study investigated whether information such as the hr and distance could potentially be used to assess the match play load . therefore , the study aimed to examine a novel approach of integrating external and internal workload variables as a ratio during hurling specific simulated match play . finally , the study aimed to quantify the relationship of integrated tl ratios and aerobic fitness measures in hurling players . twenty - five hurling players ( age : 24 4 yrs ; body height : 1.80 0.02 m ; body mass : 78 3 kg ; vo2max : 57.78 3.05 mlkgmin ) took part in this study . all subjects provided written informed consent to the experimental procedures after all possible benefits and risks were explained to them . ethical consent for the study was granted by the research ethics committee of the institute of technology tallaght . the study conformed to the codes of ethics of the world medical association ( declaration of helsinki , 1964 ) the study was completed over a 3 week period . during the 1 week an incremental vo2max and lactate threshold test ( vo2/lt ) was conducted . the vo2/lt consisted of five ( 8 , 10 , 12 , 14 & 16 kmh ) 4 min stages followed by an increase in speed at a rate of 0.2 kmh every 12 s until exhaustion was reached . the treadmill gradient was set at 1% for the entire test to reflect the energetic cost of outdoor running ( jones and doust , 1996 ) . during the incremental test , each stage was separated by a 1 min recovery period where capillary blood lactate was taken ( lactate plus , nova medical ) in duplicate , with the mean value for each stage recorded . the velocity at 2 mmoll ( vlt ) and velocity at 4 mmoll ( vobla ) were considered as markers of aerobic fitness . maximal aerobic capacity ( vo2max ) was assessed during the vo2/lt through breath by breath analysis ( cosmed k4b , rome , italy ) . vo2max was recorded as the highest mean vo2 obtained for a 1 min period with the following criterion met : ( 1 ) a plateau in vo2 despite increasing treadmill speed ( 2 ) a respiratory ratio above 1.10 ( 3 ) attainment of the age predicted hr ( lucia et al . , 2004 ) . the hr was recorded using hr belts ( polar team system , polar electro , oy , finland ) . the mean hr during the last minute of each stage was utilised to ascertain the individualized hr blood lactate relationship for the calculation of itrimp ( au ) weightings . finally , the peak hr during the incremental treadmill test was considered the peak hr of the participant . during weeks 2 and 3 and at least 5 days after the vo2max / lt test , participants completed a hurling match play simulation protocol . the hurling simulation protocol was chosen given its accurate physiological replication of hurling match play ( collins et al . before conducting the study , test retest reliability analysis was conducted on the hurling match play simulation protocol to ensure that the test provides external validity and reliability . results showed that there was no significant difference between the 1 and 2 trial of the hurling simulation protocol ( 7411 354 m ; 7311 362 m ; p = 0.19 ; trivial ) . the coefficient of variation ( cv ) was 1.9% ( 95% ci : 1.1 - 4.8 ) highlighting the reliability of the simulation . the hurling match play simulation protocol was completed at the same time of the day ( 10.30 14.00 hr ) to account for any circadian variation in performance . during the simulation participants wore an individual gps unit ( vxsport , lower hutt , new zealand , issue : 330a , firmware : 3.26.7.0 ) sampling at 4 hz and containing a triaxial acceloremter and a magnetometer in a total of 30 games . the gps unit ( mass : 76 g ; 48 mm x 20 mm x 87 mm ) was encased within a protective harness between the player s shoulder blades in the upper thoracic - spine region what ensured that players range of movement in the upper limbs and torso was not restricted . the system had been validated , deemed accurate and reliable during intermittent exercise ( buchheit et al . , 2014 ) . following each match gps data were downloaded using the same proprietary software ( vxsport view , new zealand ) . each file was trimmed so that only data recorded when the player was on the field was included for further analysis . the proprietary software provided instantaneous raw velocity data at 0.25 s intervals , which were then exported and placed into a customised microsoft excel spreadsheet ( microsoft , redmond , usa ) . the spreadsheet allowed analysis of distance covered ( m ) in the following categories : total distance ( td ) , high speed distance ( hsd : 17 kmh ) and sprint distance ( sd : 22 kmh ) . internal loading was measured as individualised training impulses ( itrimp ) ( manzi et al . , 2013 ) . pearson correlation coefficients were used to assess the relationships between ( 1 ) aerobic fitness and performance , and ( 2 ) distance : itrimp and fitness . qualitative interpretations of the correlation coefficients and the 95% confidence intervals ( ci ) as defined by hopkins ( 2004 ) ( 0.09 trivial , .10.29 small , .30.49 moderate , .50.69 large , .70.89 very large , .90.99 nearly perfect , 1 perfect ) are provided for all correlations . all statistical analyses were performed using spss for windows ( version 22 , spss inc . chicago , il , usa ) with statistical significance set at an accepted level of p<0.05 . twenty - five hurling players ( age : 24 4 yrs ; body height : 1.80 0.02 m ; body mass : 78 3 kg ; vo2max : 57.78 3.05 mlkgmin ) took part in this study . all subjects provided written informed consent to the experimental procedures after all possible benefits and risks were explained to them . ethical consent for the study was granted by the research ethics committee of the institute of technology tallaght . the study conformed to the codes of ethics of the world medical association ( declaration of helsinki , 1964 ) the vo2/lt consisted of five ( 8 , 10 , 12 , 14 & 16 kmh ) 4 min stages followed by an increase in speed at a rate of 0.2 kmh every 12 s until exhaustion was reached . the treadmill gradient was set at 1% for the entire test to reflect the energetic cost of outdoor running ( jones and doust , 1996 ) . during the incremental test , each stage was separated by a 1 min recovery period where capillary blood lactate was taken ( lactate plus , nova medical ) in duplicate , with the mean value for each stage recorded . the velocity at 2 mmoll ( vlt ) and velocity at 4 mmoll ( vobla ) were considered as markers of aerobic fitness . maximal aerobic capacity ( vo2max ) was assessed during the vo2/lt through breath by breath analysis ( cosmed k4b , rome , italy ) . vo2max was recorded as the highest mean vo2 obtained for a 1 min period with the following criterion met : ( 1 ) a plateau in vo2 despite increasing treadmill speed ( 2 ) a respiratory ratio above 1.10 ( 3 ) attainment of the age predicted hr ( lucia et al . , 2004 ) . the hr was recorded using hr belts ( polar team system , polar electro , oy , finland ) . the mean hr during the last minute of each stage was utilised to ascertain the individualized hr blood lactate relationship for the calculation of itrimp ( au ) weightings . finally , the peak hr during the incremental treadmill test was considered the peak hr of the participant . during weeks 2 and 3 and at least 5 days after the vo2max / lt test , participants completed a hurling match play simulation protocol . the hurling simulation protocol was chosen given its accurate physiological replication of hurling match play ( collins et al . before conducting the study , test retest reliability analysis was conducted on the hurling match play simulation protocol to ensure that the test provides external validity and reliability . results showed that there was no significant difference between the 1 and 2 trial of the hurling simulation protocol ( 7411 354 m ; 7311 362 m ; p = 0.19 ; trivial ) . the coefficient of variation ( cv ) was 1.9% ( 95% ci : 1.1 - 4.8 ) highlighting the reliability of the simulation . the hurling match play simulation protocol was completed at the same time of the day ( 10.30 14.00 hr ) to account for any circadian variation in performance . during the simulation participants wore an individual gps unit ( vxsport , lower hutt , new zealand , issue : 330a , firmware : 3.26.7.0 ) sampling at 4 hz and containing a triaxial acceloremter and a magnetometer in a total of 30 games . the gps unit ( mass : 76 g ; 48 mm x 20 mm x 87 mm ) was encased within a protective harness between the player s shoulder blades in the upper thoracic - spine region what ensured that players range of movement in the upper limbs and torso was not restricted . the system had been validated , deemed accurate and reliable during intermittent exercise ( buchheit et al . , 2014 ) . following each match gps data were downloaded using the same proprietary software ( vxsport view , new zealand ) . each file was trimmed so that only data recorded when the player was on the field was included for further analysis . the proprietary software provided instantaneous raw velocity data at 0.25 s intervals , which were then exported and placed into a customised microsoft excel spreadsheet ( microsoft , redmond , usa ) . the spreadsheet allowed analysis of distance covered ( m ) in the following categories : total distance ( td ) , high speed distance ( hsd : 17 kmh ) and sprint distance ( sd : 22 kmh ) . internal loading was measured as individualised training impulses ( itrimp ) ( manzi et al . , 2013 ) . pearson correlation coefficients were used to assess the relationships between ( 1 ) aerobic fitness and performance , and ( 2 ) distance : itrimp and fitness . qualitative interpretations of the correlation coefficients and the 95% confidence intervals ( ci ) as defined by hopkins ( 2004 ) ( 0.09 trivial , .10.29 small , .30.49 moderate , .50.69 large , .70.89 very large , .90.99 nearly perfect , 1 perfect ) are provided for all correlations . all statistical analyses were performed using spss for windows ( version 22 , spss inc . chicago , il , usa ) with statistical significance set at an accepted level of p<0.05 . the td , hsd and sd covered was 7604 510 m , 1623 149 m and 300 47 m , respectively . the internal tl ( itrimp ) of simulated match play was 534 74 au . the relationships between external load measures and fitness are shown in table 1 . specifically , td covered during the match simulation was not significantly correlated to aerobic fitness measures i.e. vlt ( r = -0.066 ; p = 0.747 ; 95% ci : 0.014 to 0.081 ; trivial ) , vo2max ( r = 0.152 ; p = 0.458 ; 95% ci : 0.114 to 0.261 ; small ) and vobla ( r = 0.014 ; p = 0.945 ; 95% ci : -0.014 to 0.084 ; trivial ) . hsd was not related to players vo2max ( r = 0.086 ; p = 0.677 ; 95% ci : 0.014 to 0.092 ; trivial ) and vobla ( r = 0.076 ; p = 0.712 ; 95% ci : 0.034 to 0.090 ; trivial ) . in contrast to previous observations , hsd was significantly related to vlt ( r = 0.485 ; p = 0.012 ; 95% ci : 0.314 to 0.619 ; moderate ) . finally , sd showed no significant correlation with vlt ( r = -0.189 ; p = 0.356 ; 95% ci : 0.014 to 0.091 ; trivial ) , vo2max ( r = 0.094 ; p = 0.647 ; 95% ci : 0.074 to 0.102 ; trivial ) and vobla ( r = 0.012 ; p = 0.955 ; 95% ci : 0.004 to 0.074 ; trivial ) . the integration of tl measures was shown to provide significant correlations with aerobic fitness measures . the relationship between tl integration ratios and aerobic fitness measures is shown in table 2 . specifically as shown in figure 1 , td : itrimp showed significant correlations with vo2max ( r = 0.524 ; p = 0.006 ; 95% ci : 0.224 to 0.754 ; large ) and vobla ( r = 0.559 ; p = 0.003 ; 95% ci : 0.314 to 0.774 ; large ) . similar trends were shown in figure 2 for hsd : itrimp and aerobic markers for fitness measures . p = 0.009 ; 95% ci : 0.214 to 0.861 ; large ) and vobla ( r = 0.407 ; p = 0.039 ; 95% ci : 0.204 to 0.641 ; moderate ) . finally , the integration of sd : itrimp shown in figure 3 was significantly related to vlt ( r = 0.611 ; p = 0.001 ; 95% ci : 0.414 to 0.841 ; large ) . table 1the correlation of the external tl with measures of aerobic fitnessexternal tlvo2max ( mlkgmin)vlt ( kmh)vobla ( kmh)total distance ( m)-0.158 - 0.0660.014high speed distance ( m)0.0860.4850.076sprint distance ( m)0.094 - 0.1890.012high speed distance ( m ; 17 kmh ) ; sprint distance ( m ; 22 kmh ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation**correlation is significant p= 0.012 table 2the correlation of external - internal tl ratios with measures of aerobic fitnessexternal : internal tl ratiovo2max ( mlkgmin)vlt ( kmh)vobla ( kmh)td : itrimp0.524 0.3300.599 hsd : itrimp0.3250.502 0.407 sprint : itrimp0.3580.611 0.170td : itrimp = total distance ( m):itrimp ( au ) ; hsd : itrimp = high speed distance ( m ) : itrimp ( au ) ; sprint : itrimp = sprint distance ( m):itrimp ( au ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation**correlation is significant p 0.05**correlation is significant p 0.05**correlation is significant p 0.05**correlation is significant p 0.05**correlation is significant p 0.05 figure 1a scatter plot showing the relationship between ( a ) td : itrimp and vobla ( kmh ) ( b ) td : itrimp and vo2max ( mlkgmin ) figure 2a scatter plot showing the relationship between ( a ) hsd : itrimp and vobla ( kmh ) ( b ) hsd : itrimp and vlt ( kmh ) figure 3a scatter plot showing the relationship between sd : itrimp and vlt ( kmh ) the correlation of the external tl with measures of aerobic fitness high speed distance ( m ; 17 kmh ) ; sprint distance ( m ; 22 kmh ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation correlation is significant p= 0.012 the correlation of external - internal tl ratios with measures of aerobic fitness td : itrimp = total distance ( m):itrimp ( au ) ; hsd : itrimp = high speed distance ( m ) : itrimp ( au ) ; sprint : itrimp = sprint distance ( m):itrimp ( au ) ; vlt = velocity at the lactate threshold ; vobla = velocity at onset of blood lactate accumulation correlation is significant p 0.05 correlation is significant p 0.05 correlation is significant p 0.05 correlation is significant p 0.05 correlation is significant p 0.05 a scatter plot showing the relationship between ( a ) td : itrimp and vobla ( kmh ) ( b ) td : itrimp and vo2max ( mlkgmin ) a scatter plot showing the relationship between ( a ) hsd : itrimp and vobla ( kmh ) ( b ) hsd : itrimp and vlt ( kmh ) a scatter plot showing the relationship between sd : itrimp and vlt ( kmh ) the current study investigated a novel approach of integrating external and internal workload variables as a ratio during hurling specific simulated match play . the results of the study show that measures of match running performance relate poorly to aerobic fitness measures . interestingly , there appears to be a relationship between markers of aerobic fitness and high speed running performance . the relationship has been previously reported in soccer populations ( akubat et al . , 2014 ) , but this is the first study to report this relationship in a hurling population . caution should be taken when utilising high speed running as a marker of performance , the training load and fatigue , given the match to match variability in this performance measure . gregson and colleagues ( 2010 ) reported that high speed running performance could vary by as much as 16% ( cv : 16.2 6.4% , 95% ci : 15.6 - 16.7% ) between competitive games 2015 ) observed large between - match ( within - player ) variation for high speed running distance ( 27.6% ; 90% confidence limits 6.9% [ forwards ] , 20.1% ; 4.1% [ backs ] ) , very high speed running distance ( 68% ; 19% , 34.1% ; 7.5% ) within rugby union . these findings question the reliability of these measures as accurate determinants of a player training load . the non - significant relationship between external load measures and measures of fitness seen in this study is in contrast to many studies that have analysed performance in other sports such as endurance running where the relationships between similar aerobic fitness variables in endurance events show very large to nearly perfect relationships ( mclaughlin et al . , 2010 ) . possible explanations of the non - significant relationships between the external measure of performance and fitness measures seen in this study can be explained by the simulated nature of match play . during competitive hurling match play greater effort may be required at a given time due to external stimuli such as standard of opposition , tactical formation or contextual factors ( paul et al . , 2015 ) . match simulations such as the hurling match play simulation protocol have been previously questioned as they allow players to pace themselves at levels that fail to replicate match play accurately . this pacing strategy can be related to the absence of opposition , tackling , tactical responsibility and game situation , all of which impact the locomotor performance and result in variability that may influence performance ( akubat et al . , 2014 ) . the impact of the above may explain the limited relationships between running performance and aerobic fitness variables observed during the current investigation . with the known limitations of analysing running performance , it may be more valuable to integrate both the external ( distance ) and internal ( itrimp ) load to provide a ratio that considers the internal load of exercise and the external output ( performance ) , thus providing a value representative of match day performance and fatigue ( akubat et al . , 2014 ) . previous studies have shown the value of the basic arbitrary itrimp unit as a marker of assessing adaptations in fitness throughout a pre - season period within professional soccer populations ( manzi et al . , 2013 ) . adding to this the integration method has been shown to have moderate to large correlations with aerobic fitness markers in a soccer population ( akubat et al . , 2014 ) . however , this study is the first to begin the process of integrating tl values in hurling . during the analysis td , hsd and sd athletes are provided with a valuable training and match day marker of the tl given banister s ( 1991 ) proposal that performance at a given time point is a result of fitness of an individual minus the accrued fatigue . the ratio provides the practitioner with a measure of the accrued fatigue at a given time point ( distance covered during the match or training session ) and the fitness measures ( itrimp ) . in addition , the temporal pattern of performance decrement has often been attributed to fatigue ( paul et al . , 2015 ) , therefore , this measure appears to have merits as a tl marker considering that it accounts for performance decrement and provides integration of this decrement with fitness measures . given the amateur nature of hurling players , frequent laboratory testing of fitness may be difficult . in addition , considering the known match to match variability in traditional external load measures such as td and hsd , the tl integration method may provide advancement in the assessment of the fitness if these measures of fitness predetermine a given performance and are reduced due to fatigue , it is reasonable to expect a performance decline . black and dobson ( 2011 ) showed that exercise induced fatigue reduced vo2peak and the ventilatory threshold showing a temporary reduction in measures of aerobic fitness . future research should be conducted during actual match play situations to assess if the integration ratio relationships hold true . moreover , there is a need for the assessment of these ratios as a marker of fatigue . by determining this , we can assess the validity of this ratio as a marker of the dose - response relationship as well as allow practitioners to become more proactive and manipulate the dose rather than reacting to the response . the current study presents additional findings to support the utilisation of integrated tl ratios in intermittent team sport environments . the findings suggest that the use of tl ratios provides data to practitioners that are correlated to aerobic fitness measures . in the current investigation , td was not significantly correlated to fitness measures i.e. vlt ( r = -0.066 ; p = 0.747 ; 95% ci : 0.014 to 0.081 ; trivial ) , vo2max ( r = 0.152 ; p = 0.458 ; 95% ci : 0.114 to 0.261 ; small ) and vobla ( r = 0.014 ; p = 0.945 ; 95% ci : -0.014 to 0.084 ; trivial ) . similar trends were seen with hsd with this external load measure not correlated to vo2max ( r = 0.086 ; p = 0.677 ; 95% ci : 0.014 to 0.092 ; trivial ) and vobla ( r = 0.076 ; p = 0.712 ; 95% ci : 0.034 to 0.090 ; trivial ) . interestingly , tl integration ratios were correlated to fitness measures more closely than external load measures . td : itrimp correlated with aerobic fitness measures i.e. vo2max ( r = 0.524 ; p = 0.006 ; 95% ci : 0.414 to 0.724 ; large ) and vobla ( r = 0.559 ; p = 0.003 ; 95% ci : 0.314 to 0.774 ; large ) . similar trends were shown for hsd : itrimp and aerobic markers for fitness measures i.e. vlt ( r = 0.502 ; p = 0.009 ; 95% ci : 0.214 to 0.861 ; large ) and vobla ( r = 0.407 ; p = 0.039 ; 95% ci : 0.204 to 0.641 ; moderate ) . the sd : itrimp showed significant correlations with vlt ( r = 0.611 ; p = 0.001 ; 95% ci : 0.414 to 0.841 ; large ) . overall , these measures support the use of integrated ratios as a method of monitoring training loads . integrated ratio measures were correlated to fitness measures , as such the use of these ratios presents advancement from the use of external load measures alone in the assessment of aerobic fitness of players .

abstractthe current study aimed to assess the relationship between the hurling player s fitness profile and integrated training load ( tl ) metrics . twenty - five hurling players performed treadmill testing for vo2max , the speed at blood lactate concentrations of 2 mmoll-1 ( vlt ) and 4 mmoll-1 ( vobla ) and the heart rate - blood lactate profile for calculation of individual training impulse ( itrimp ) . the total distance ( td ; m ) , high speed distance ( hsd ; m ) and sprint distance ( sd ; m ) covered were measured using gps technology ( 4-hz , vx sport , lower hutt , new zealand ) which allowed for the measurement of the external tl . the external tl was divided by the internal tl to form integration ratios . pearson correlation analyses allowed for the assessment of the relationships between fitness measures and the ratios to performance during simulated match play . external measures of the tl alone showed limited correlations with fitness measures . integrated tl ratios showed significant relationships with fitness measures in players . td : itrimp was correlated with aerobic fitness measures vo2max ( r = 0.524 ; p = 0.006 ; 95% ci : 0.224 to 0.754 ; large ) and vobla ( r = 0.559 ; p = 0.003 ; 95% ci : 0.254 to 0.854 ; large ) . hsd : itrimp also correlated with aerobic markers for fitness vlt ( r = 0.502 ; p = 0.009 ; 95% ci : 0.204 to 0.801 ; large ) ; vobla ( r = 0.407 ; p = 0.039 ; 95% ci : 0.024 to 0.644 ; moderate ) . interestingly sd : itrimp also showed significant correlations with vlt ( r = 0.611 ; p = 0.001 ; 95% ci : 0.324 to 0.754 ; large ) . the current study showed that tl ratios can provide practitioners with a measure of fitness as external performance alone showed limited relationships with aerobic fitness measures .

Introduction Material and Methods Participants Procedures Statistical Analysis Results Discussion Conclusions

PMC3899549

fistula - in - ano is an abnormal hollow tract lined by unhealthy granulation tissue connecting a primary opening in the anal canal to a secondary opening in the perianal skin . the fistula tract may be single or multiple and may extend from the same primary opening . it is nearly always caused by a previous anorectal abscess , following either poor surgical or spontaneous drainage in the perianal skin . men are involved in 70% of cases and the majority of cases present initially in the 3 to the 6 decade , which incidentally coincide with the mean age occurrence of fournier 's gangrene . fournier 's gangrene , which was first described by jean alfred fournier in 1883 , is an infective necrotizing fasciitis of the perineal , genital or perianal regions as a result of subcutaneous vascular thrombosis and resulting in gangrene of the overlying scrotal skin . fistula - in - ano and fournier 's gangrene still continue to pose management challenges to surgeons . however , there has been no report on management of this concomitant occurrence of these diseases . this is a retrospective study of all fistulas - in - ano complicated by fournier 's gangrene managed in university of maiduguri teaching hospital and federal medical center yola and gombe within a period of 5 years ( january 2007 to december 2011 ) highlighting the causal relationship . the case files of all patients with the diagnosis of fistula - in - ano complicated by fournier 's gangrene during the study period were retrieved from the central medical records department of these hospitals . information retrieved includes the demographic data , mode of presentation , type of infecting organisms , co - morbidity illnesses , nature and outcome of treatment . data analysis was carried out using the statistical package for the social sciences version 16 ( spss 16 ) to determine the level of significance and correlations with the variables . a total of 10 male patients who developed fistula - in - ano and then complicated by fournier 's gangrene with a mean age of 50.5 ( 50 - 59 ) years were managed within the period of 5 years [ table 1 ] . nearly , 50% of patients were peasant farmers , 30% businessmen and 20% were civil servant . all patients presented with initial perianal pain , external opening and discharge and then followed by scrotal swelling and high grade fever [ table 2 ] . nearly , 70% of these patients presented with fournier 's gangrene within 1 - 4 weeks of development of fistula - in - ano , which were preceded by perianal abscess and the rest within 8 weeks [ table 3 ] . fistula - in - ano was submuscular ( intersphincteric ) , 30% subcutaneous with straight anterior external opening and 10% were complex or recurrent running curved course from internal posterior to anterior external opening [ figure 1 ] . age distribution of 10 patients with fistula - in - ano and fournier 's gangrene major clinical features of 10 patients with fistula - in - ano and fournier 's gangrene duration of symptoms prior to presentation of 10 patients with fistula - in - ano and fournier 's gangrene types of fistula - in - ano with fournier 's gangrene at presentation of the 10 patients nearly , 40% of these patients had initial nick for drainage of their perianal abscess and 20% incision and drainage ( i and d ) , but 40% came with spontaneously ruptured perianal abscess , which developed fistula - in - ano and then fournier 's gangrene with no initial treatment [ table 4 ] . types of treatment prior to presentation of 10 patients with fistula - in - ano and fournier 's gangrene all patients were given adequate intravenous fluid and parenteral antibiotics before definitive treatment . 20% of patients had fistulotomy and seton ( rubber band ) application for adequate drainage . in 50% of patients mucosal advancement flap was done to close the internal opening of the fistula , but 30% had fistulotomy and sitz bath [ table 5 ] . types of procedure and surgery for 10 patients with fistula - in - ano and fournier 's gangrene there is a significant relationship between initial treatments , type of fistula - in - ano , durations of the clinical features type of definitive surgical procedure carried out and the outcome [ table 6 ] . the paired t samples tests of association of durations and the clinical features with other variables if the cryptoglandular abscess is either poorly drained or spontaneously ruptures through the perianal skin , then becomes fistula in 50% of patients . severe polymicrobial synergistic infection from the gut flora can cause an extensive subfascial tissue necrosis involving the scrotum , fournier 's gangrene [ figure 2 ] , which is an unusual complication of fistula - in - ano . other common causes of anal fistula include chronic ulcerative colitis , crohn 's disease , tuberculosis , carcinoma of the rectum or anal canal , benign rectal strictures , foreign bodies or diverticulitis . cryptoglandular fistula with fournier 's gangrene fournier 's gangrene , which was thought to be idiopathic by jean alfred fournier ( 1883 ) is now known to have definite etiology and is characterized by obliterative endarteritis causing cutaneous and subcutaneous vascular thrombosis and tissue necrosis , then invaded by polymicrobial enterobacteria . in our region , staphylococcus and proteus species are the commonest mixed growth infection and quinolones ( ciprofloxacin and ofloxacin ) are the most potent antibiotics in treating these infection . urogenital foci such as urethral stricture , indwelling urethral catheter , prostatic message and biopsy are the most common primary adjacent focus of fournier 's gangrene and in africa immunosuppressive illnesses such as diabetes mellitus , human immunodeficiency virus infection , filarial infestations and low socio - economic status contributed to the high prevalence of the disease . in these patients , 50% are in the low socio - economic category , however , 80% of them were otherwise healthy without immunosuppressive illnesses . usually , fournier 's gangrene is presented in the acute form , but recently it runs an indolent course , which some studies attributed it to immunosuppressive comorbidities ; however in our series , it 's probably due to suboptimal antibiotic therapy and delay in referral . these patients presented with high grade fever associated with rigors and sweating , some of them delirious . nearly , 60% of the patients either had a perianal nick [ figure 3 ] or inadequate i and d [ figure 4 ] and 40% had a prior history of spontaneous rupture and discharge from a perianal abscess with worse clinical symptoms . nick of perianal abscess and fournier 's gangrene inadequate perineal abscess drainage and fournier 's gangrene the parks classification system divided fistula - in - ano resulting from cryptoglandular infections as intersphincteric , transsphincteric , suprasphincteric and extrasphincteric , however , the current procedural terminology codes classification include subcutaneous fistula resulting from unhealed anal fissure or anorectal procedures such as hemorrhoidectomy , submuscular fistula ( intersphincteric , low transsphincteric ) and complex fistular , recurrent ( high transsphincteric , suprasphincteric and extrasphinteric , multiple tracts , recurrent ) . majority of fistula - in - ano can be diagnosed based on clinical history and physical examination , however , in cases associated with multiple or complex problems such as a horse shoe abscesses or recurrent complex fistula - in - ano , rectal cancer or crohn 's disease , diagnostic imaging such as transrectal ultrasound , computed tomography scan or magnetic resonance imaging would be required . the parenteral antimicrobials ciprofloxacin and metronidazole were given based on the sensitivity pattern of the microbial pathogens in our region . some of the patients had extensive debridement from the perianal region up to the scrotum and groins leaving the testes bare [ figure 5 ] . hypertonic saline soaking ( sitz bath ) and wound dressing were continued mornings and evenings until the wounds were cleaned and granulated in 2 - 4 months [ figures 6 and 7 ] . if patient presented early with cryptoglandular infection and perianal abscess , the abscess is immediately drained by either radial perianal incision or adequate cruciate incision with unroofing the abscess cavity . these procedures allow adequate abscess drainage and are performed close to anal verge in order to allow fast healing . it is rare for fistula to heal spontaneously even with antibiotic therapy . in order to heal , the internal opening ( crypt ) should be obliterated and therefore operative procedure is necessary . if the internal opening identified , the tract should be probed to clarify and a seton suture of silk passed through to serve as a drain until definitive treatment can be done . nearly , 40% of these patients had a nick of the abscess cavity as primary treatment , which was inadequate i and d and 40% had spontaneous rupture of the abscess cavity . these made perineal spread of the infection rapid and thus prolonged period of patient 's management and increased risk of mortality . although 20% of the patients had adequate i and d , but yet developed a complication of fournier 's gangrene , probably would have benefited from closure of the internal opening as part of primary treatment . extensive perineal and scrotal debridement with bare testes cleaned granulated scrotal and perineal wound ready for grafting healing anal fistula and scrotal wound with continuing sitz bath the operative goals for subcutaneous and submuscular fistulae are to open the tract and remove tract lining either by curettage or electrocautery . the complex or recurrent fistulae may be treated by either non - cutting setons , fibrin glue , ablation , rectal mucosal advancement flap , anal fistula plugs or combination of these in order to avoid sphincter muscle division and its attendant fecal incontinence complication . fistulotomy is a preferred procedure for subcutaneous and submuscular fistulae whether complicated or not and has 3 - 5% risk of flatus and stool leakage and with more muscle cutting fecal incontinence may develop . majority of the patients had their scrotal wounds healed spontaneously with continuing sitz bath [ figure 7 ] , but those with more extensive perineal wound had mesh skin grafting [ figure 8 ] . patients were followed - up over a year with no history of fecal incontinence or fistula recurrence . cryptoglandular infection is an important cause of perianal abscess and fistula - in - ano when poorly managed can lead to fournier 's gangrene , which is a urological emergency . early adequate parenteral antibiotic therapy , primary surgical treatment including closure of internal opening ( crypt ) can prevent serious complications like fournier 's gangrene .

background : fistula - in - ano when complicated by fournier 's gangrene is an unusual finding and always carries high morbidity . this study details our experience in managing 10 cases.methods of study : case files of all patients managed in university of maiduguri teaching hospital and federal medical center of yola and gombe from january , 2007 to december , 2011 were retrieved from medical record departments and other hospital records . these were analyzed for demographic , clinical and pathological variables , the type of treatment and follow-up.results:a total of 10 men with a mean age of 50.5 years ( 35 - 60 ) were managed in the period of study . nearly , 50% of the patients were farmers , 30% businessmen and 20% were civil servant . 7 ( 70% ) of these patients presented with fournier 's gangrene within 4 weeks of development of fistula - in - ano and the rest within 8 weeks . 4 ( 40% ) of these patients had inadequate drainage of their perianal abscess and 2 ( 20% ) had incision and drainage . another 4 ( 40% ) had spontaneously rupture of the perianal abscess . 6 ( 60% ) of the fistula - in - ano was submuscular , 30% subcutaneous and 10% were complex or recurrent . nearly , 20% of patients had fistulotomy and seton application for adequate drainage . mucosal advancement flap was performed in 5 ( 50% ) and fistulotomy in 3 ( 30% ) patients . another 30% had fistulotomy and continuing sitz bath.conclusion:cryptoglandular infection is an important cause of perianal abscesses and fistula - in - ano and if poorly managed results in fournier 's gangrene . early broad spectrum parenteral antibiotic therapy and primary surgical treatment can prevent fournier 's gangrene .

I M R D C

PMC4745447

bacteriocins are toxins produced by the bacteria to inhibit the growth of similar or closely related bacterial strain(s ) during stress conditions . they are structurally , functionally , and ecologically diverse , produced by almost all major lineages of eubacteria and archaebacteria . ribosomal encoded bacteriocins are generally secreted in the extracellular milieu by the producers where they recognize specific receptors on the surface of susceptible or target cells . they induce toxicity in the target cells by different mechanisms like enzymatic nuclease ( dnase or rnase ) or pore formation in cytoplasmic membrane . their structure comprises of three distinct domain organizations : ( i ) a domain involved in recognition of specific receptor r , ( ii ) a domain involved in translocation t , and ( iii ) a domain responsible for their toxic activity c. molecular mass of ribosomal encoded bacteriocins vary from ~25 to 80 kda and are broadly classified into two groups , group a and b , based on their cross - resistance . these proteins have received increasing attention due to their potential use as preservatives in the food industry or in the therapeutic applications for clinical usage . xenorhabdus nematophila is a motile gram - negative bacterium belonging to the family enterobacteriaceae , and forms a symbiotic association in the gut of soil nematode from the family steinernematidae [ 6 , 7 ] . free - living forms of the bacterium have not yet been isolated from soil or water sources , which suggests that the symbiotic association may be essential for the survival of the bacteria in the environment . juvenile ( ijs ) enters digestive tract of the insect larva and subsequently penetrates into hemocoel of the host insect . the nematode can also gain access to the hemocoel via the respiratory spiracles or by penetrating directly through insect cuticle . bacteria , in turn , are essential for effective killing of the insect host and are required by the nematode to complete its life cycle [ 8 , 9 ] . growth in vitro is probably supported by the rich nutrient supply of the laboratory growth media and lack of competition that normally exists in the soil environment . as the bacteria enter stationary phase of growth cycle , they secrete several extracellular products , which include lipase(s ) , phospholipase(s ) , and protease(s ) , and several broad spectrum antibiotics that can be assayed in the culture media [ 10 , 11 ] . these extracellular products are believed to be secreted in the insect hemolymph when the bacteria enter stationary phase . these degradative enzymes break the macromolecules from insect cadaver to provide the developing nematode with nutrient supply , while the antibiotics suppress contamination of the cadaver by other microorganisms . cytoplasmic inclusion bodies , composed of highly expressed crystalline proteins , are also produced by the bacterium during stationary - phase growth . in our earlier study recombinant xenocin - immunity protein complex is toxic to six bacterial genus like bacillus , enterobacter , enterococcus , citrobacter , serratia , and stenotrophomonas . xenocin - immunity protein complex has atypical features which include the following . ( 1 ) tol box which has been replaced by ton box from n terminal end of translocation domain of xenocin . ( 2 ) there is only 30% similarity of xenocin with other bacteriocins . ( 3 ) size of its cognate immunity protein is 42 kda , whereas 1016 kda have been reported in other prokaryotic systems . immunity protein of x. nematophila is a fusion of two different domains , immunity domain and hemolysin domain . ( 4 ) immunity protein has atpase activity , although walker motif is missing in its primary amino acid sequence . ( 5 ) xenocin - immunity protein complex is secretory in nature without any signal sequence . in this study we have cloned , expressed , and purified all the possible domains of xcina and ximb genes . exogenous toxicity assays were performed with purified recombinant xenocin - immunity domain protein complex and other domains . in silico study of the immunity protein showed its similarity with hemolysin and purine ntpase like protein ; therefore , hemolysis and atpase assays were performed . finally , secondary structural analysis of recombinant xenocin - immunity domain protein complex , catalytic - immunity domain protein complex , immunity protein , and its hemolysin domains were performed with circular dichroism . all the chemicals were purchased from sigma ( sigma - aldrich ) except where otherwise mentioned . vector pqe30 , ni - nta agarose resin and qia quick spin columns were from qiagen ( germany ) . e. coli strains dh5 ( bethesda research laboratories ) were used as the host for cloning . e. coli bl 21(de3 ) plyss strain from novagen and m 15 strain from qiagen were used in the expression studies . the plasmid vector pgem - t easy from promega ( madison , usa ) were used for pcr cloning . ampicillin , kanamycin , and chloramphenicol were used in the concentration of 100 , 35 , and 25 g ml , respectively . phylogenetic analysis of immunity protein was done by a method as described earlier . briefly , amino acids of all the protein sequences that matched with immunity protein were aligned by clustalw ( mac vector 7.0 ) , and a tree was constructed using neighbour - joining method , with the best tree mode in the mac vector version 7.0 ( oxford molecular , england ) program . all the constructs were amplified from the 4.3 kb genomic dna fragment of x. nematophila . the orf 1 encoding xcina gene and partial orf 2 encoding immunity domain of ximb gene were obtained by pcr amplification using primer 1 with a bamhi site at the 5 end and a reverse primer 6 with hindiii site at the 3 end . the amplified product ( 2 kb ) was ligated in pgem - t easy and pqe30 vector , producing pjc5 and pjc6 plasmids , respectively . the catalytic domain ( 318 bp ) of xcina gene was cloned with the immunity domain ( first 270 bp of ximb gene ) of ximb gene using primer pair 2 and 6 as described earlier . primer 1 with a bamhi site at the 5 end and a reverse primer transb with ncoi site at the 3 end were used to amplify the translocation domain of xcina gene . amplified product of 1 kb was ligated in pgem - t easy vector and pqe30 vector , producing pjc7 and pjc8 plasmids , respectively . primer 1 with a bamhi site at the 5 end and a reverse primer recb with hindiii site at the 3 end were used to amplify the translocation - receptor domain of xcina gene . the amplified product ( 1.5 kb ) was ligated in pgem - t easy vector and pqe30 vector producing pjc9 and pjc10 plasmids , respectively . primer 3 with bamhi site at the 5 end and a reverse primer 5 with hindiii site at the 3 end used for ximb cloning and forward primer 4 without any restriction site and backward primer 7 without any restriction site were used for cloning ximb hemolysin domain . the amplified products of 1 kb and 700 bp were ligated in pgem - t easy vector producing pjc11 and pjc12 plasmids . the 1 kb amplified product was further ligated in pqe30 and 700 bp amplified product was ligated in pqe31 expression vector producing pjc13 and pjc14 plasmids , respectively . a 2.330 kb dna fragment containing both xcina and immunity domain of ximb gene with native promoters was amplified using xenocinf1 ( 300 bp upstream of start codon of xcina locus ) and primer 6 and cloned in pgem - t easy vector producing pxim construct . the empty pgem - t easy vector designated as pgem was used as control . the plasmids pjc6 , pjc8 , pjc10 , pjc13 , and pjc14 were transformed in m15 cells where as pjc4 was transformed in e. coli bl 21(de3 ) plyss cells . the resulting strains jc4 , jc6 , jc8 , jc10 , jc13 , and jc14 were used for expression and purification of recombinant proteins under the control of iptg inducible t7 promoter as per the protocol described earlier . briefly , overnight grown cultures were diluted 100 fold in fresh 50 ml lb medium and grown till the od600 reached 0.5 . culture was induced by adding 1 mm final concentration of iptg and incubated at 30c for 6 hours . cells were harvested and washed with 40 ml of cold and 50 mm sodium phosphate buffer , ph 8 , containing 300 mm nacl and 50 mm benzamidine ( buffer a ) . the cell pellet was suspended in 25 ml of buffer a and cells were disrupted by sonication at 4c . the cell lysate was centrifuged at 12000 g for 30 min at 4c in a rc5 plus centrifuge , and the 6xhis - tagged recombinant proteins or protein complexes in the soluble fractions were purified as follows . the supernatant from the previous step was loaded on ni - nta agarose column preequilibrated with buffer a at 4c . the column was washed extensively with buffer a , containing 2550 mm imidazole , and the protein / protein complex was eluted with buffer a containing 300 mm imidazole . fractions containing pure protein or protein complex were concentrated using centricon ( millipore pm10 ) . recombinant protein or protein complexes were dialyzed overnight against 100 volumes of 50 mm sodium phosphate buffer , ph 8 , and the final preparations were stored at 20c in the presence of 15% glycerol . to study the neutralizing effect of the immunity domain protein , xcina gene was cloned with its native promoter along with immunity domain of ximb gene which gave rise to pxim . empty pgem - t easy vector was considered as control and transformed in e. coli dh5 to give rise to gem strain . overnight grown strains gem and xim were subcultured in fresh medium and incubated till the od600 reached 0.5 . the cultures were diluted in fresh medium 1 : 100 and induced with 0.3 g ml of mitomycin c ( an inducer of xenocin native promoter ) . the optical densities of the cultures were monitored at 600 nm during different intervals . the bacteriostatic activity of purified recombinant proteins / complexes were determined by the protocol as described earlier . briefly , lb agar plates without antibiotics were overlaid with 3 ml of soft nutrient agar containing indicator e. coli dh5 strain grown in m9 medium , and the protein complex was applied to sterile disks . the plates were incubated overnight at 37c , and the sizes of clearance zones were recorded . freshly isolated rabbit blood cells were washed thrice with phosphate buffer saline ( pbs ) by centrifuging at 1000 g , 4c for 10 minutes . washed erythrocytes were resuspended in pbs to make a final concentration of 4% . the same volume ( 100 l ) of protein ( 5 m ) sample dissolved in pbs and erythrocytes suspension were added into wells of 96-well plate . the plate was then incubated at 37c for 1 hr and centrifuged at 1000 g for 5 minutes . the resulting supernatant was transferred to new wells , and the absorbance was determined at 540 nm on a continuous spectrum microtitre plate reader . protein samples of different concentrations were incubated with 0.2 ci of [ -p ] labelled atp ( 6000 ci/ mmol , perkinelmer life sciences , usa ) in a buffer containing 20 mm tris - hcl ( ph 8.0 ) , 1 mm mgcl2 , 100 mm kcl , 8 mm dtt , and 80 g / ml of bsa in a total reaction volume of 10 l . samples were incubated at 37c for 30 minutes . at the end of the reaction , 1 l of the reaction mixture was spotted on a polyethyleneimine thin - layer chromatography ( tlc ) plate ( sigma - aldrich , usa ) and air - dried . chromatography was performed using 0.5 m licl and 1 m hcooh as the running solvent . the far - uv cd spectrum was recorded between 190 and 260 nm ( 500 l sample volume ) on a jasco j-810 spectropolarimeter equipped with a jasco peltier temperature controller at 25c using 1 mm optical path length quartz cells and the step size was 0.5 nm with 1 nm bandwidth at a scan speed of 20 nm minute . averages of 5 scans were obtained for blank and protein spectra , and the data was corrected for buffer contribution . the results were expressed as mean residue ellipticity in units of degree / cm / dmol . phylogenetic analysis of the xenocin and its immunity protein was done by preparing phylogenetic tree , using neighbour - joining method . results showed that immunity protein formed a separate cluster in the very beginning as shown figure 1(b ) . further , xcina gene was cloned along with n terminal immunity domain ( 10 kda ) of ximb gene . purification of recombinant xenocin - immunity domain ( 10 kda ) protein complex from jc6 strain was done by ni - nta chromatography under native conditions . one was at the position corresponding to 66 kda and another below 14 kda protein marker as shown in figure 2(a ) which corroborates with the size of xenocin and immunity domain of immunity protein , respectively . purification of recombinant catalytic - immunity domain protein complex with ni - nta chromatography under native conditions from jc4 strain also showed two bands corresponding to the size of catalytic domain of xcina and immunity domain of ximb as shown in figure 2(b ) . purification of recombinant translocation domain of xcina gene from jc8 strain with ni - nta chromatography showed multiple bands in sds - page as shown in figure 2(e ) . further , western blot was performed using purified fraction , probed with anti - his antibody , and the result showed single band at 38 kda which corresponds to the size of recombinant translocation domain protein as shown in figure 2(f ) . purification of recombinant translocation - receptor domain protein with ni - nta chromatography from jc10 strain showed two prominent bands in sds - page . upper band corresponded to ~52 kda whereas lower band corresponded to ~28 kda as shown in figure 2(g ) . western blot was performed using whole cell lysate and the purified fraction , probed with anti - his antibody , which showed a single band at 52 kda corresponding to a size of recombinant translocation - receptor domain protein as shown in figure 2(h ) . purification of recombinant full length immunity protein , as well as its hemolysin domain protein from jc13 and jc14 strains , showed less but stable expression as shown in figures 2(c ) and 2(d ) , respectively . neutralization of endogenous toxicity of xcina by immunity domain of ximb gene was determined by endogenous assay . endogenous assay with xim strain ( harboring pxim containing xcina with its native promoter and first 85 amino acid residues of ximb gene ) and gem strain ( harboring empty pgem t - easy vector ) in the presence of mitomycin c showed the same growth profile as shown in figure 3(a ) . exogenous toxicity assay was performed with purified recombinant xenocin - immunity domain ( 10 kda ) complex using e. coli dh5 as target cells . the zone of inhibition was observed as shown in figure 3(b ) ( i ) . purified catalytic - immunity domain protein complex was used to study for the bacteriostatic effect in the exogenous assays . the zone of inhibition was not observed in this case as shown in figure 3(b ) ( ii ) . similar results were observed when full length immunity protein ( 42 kda ) encoded by ximb gene or its hemolysin domain ( 32 kda ) was used for exogenous assay as shown in figure 3(b ) ( iii ) and ( iv ) , respectively . moreover , as expected zone of inhibition was not observed in buffer control experiment as shown in figure 3(b ) ( v ) . protein - protein blast results ( http://www.ncbi.nlm.nih.gov/blast/ ) of the immunity protein showed its similarity with hemolysin ( aaf42109 ) and purine ntpase like protein ( data not shown ) . hemolytic assay with fresh rabbit red blood cells was performed with purified full length immunity protein as well as its immunity and hemolysin domain . results showed that none of the protein had hemolytic activity ( data not shown ) . atpase assay was performed with purified recombinant full length immunity protein , its immunity domain , and hemolysin domain . atpase activity was not detected in recombinant immunity domain , hemolysin domain protein , and even in the purified bsa which was used as negative control as shown in figure 4(a ) lane 1 . however , full length recombinant immunity protein ( 42 kda ) showed atpase activity with increasing concentration of protein and was comparable to the atpase activity of purified groel protein of x. nematophila which was used as the positive control , as shown in figure 4(a ) lane 2 . the far uv spectra of purified recombinant xenocin - immunity domain ( 10 kda ) protein complex , catalytic - immunity domain protein complex , immunity , and its hemolysin domain were recorded at 25c as shown in figure 5 . recombinant xenocin - immunity domain protein complex was found to contain 41% -helical structure and 21% -sheet . catalytic - immunity domain protein complex was found to contain 51% -sheet and only 7% -helical structure . in case of full length immunity protein 30% of the secondary structure hemolysin domain of the immunity protein also had the same secondary conformation with 30% -helical and 11% -sheet content . recombinant xenocin-(66 kda- ) immunity ( 42 kda ) protein complex has a broad range bacteriostatic property , inhibiting the growth of six insect gut resident bacterial species . due to only 30 - 31% primary sequence similarity with other bacteriocins , xenocin from the x. nematophila forms a distinct cluster in phylogenetic tree . phylogenetic analysis of immunity protein showed similar results , in which immunity protein from x. nematophila forms a separate cluster in the very beginning . this could be due to variable length of immunity protein from x. nematophila . cognate immunity protein of xenocin consists of 368 amino acid residues and is a unique fusion of two different domains . its n terminal ( first 85 amino acid residues ) showed similarity with immunity protein from other prokaryotic systems , whereas the c terminal showed similarity with hemolysin ( n. meningitidis accession no . three - dimensional structure of xenocin has been recently deciphered by homology modelling in my lab . it is a multidomain protein which consists of 576 amino acid residues . from its n terminal 1327 amino acid residues form translocation domain ( t ) , 328476 amino acid residues form the middle receptor domain ( r ) , and amino acid residues from 477576 form the catalytic domain ( c ) at the c terminal . while cloning xcina gene alone in expression vector , not a single transformant was observed . one reason for this result could be the leaky expression of toxic xcina gene . to address this question , xcina gene was cloned along with n terminal immunity domain ( 10 kda ) of ximb gene . when the recombinant protein from jc6 strain was purified by ni - nta chromatography , xenocin was visible in sds - page at position corresponding to 66 kda whereas immunity domain of immunity protein was observed below 14 kda protein marker . this result showed that n terminal immunity domain ( 10 kda ) of immunity protein ( 42 kda ) encoded by ximb gene is enough to bind with and neutralize the in vivo toxic effect of xcina gene . to confirm this result and to map the minimum functional domain of immunity protein required to abolish the xcina gene toxicity in vivo , first 85 amino acids of immunity protein were cloned along with xcina gene under its native promoter . same growth profile of xim strain ( harboring pxim ) and gem strain ( harboring empty pgem t - easy vector ) in the presence of mitomycin c confirmed that first 85 amino acid residues of ximb gene were able to neutralize the toxic activity of xenocin in vivo . further , purification of recombinant catalytic - immunity domain protein complex with ni - nta chromatography under native conditions from jc4 strain confirmed the minimal domains of xcina and ximb genes that could be expressed and purified . as we were unable to express xcina gene alone which is composed of translocation , receptor , and catalytic domain an attempt had been made to clone , express , and purify the translocation domain alone or along with receptor domain of xcina gene . in native conformation , translocation domain of bacteriocin like colicin e3 interacts with catalytic domain of e3 and immunity protein via receptor domain , and this interaction further provides stability to the translocation domain . however , in recombinant translocation domain of xcina , receptor and catalytic domains as well as immunity protein were missing which may be probably made it to attain an open conformation and be susceptible to proteases from the host cells . hence , during the purification of translocation domain from pjc8 , multiple bands were observed in sds - page . however , western blot with purified fraction when probed with anti - his antibody showed a single band corresponding to the size of translocation domain which confirmed the expression of translocation domain alone . further , purification of recombinant translocation - receptor domain protein with ni - nta chromatography from jc10 strain , showed two prominent bands in sds - page rather than multiple bands . upper band corresponded to ~52 kda whereas lower band corresponded to ~28 kda . western blot using whole cells and purified fraction when probed with anti - his antibody showed a single band at 52 kda which is corresponding to a size of recombinant translocation - receptor domain protein . therefore , we inferred that recombinant translocation domain along with receptor domain of the xcina gene was more stable as compared to the recombinant translocation domain alone , but due to the absence of catalytic domain and immunity protein it was still prone to proteases of the host cell . endogenous toxicity assays with xim and gem strains were performed to identify the minimum domain required to neutralize the xcina toxicity . results showed that first 85 amino acids of the immunity protein were able to neutralize xenocin endogenous toxicity as both strains had the same growth profile . moreover , from this experiment we inferred that expression of cognate immunity protein domain and its binding to the catalytic domain of xenocin occurs in the host cell simultaneously . receptor domain in bacteriocin plays a major role in the exogenous toxic effect , as it is the first domain which binds to ligand present on the surface of its target cells . binding is followed by import of bacteriocin into the cells with the assistance of either ton proteins ( exbb , exbd , and tonb ) or tol proteins ( tola , -b , -q , and -r ) of periplasm and facilitate the translocation of the catalytic domain of bacteriocins into the periplasmic space of the target cell for further processing [ 5 , 1618 ] . interaction of bacteriocins either with ton or tol proteins of periplasm occurs via ton or tol box present at their n terminal end of the translocation domain . interestingly , although xenocin has rnase activity , tol box in its translocation domain has been replaced by ton box . this could be due to the domain swapping which generally occurs during the horizontal gene transfer in prokaryotes . in the colicin e3-immunity complex , binding of the receptor domain of e3 to the surface protein on target cells leads to the conformational changes which assisted the immunity protein to dissociate from the protein complex [ 4 , 18 ] . the immunity protein from x. nematophila is a unique fusion of immunity domain ( 10 kda ) and hemolysin domain ( 32 kda ) protein , and endogenous assay confirmed that immunity domain of ximb gene is enough to neutralize the detrimental effect of xcina gene . therefore , exogenous assay was performed with purified recombinant xenocin - immunity domain ( 10 kda ) complex using e. coli dh5 as target cells to determine translocational ability of xenocin - immunity domain ( 10 kda ) complex into the cytoplasm of target cells . clear zone of inhibition was observed in the exogenous toxicity assay due to the detrimental effect of xenocin - immunity domain ( 10 kda ) protein complex . this result confirmed the functionality of translocation as well as receptor domain of xenocin and their individual roles in internalization of xenocin into the cytoplasm of target cells . moreover , it also confirmed that hemolysin domain ( 32 kda ) of ximb gene has no role in the internalization of xenocin as well as in exogenous toxicity . catalytic domain of xcina gene along with immunity domain of ximb gene was the minimum domain which could be expressed and purified . therefore , purified catalytic - immunity domain protein complex was studied for the bacteriostatic effect in the exogenous assays . results showed that zone of inhibition was not observed , which again confirmed the role of translocation as well as receptor domain of xcina gene for its internalization into the cytoplasm on the target cells . similar results were observed when full length immunity protein ( 42 kda ) encoded by ximb gene or its hemolysin domain ( 32 kda ) was used for exogenous assay . moreover , as expected zone of inhibition was not observed in the experiment in which buffer was used as a negative control . to neutralize the detrimental effect of bacteriocins , the molecular weight of the immunity protein in other prokaryotic systems is between 10 and 16 kda ; however , immunity protein encoded by ximb gene corresponds to 42 kda . protein - protein blast using blastp ( http://www.ncbi.nlm.nih.gov/blast/ ) of the immunity protein showed similarity with hemolysin ( aaf42109 ) and purine ntpase like protein . hemolytic assay with fresh rabbit red blood cells was performed with purified full length immunity protein as well as its immunity and hemolysin domains . due to the partial primary amino acid sequence similarity with hemolysin protein , none of the protein / protein domains showed hemolytic activity . as immunity protein also showed similarity with protein like purine ntpase 's atpase assay was performed with purified recombinant full length immunity protein , its immunity domain , and hemolysin domain . although full length immunity protein showed atpase activity , in silico analysis did not show walker motif in its primary amino acid sequence , which is generally present in the proteins having atpase activity . possibility of atpase activity in immunity protein towards the secretion of recombinant xenocin - immunity protein complex can not be ruled out at this stage as none of the proteins have any signal sequence at their either ends . secondary structure analysis of purified recombinant xenocin - immunity domain ( 10 kda ) protein complex , catalytic - immunity domain protein complex , immunity , and its hemolysin domain was done with far uv spectra . high percentage of -helical structure of xenocin - immunity complex is attributed due to the helix turn helix structure of receptor domain of xenocin which corroborates with its recently deciphered three - dimensional structure . catalytic - immunity domain protein complex was found to contain 51% -sheet and only 7% -helical structure . this open conformation with high -sheet might be beneficial to bind with its substrate ( rna ) and act upon it [ 20 , 21 ] . in case of full length immunity protein 30% of hemolysin domain of the immunity protein also had the same secondary conformation with 30% -helical and 11% -sheet content . since immunity protein is a novel fusion of two different domains the role of its secondary is not deciphered yet .

xenorhabdus nematophila , a gram - negative bacterium belonging to the family enterobacteriaceae is a natural symbiont of a soil nematode from the family steinernematidae . in this study cloning , expression , and purification of broad range iron regulated multidomain bacteriocin called xenocin from x. nematophila ( 66 kda , encoded by xcina gene ) and its multidomain immunity protein ( 42 kda , encoded by ximb gene ) have been done . xcina - ximb ( n terminal 270 bp ) , translocation , and translocation - receptor domain of xcina , ximb , and its hemolysin domain were cloned , expressed , and purified by single step ni - nta chromatography under native conditions . in the functional characterization , neutralization of xcina toxicity by immunity domain of ximb gene was determined by endogenous assay . exogenous toxic assays results showed that only the purified recombinant xenocin - immunity domain ( 10 kda ) protein complex had toxic activity . atypical cognate immunity protein ( 42 kda ) of xenocin was fusion of immunity domain ( 10 kda ) and hemolysin domain ( 32 kda ) . in silico analysis of immunity protein revealed its similarity with hemolysin and purine ntpase like proteins . hemolytic activity was not observed in immunity protein or in its various domains ; however , full - length immunity protein lacking walker motif showed atpase activity . finally , using circular dichroism performed secondary structural analyses of all the recombinant proteins / protein complexes .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

PMC3836686

in an era of finite resources and ever - increasing medical possibilities , every health care system faces challenges in determining which new health technologies , including medical devices , should be introduced into clinical practice.1 while technology can improve safety2 and have other benefits , it can also bring new risks35 and contribute to the increasing cost of care.6,7 ideally , health organizations should have a systematic process both to gather relevant scientific information about a technology s safety and effectiveness and to decide whether the technology is suitable for the local setting . however , several studies have concluded that the decision - making process for the adoption of new technologies could be improved at the institutional level.810 the international network of agencies for health technology assessment defines health technology assessment ( hta ) as : [ ] the systematic evaluation of properties , effects , and/or impacts of health care technology . it may address the direct , intended consequences of technologies as well as their indirect , unintended consequences . its main purpose is to inform technology - related policymaking in health care.11 hta reports from various international , national , and provincial agencies provide comprehensive , objective , evidence - informed analyses about the safety , clinical effectiveness , cost - effectiveness , and the broader impact of health technologies , including devices , drugs , and procedures . despite the exponential growth in the numbers and the types of hta reports over the past decade and their documented impact on health care,1215 it has been observed that recommendations are not put into practice as often as their envisioned potential.1618 there are several possible reasons for this . first , local decision makers may be unaware of the wealth of hta information available to them.19,20 second , external hta agencies may not be able to consider operational factors that are critical for local decision - making , such as local needs , financial impact , and the presence of local alternatives , trained personnel , or sufficient resources . third , health care organizations may lack a systematic process by which to integrate and translate context - free hta reports with context - sensitive considerations , particularly as decision making on technology adoption at the local level is a complex process , involving many disciplines and players within an organization.2123 indeed , it has been shown that a key determinant of successful hta uptake is a clear , fair , and consistent decision - making process for the approval and introduction of new health technologies.24 to address this issue , the department of surgery and surgical services in the former calgary health region ( calgary , ab , canada ) developed and implemented a systematic decision - making process to introduce new health technologies called the local hta decision support program , which comprises sets of forms and tools.25 the forms gather context - free , scientific evidence about the technology , such as hta reports , and context - sensitive information about local needs and constraints . the tools provide decision guides based on explicit criteria26 to assist an interdisciplinary advisory committee in translating the evidence to make a recommendation to the surgical executive committee , who then makes the final decision on whether and under what conditions the technology will be used . over a five - year period , 68 technology requests were reviewed using this program . as well as producing yes or no decisions on some technologies , the local hta decision support program gave other technologies restricted approval , with full approval contingent on satisfying clinical outcomes reporting , training protocol development , or funding.25 thus , the program provides a strong link between hta , outcomes measures and research , and improvement in the quality of care . we have observed considerable interest on the part of health care organizations in developing a similar program to combine the evidence of hta reports with local operational considerations to produce technology adoption decisions for their own needs . an alternative would be to adapt a preexisting program to the needs of the new setting rather than developing one de novo . the present project was launched to develop a framework and tools for adapting the local hta decision support program for use by other departments in the calgary health region . the operation of the program itself has been described in detail elsewhere.25 the purpose of this article is to describe the adaptation framework and tools , identify the key elements required for successful participation , and describe the lessons learned to support others who wish to use this framework for adapting our local hta decision support program , or a similar one , for their local needs . at the time of this project , the calgary health region was an integrated , primarily urban health authority in alberta , canada . it provided services across the continuum of care , from community health services to acute tertiary care , and served about one million people . its administrative structure comprised 14 major regional clinical departments , which worked within a portfolio structure . each portfolio was overseen by an executive director ( administrator ) and a medical director ( physician ) , who worked with the senior management team in the region . since then , the calgary health region has been restructured as part of a single provincial health services organization ( alberta health services ) . the department of surgery and surgical services in the calgary area comprises 286 surgeons in 14 divisions . following an extensive hta education initiative , there was a desire to see the local hta decision support program originally developed by the department of surgery and surgical services adopted by other departments within the calgary health region.19,27 with strong support by senior management , the present project was launched to develop a framework and tools for adapting the local hta decision support program for use by other departments in the region . adaptation of the local hta decision support program involved six steps organized into three main phases : set - up , review and adaptation , and finalization . each phase included a set of objectives , tasks , and documents ( table 1 ) modified from the adapte framework.28 the set - up phase consisted of tasks to be completed before the adaptation process . step 1 was the development of a local health technology assessment decision support program : review and adaptation manual,29 which contained a description of the local hta decision support program , hta - related reference materials for education purposes ( for example , table 2 ) , a project time line , and tips on program administration and evaluation . step 2 involved identification , education , and selection of departments ready and willing to review and adapt the program . in addition to surgery and surgical services , we approached ten other departments within the calgary health region we scheduled semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees to introduce the project , gather initial feedback , and gain approval for their participation in the adaptation process . each department s readiness for the project was determined using an assessment of readiness tool ( table 3 ) . outcomes required to proceed included the department s desire to change its manner of introducing new health technologies and a commitment of resources and personnel , namely the appointment of the local hta leaders ( a physician and administrator team ) to oversee the review and adaptation phase . we then met with these leaders to ensure they understood the local hta decision support program , the material in the review and adaptation manual,29 and what would be required for the review and adaptation process . the review and adaptation phase involved independent and joint reviews of the source program by participating departments . for step 3 , each department s appointed local hta leaders were charged with conducting an independent review with their membership either in principle by evaluating the structure , forms , and processes of the program or in practice by evaluating actual requests for new technologies from their members . each participating department was asked to provide written feedback using the points to consider questionnaire ( table 4 ) . step 4 consisted of a joint review by all participating departments in the form of full - day retreats . the retreat objectives were to learn from each department s experience in reviewing the program , to share input and ideas for improvement , to explore the need for consistency and the need for flexibility , and to determine future directions and next steps . retreats were divided into five sessions that followed the outline of the points to consider questionnaire ( table 4 ) to systematically review the program s policy , flow , structure , content , and funding . participants were asked to share their observations and to make joint recommendations to improve the program . retreat reports were produced that incorporated structured feedback from participants during working sessions and notes made by the facilitator and research team members . the finalization phase involved revision of the local hta decision support program and review of the adaptation process itself . for step 5 , feedback was compiled from the semi - structured interviews and meetings held with each of the local hta leaders from individual departments , written comments provided on the points to consider questionnaire reporting form , and the retreat reports . data were analyzed using a content analysis - type approach.30 major themes and subthemes were developed through comparison and categorization of information gathered . the team met to discuss the outcomes and reached consensus in cases where discrepancies arose . the outcomes were then interpreted into meaningful concepts pertaining to the local hta decision support program , and the program was revised accordingly . for step 6 , we developed a project evaluation questionnaire and gave it to all participants at the end of each retreat to evaluate the review and adaptation process used in this project . at the time of this project , the calgary health region was an integrated , primarily urban health authority in alberta , canada . it provided services across the continuum of care , from community health services to acute tertiary care , and served about one million people . its administrative structure comprised 14 major regional clinical departments , which worked within a portfolio structure . each portfolio was overseen by an executive director ( administrator ) and a medical director ( physician ) , who worked with the senior management team in the region . since then , the calgary health region has been restructured as part of a single provincial health services organization ( alberta health services ) . the department of surgery and surgical services in the calgary area comprises 286 surgeons in 14 divisions . following an extensive hta education initiative , there was a desire to see the local hta decision support program originally developed by the department of surgery and surgical services adopted by other departments within the calgary health region.19,27 with strong support by senior management , the present project was launched to develop a framework and tools for adapting the local hta decision support program for use by other departments in the region . adaptation of the local hta decision support program involved six steps organized into three main phases : set - up , review and adaptation , and finalization . each phase included a set of objectives , tasks , and documents ( table 1 ) modified from the adapte framework.28 the set - up phase consisted of tasks to be completed before the adaptation process . step 1 was the development of a local health technology assessment decision support program : review and adaptation manual,29 which contained a description of the local hta decision support program , hta - related reference materials for education purposes ( for example , table 2 ) , a project time line , and tips on program administration and evaluation . step 2 involved identification , education , and selection of departments ready and willing to review and adapt the program . in addition to surgery and surgical services , we approached ten other departments within the calgary health region we scheduled semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees to introduce the project , gather initial feedback , and gain approval for their participation in the adaptation process . each department s readiness for the project was determined using an assessment of readiness tool ( table 3 ) . outcomes required to proceed included the department s desire to change its manner of introducing new health technologies and a commitment of resources and personnel , namely the appointment of the local hta leaders ( a physician and administrator team ) to oversee the review and adaptation phase . we then met with these leaders to ensure they understood the local hta decision support program , the material in the review and adaptation manual,29 and what would be required for the review and adaptation process . the review and adaptation phase involved independent and joint reviews of the source program by participating departments . for step 3 , each department s appointed local hta leaders were charged with conducting an independent review with their membership either in principle by evaluating the structure , forms , and processes of the program or in practice by evaluating actual requests for new technologies from their members . each participating department was asked to provide written feedback using the points to consider questionnaire ( table 4 ) . step 4 consisted of a joint review by all participating departments in the form of full - day retreats . the retreat objectives were to learn from each department s experience in reviewing the program , to share input and ideas for improvement , to explore the need for consistency and the need for flexibility , and to determine future directions and next steps . retreats were divided into five sessions that followed the outline of the points to consider questionnaire ( table 4 ) to systematically review the program s policy , flow , structure , content , and funding . participants were asked to share their observations and to make joint recommendations to improve the program . retreat reports were produced that incorporated structured feedback from participants during working sessions and notes made by the facilitator and research team members . the finalization phase involved revision of the local hta decision support program and review of the adaptation process itself . for step 5 , feedback was compiled from the semi - structured interviews and meetings held with each of the local hta leaders from individual departments , written comments provided on the points to consider questionnaire reporting form , and the retreat reports . data were analyzed using a content analysis - type approach.30 major themes and subthemes were developed through comparison and categorization of information gathered . the team met to discuss the outcomes and reached consensus in cases where discrepancies arose . the outcomes were then interpreted into meaningful concepts pertaining to the local hta decision support program , and the program was revised accordingly . for step 6 , we developed a project evaluation questionnaire and gave it to all participants at the end of each retreat to evaluate the review and adaptation process used in this project . comments provided described how some technologies were introduced , removed , or replaced with alternatives without adequate evidence review or input from clinicians . to give some examples , however , the upgraded device was incompatible with existing hand pieces and added a large generator to an already tight space . although hta reports indicated good evidence for efficacy , the device failed to improve patient outcomes in our local setting , due to the lack of lead time to allow for appropriate training . however , no funds were given for its high operating cost , and this unbudgeted expense then fell to the health care system . new endoscopes were purchased , but they required specialized sterilization equipment , which was not available in all operating rooms . numerous examples were also given of requests for technologies that were based only on information obtained from the manufacturers representatives . consequently , department administrators and physician leaders wanted a system that would provide greater accountability and broader review for technology adoption . there was also some resistance to change particularly around the perceived time and work required to run a local hta decision support program . in addition to the department of surgery and surgical services , we were successful in recruiting eight out of the ten departments originally approached for a total of nine departments : six clinical departments and three administrative departments . all the clinical departments appointed a physician and an administrator as local hta coleaders , while the administrative departments appointed only an administrator as their hta leader . the department of surgery and surgical services already had a functioning physician - administrator team as the hta coleaders . of the two departments that declined to participate in the project , one was in the midst of organizational transition after hiring a new department head and the other was not interested in developing its own program as its technology purchasing was overseen by the department of surgery and surgical services . four departments reviewed the local hta decision support program in practice ( three invited departments as well as surgery and surgical services ) , and five departments reviewed the program in principle . all participating departments submitted their individual review feedback questionnaire and participated in the joint retreats . in total , we met with 67 individuals during the semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees , including physicians , program managers , nurse clinicians , patient care managers , health services directors , health services managers , clinical product specialists , clinical safety leaders , researchers , and financial analysts , as well as the senior administrators . thirteen individuals participated in the retreat for the first review cycle ; 16 participated in the retreat for the second review cycle . during the joint retreats , a significant amount of time what section of the points to consider questionnaire ( table 4 ) ; that is , what information needs to be gathered to ensure that the department s local hta committee would have enough information to decide if a technology could be simply approved or if it needed further assessment ? this was a contentious issue , because it is difficult to strike a balance between avoiding unnecessary work while still being able to protect patient safety and reduce risk when introducing new technologies . there was good consensus on the why section of the questionnaire ; that is , the need for a systematic and transparent decision support program for technology review and adoption and a clear policy statement . there was also good consensus on the how , who , and funding sections of the questionnaire , with the general recommendation that the details of program operation should be left to the discretion of individual departments . similar comments were brought forward by those who reviewed the program in practice as compared with those who reviewed in principle . to evaluate the review and adaptation process used in this project , the research team reviewed the responses to the project evaluation questionnaire that was given to participants after each of the two retreats . for the may 2007 retreat , 89%100% of respondents ( n=9 ) agreed or strongly agreed that the retreat objectives were met and 100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were the revised local hta decision - support program is adaptable to the needs of various clinical departments and i am committed to the next steps . for the november 2007 retreat , 80%100% of respondents ( n=10 ) agreed or strongly agreed that the retreat objectives were met , and 90%100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were : the revised local hta decision support program is adequate to go live for january 2008 implementation , and i am committed to continued participation . one person disagreed with the first outcome statement , but no other participants scored disagree or strongly disagree for any of the other objective or outcome statements . open - ended comments on the project evaluation questionnaire revealed an overwhelming desire to continue face - to - face meetings . participants found value in hearing the experiences of others using or reviewing the program , gathering feedback from different perspectives , and working collaboratively to come to consensus . as well , participants affirmed the value in having a fair , standardized , and consistent process for technology adoption across the various specialties within the calgary health region . using the above described methodology , two cycles of the review and adaptation and finalization phases were performed with invited departments . as part of another project,26 an additional cycle of review comments provided described how some technologies were introduced , removed , or replaced with alternatives without adequate evidence review or input from clinicians . to give some examples , however , the upgraded device was incompatible with existing hand pieces and added a large generator to an already tight space . although hta reports indicated good evidence for efficacy , the device failed to improve patient outcomes in our local setting , due to the lack of lead time to allow for appropriate training . however , no funds were given for its high operating cost , and this unbudgeted expense then fell to the health care system . new endoscopes were purchased , but they required specialized sterilization equipment , which was not available in all operating rooms . numerous examples were also given of requests for technologies that were based only on information obtained from the manufacturers representatives . consequently , department administrators and physician leaders wanted a system that would provide greater accountability and broader review for technology adoption . there was also some resistance to change particularly around the perceived time and work required to run a local hta decision support program . in addition to the department of surgery and surgical services , we were successful in recruiting eight out of the ten departments originally approached for a total of nine departments : six clinical departments and three administrative departments . all the clinical departments appointed a physician and an administrator as local hta coleaders , while the administrative departments appointed only an administrator as their hta leader . the department of surgery and surgical services already had a functioning physician - administrator team as the hta coleaders . of the two departments that declined to participate in the project , one was in the midst of organizational transition after hiring a new department head and the other was not interested in developing its own program as its technology purchasing was overseen by the department of surgery and surgical services . four departments reviewed the local hta decision support program in practice ( three invited departments as well as surgery and surgical services ) , and five departments reviewed the program in principle . all participating departments submitted their individual review feedback questionnaire and participated in the joint retreats . in total , we met with 67 individuals during the semi - structured interviews and meetings with each department s clinical and administrative heads and their executive committees , including physicians , program managers , nurse clinicians , patient care managers , health services directors , health services managers , clinical product specialists , clinical safety leaders , researchers , and financial analysts , as well as the senior administrators . thirteen individuals participated in the retreat for the first review cycle ; 16 participated in the retreat for the second review cycle . during the joint retreats , a significant amount of time what section of the points to consider questionnaire ( table 4 ) ; that is , what information needs to be gathered to ensure that the department s local hta committee would have enough information to decide if a technology could be simply approved or if it needed further assessment ? this was a contentious issue , because it is difficult to strike a balance between avoiding unnecessary work while still being able to protect patient safety and reduce risk when introducing new technologies . there was good consensus on the why section of the questionnaire ; that is , the need for a systematic and transparent decision support program for technology review and adoption and a clear policy statement . there was also good consensus on the how , who , and funding sections of the questionnaire , with the general recommendation that the details of program operation should be left to the discretion of individual departments . similar comments were brought forward by those who reviewed the program in practice as compared with those who reviewed in principle . to evaluate the review and adaptation process used in this project , the research team reviewed the responses to the project evaluation questionnaire that was given to participants after each of the two retreats . for the may 2007 retreat , 89%100% of respondents ( n=9 ) agreed or strongly agreed that the retreat objectives were met and 100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were the revised local hta decision - support program is adaptable to the needs of various clinical departments and i am committed to the next steps . no participant scored disagree or strongly disagree for any of the objective or outcome statements . for the november 2007 retreat , 80%100% of respondents ( n=10 ) agreed or strongly agreed that the retreat objectives were met , and 90%100% agreed or strongly agreed that the desired outcomes were achieved . these outcomes were : the revised local hta decision support program is adequate to go live for january 2008 implementation , and i am committed to continued participation . one person disagreed with the first outcome statement , but no other participants scored disagree or strongly disagree for any of the other objective or outcome statements . open - ended comments on the project evaluation questionnaire revealed an overwhelming desire to continue face - to - face meetings . participants found value in hearing the experiences of others using or reviewing the program , gathering feedback from different perspectives , and working collaboratively to come to consensus . as well , participants affirmed the value in having a fair , standardized , and consistent process for technology adoption across the various specialties within the calgary health region . using the above described methodology , two cycles of the review and adaptation and finalization phases were performed with invited departments . as part of another project,26 an additional cycle of review decision making is a complex process , involving many disciplines and players within an organization when introducing new health technologies , including medical devices.23 because of cultural and organizational differences , a recommendation for technology adoption in one local setting may not be appropriate in another , even when the scientific evidence , such as that supplied by hta reports , is the same . thus , careful consideration of the specific questions relevant to local needs , priorities , legislation , policies , values , norms , as well as human and material resources , is as necessary as acquiring impartial scientific evidence . the local hta decision support program developed initially by the department of surgery and surgical services of the calgary health region is one attempt to provide a model ( structure , processes , criteria , and decision tools ) for decision making that systematically gathers scientific , operational , and value - based information for consideration when introducing new health technologies . similar programs exist elsewhere.3134 any program that attempts to integrate context - free scientific evidence about a technology with context - sensitive information about the setting in which it will be used must be appropriate and relevant for each local organizational body . however , small rural and community hospitals or health authorities with fewer financial , personnel , and community resources may be poorly positioned for the investment required for de novo decision support program development . programs developed by large health care systems can be adapted and applied successfully to rural settings , provided that needed tools are provided for the adaptation process.35 this paper provides an adaptation process that can be used in lieu of de novo program development , much like processes for adapting clinical practice guidelines.36,37 the adaptation process described and used in this article involved six steps organized into three main phases : the set - up phase involved the development of a manual and identifying and educating interested departments , the review and adaptation phase involved assisting the departments to conduct an actual review and adaptation of the program , and the finalization phase involved program revision and evaluation of the adaptation process . during the set - up phase , we found a general dissatisfaction of each department with its traditional decision - making processes when introducing new health technologies . similar dissatisfaction has been observed by others10 and provides a good opportunity to restructure these processes . to do this , we found that provision of a clearly written document , the local health technology assessment decision support program : review and adaptation manual , was vital to educate members about the value of hta and a structured decision support program and to capture interest and buy - in . hta , therefore having well - written program documents and educational reference materials was vital to ensure their learning and understanding . a key document proved to be the table that explained the distinction between hta producers and hta users . we found that understanding this difference was important for understanding the rationale for a local hta decision support program . the program does not duplicate the efforts of hta producers ; it complements their work by acting as the receptor to use hta reports . the assessment of readiness tool was a critical element of the set - up phase . it required that participating departments demonstrate readiness for change and appoint local hta physician and administrative leaders . during the review and adaptation phase , the research team provided ongoing assistance to participating departments by means of phone , email , and face - to - face meetings and education . we ensured that we provided a variety of methods to solicit feedback , as recommended by others.38 we found that the points to consider questionnaire ( why , how , who , what , funding ) was essential to ensure that all important program review questions would be systematically discussed by reviewing departments . the request that each department appoint an hta physician leader and an hta administrative leader was crucial to the success of the project . while physicians were more focused on clinical evidence , training and credentialing issues , and other such clinically relevant topics , their administrative counterparts ensured that infrastructure implications , cost , and organizational impact were reviewed . similarly , the presence of relevant stakeholders , such as physicians , program managers , nurse clinicians , patient care managers , health services directors , health services managers , clinical product specialists , clinical safety leaders , researchers , financial analysts , and high - level health services administrators in the review and adaptation process can provide critical advantages . first , it ensures that those most likely to use the program have an opportunity to offer feedback and to identify problems before it is finalized . second , it gives administrators the opportunity to consider the impact on the organization of implementing the program and to begin preparing for its future adoption . third , the solicitation of practitioner feedback serves as the first wave of dissemination of the proposed program . fourth , experience elsewhere has shown that when surgeons guide the appraisal of new and emerging surgical technologies , the benefits of an evidence - informed approach in the provision of high - quality patient care are realized.39 the recommendation taken forward to the finalization phase was that the content ( what ) should be standardized across departments and an interdisciplinary team should review the application , but that administrative details ( who and how ) should be left to the discretion of each department . this reflects the importance participants saw in having a fair , standardized , and consistent decision support program for technology adoption across specialties within the institution while acknowledging flexibility of operation . participants demonstrated a strong desire to adopt the decision support program and to keep the review process going by engaging in regular face - to - face group collaboration and interaction . in fact , the review and adaptation and finalization phases can be repeated on a regular scheduled basis in a retreat setting , resulting in a continuous improvement cycle , as well as developing a community of practice , which is known to be effective in sustaining the viability and improvement of programs of interest.40 we identified several obstacles to long - term implementation of the program . first , staff turnover during the course of implementation can result in a continual need to educate and update decision makers . second , there is yet no process by which any cost savings realized by the adoption of new technologies can be attributed back to the operation of the program . third , shifting priorities of a health care system ( for example , funds diverted to pandemic flu inoculations ) may result in the cancellation or delay in the implementation of a recommended new technology . these obstacles are in addition to challenges encountered during the project itself , including finding common meeting times among all stakeholders , some resistance to change ( ie , the perceived fear that running a decision support program when introducing new health technologies may be labor intensive ) , and finding funding for the appointment of hta leaders and support personnel . benefits of the project included increased research - mindedness among clinicians , staff , and administrators who participated in the decision support program review and adaptation process , increased awareness of hta , increased understanding and appreciation of clinicians and administrators in the complexity of evidence - informed technology introduction process , and reduced barriers to implementation of the proposed program as a result of buy - in from the participating stakeholders . these observations will need to be confirmed as others use the decision - making program review and adaptation framework . in summary , we present a three - phase framework for reviewing and adapting a well - developed local hta decision support program for application in different settings . this adaptation process divides a complicated process into a discrete step - by - step approach , which can be repeated regularly to improve quality , and ensures that the adaptation process is systematic , rigorous , and interdisciplinary . we conclude that the adaptation of a preexisting program may reduce duplication of effort , save resources , raise health care providers awareness of hta , and foster constructive stakeholder engagement , which enhances the legitimacy of evidence - informed recommendations for introducing new health technologies . we encourage others to use this framework for decision support program adaptation and report their experiences .

purposeintroducing new health technologies , including medical devices , into a local setting in a safe , effective , and transparent manner is a complex process , involving many disciplines and players within an organization . decision making should be systematic , consistent , and transparent . it should involve translating and integrating scientific evidence , such as health technology assessment ( hta ) reports , with context - sensitive evidence to develop recommendations on whether and under what conditions a new technology will be introduced . however , the development of a program to support such decision making can require considerable time and resources . an alternative is to adapt a preexisting program to the new setting.materials and methodswe describe a framework for adapting the local hta decision support program , originally developed by the department of surgery and surgical services ( calgary , ab , canada ) , for use by other departments . the framework consists of six steps : 1 ) development of a program review and adaptation manual , 2 ) education and readiness assessment of interested departments , 3 ) evaluation of the program by individual departments , 4 ) joint evaluation via retreats , 5 ) synthesis of feedback and program revision , and 6 ) evaluation of the adaptation process.resultsnine departments revised the local hta decision support program and expressed strong satisfaction with the adaptation process . key elements for success were identified.conclusionadaptation of a preexisting program may reduce duplication of effort , save resources , raise the health care providers awareness of hta , and foster constructive stakeholder engagement , which enhances the legitimacy of evidence - informed recommendations for introducing new health technologies . we encourage others to use this framework for program adaptation and to report their experiences .

Introduction Materials and methods Setting and overview Set-up phase Review and adaptation phase Finalization phase Results Set-up phase Review and adaptation phase Finalization phase Discussion Conclusion

PMC1160143

rna can play an important functional role in catalysis , e.g. ribozymes are rna enzymes that cleave rna phosphodiester bonds at specific sites ( 1 ) ; see ( 2 ) for an overview of potential therapeutic applications of ribozymes to cleave mrnas of oncogenes ( ras or bcr - abl ) and viral transcripts ( hiv-1 ) , to overcome drug resistance , control arthritis , etc . such is the case for the aminoglycoside and macrolide families of antibiotics , which disrupt rna translation in prokaryotes by targeting ribosomal ( rrna ) ( 3 ) . in contrast to mrna , noncoding rna ( ncrna ) is transcribed from genomic dna and plays a biologically important role , although it is not translated into protein . examples of ncrna include ribozymes , riboswitches , micro rna , small interfering rna ( 4 ) , trna , rrna , etc . riboswitches have recently been discovered to interact with small ligands and up- or down - regulate certain genes . breaker and co - workers ( 5 ) report the crystal structures of the add a - riboswitch and xpt g - riboswitch aptamer modules , which distinguish between bound adenine and guanine ; see ( 6 ) for an overview of bacterial riboswitches , and ( 7 ) for the structure , as given in the pdb code 1u8d of a guanine - responsive riboswitch with the metabolite hypoxanthine . rnaomics ( 8) , analogous to proteomics , concerns aspects of the secondary and tertiary structure , folding pathway , kinetics , comparison , function and regulation of all rna in a living organism . rnaomics requires the application of numerous existent tools , as well as the development of new computational methods . well - known rna computational tools include secondary structure prediction web servers mfold ( 9 ) and vienna rna package ( 10 ) , the sfold web server ( 11 ) to sample secondary structures according to the boltzmann probability distribution , the trnascan - se gene finder for trna ( 12 ) , multiple sequence alignment for the statistical detection of rna secondary structure msari ( 13 ) , dynamic programming pairwise sequence - structure alignment dynalign ( 14 ) , tertiary structure modeling tool mc - sym ( 15 ) , etc . only a few of the many important computational tools for rna structure prediction , gene finding , alignment , etc . have been listed . in this paper , we describe the web server rnaloss , based on the algorithm of clote ( 16 ) , which computes an aspect of the folding landscape of an rna nucleotide sequence s = s1 , , sn . given s , this algorithm runs in time o(n ) and space o(n ) , and computes for each k , the number of k - locally optimal secondary structures ( explained below ) . work by clote ( 16 ) was motivated by the following question , as has been suggested for proteins ( 17 ) : is it the case that rna has been under selective pressure to fold rapidly ? using the algorithm of the web server rnaloss , it appears that structural rna has a different folding landscape than random rna of the same dinucleotide frequency ; specifically , for small values of k , there appear to be fewer k - locally optimal secondary structures than in random rna . related , but distinct work has appeared in ( 1821 ) , for discussion see ( 16 ) . a secondary structure for an rna sequence s = s1, ,sn is an expression s = s1, ,sn involving dot , left and right parenthesis , which is well balanced , such that nucleotides corresponding to matching parentheses are either watson crick complements or gu wobble pairs . a secondary structure s on rna sequence s = s1, ,sn is defined to be a set of ordered pairs ( i , j ) , such that i + 3 < j and the following conditions are satisfied . watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g).threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop.non-existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l.no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g ) . threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop . non - existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l. no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. a secondary structure is k - locally optimal if it has k fewer base pairs than the maximum possible number [ i.e. than in the nussinov jacobson optimal structure ( 22,23 ) ] , and yet no base pairs can be added without violating the definition of secondary structure ( e.g. without introducing a pseudoknot ) . to illustrate this notion , consider the rna sequence ggggccccc , which has three as the maximum possible number of base pairs , as given in the structure ( ( ( ) ) ) . there is only one structure having 3 bp , so the number of 0-locally optimal secondary structures is 1 . on the other hand , the latter are listed as follows : ( i ) ( ) .... ( ii ) ( .... ) .. ( iii ) ( ..... ) . the algorithm of ( 16 ) uses dynamic programming to compute , for each i < j and each k , the number of k - locally optimal secondary structures on the subsequence s = si, ,sj . additionally , the algorithm must keep track of visible nucleotides and positions , i.e. those external to any base pair [ for technical details see ( 16 ) ] . a secondary structure s on rna sequence s = s1, ,sn is defined to be a set of ordered pairs ( i , j ) , such that i + 3 < j and the following conditions are satisfied . watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g).threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop.non-existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l.no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. watson crick or gu wobble pairs : if ( i , j ) belongs to s , then pair ( ai , aj ) must be one of the following canonical base pairs : ( a , u ) , ( u , a ) , ( g , c ) , ( c , g ) , ( g , u ) and ( u , g ) . threshold requirement : if ( i , j ) belongs to s , then j i > 3 ; i.e. there must be at least three unpaired bases in a hairpin loop . non - existence of pseudoknots : if ( i , j ) and ( k , l ) belong to s , then it is not the case that i < k < j < l. no base triples : if ( i , j ) and ( i , k ) belong to s , then j = k ; if ( i , j ) and ( k , j ) belong to s , then i = k. a secondary structure is k - locally optimal if it has k fewer base pairs than the maximum possible number [ i.e. than in the nussinov jacobson optimal structure ( 22,23 ) ] , and yet no base pairs can be added without violating the definition of secondary structure ( e.g. without introducing a pseudoknot ) . to illustrate this notion , consider the rna sequence ggggccccc , which has three as the maximum possible number of base pairs , as given in the structure ( ( ( ) ) ) . there is only one structure having 3 bp , so the number of 0-locally optimal secondary structures is 1 . on the other hand , the latter are listed as follows : ( i ) ( ) .... ( ii ) ( .... ) .. ( iii ) ( ..... ) . the algorithm of ( 16 ) uses dynamic programming to compute , for each i < j and each k , the number of k - locally optimal secondary structures on the subsequence s = si, ,sj . additionally , the algorithm must keep track of visible nucleotides and positions , i.e. those external to any base pair [ for technical details see ( 16 ) ] . the web server rnaloss implements a new algorithm , described in ( 16 ) , running in o(n ) time and o(n ) space , which computes for a given rna sequence s = s1, ,sn and all k 0 , the number of k - locally optimal secondary structures for s. an rna nucleotide sequence may be input by uploading a fasta - format file or by entering a nucleotide sequence in the blank provided on the web server form . three tables are returned by rnaloss : the number of k - locally optimal secondary structures , the relative density of states ( i.e. the ratio of number of k - locally optimal structures over the total number of locally optimal structures ) and the minimum free energy ( mfe ) of a sample k - locally optimal secondary structure ( for each value of k , rnaloss computes a single k - locally optimal secondary structure , denoted here as sk , among the many possible k - locally optimal structures . since this feature was implemented for debugging purposes , the current version of rnaloss does not guarantee that sk has lowest mfe as evaluated by rnaeval , over all k - locally optimal secondary structures . for this reason , the energy of sample structures sk does not necessarily increase monotonically with increasing value of k ) . for the latter , mfe is computed using rnafold from the vienna rna package . figure 2 lists the number of k - locally optimal secondary structures as computed by rnaloss for type iii hammerhead ribozyme af170517 from rfam ( 24 ) . figure 3 presents the relative density of states for k - locally optimal secondary structures for af170517 . owing to algorithmic time and space constraints , the rnaloss web server immediately processes rna of length at most 60 nt , while for rna of length 61100 nt , the results are emailed to the user . currently , rnaloss refuses to process any sequence of length > 100 nt . current hardware supporting rnaloss web server consists of a beowulf - style cluster comprising 6 dell 1650 , 2 1300 mhz pentium iii , 2 gb ram with 4 apple xserve , 2 1333 mhz g4 , 2 gb ram and finally 6 dell 1850 , 2 2800 mhz xeon em64 t , 2 gb ram . pentium iii nodes are running redhat linux 9 , xeon em64 t nodes are running whitebox linux 3 and g4 nodes are running macos 10.2.8 . upon testing , structurally important rna , such as selenocysteine insertion sequence elements , precursor mrnas , type iii hammerhead ribozymes and trna , all have a markedly smaller number of k - locally optimal structures than that of random rna of the same dinucleotide frequency , for small and moderate values of k. since the free energy of k - locally optimal secondary structures is generally closer to that of the native state for small k , this suggests that structural rna has been optimized not only to have low folding energy ( 25 ) , but also to have relatively few potential kinetic traps . this suggests that rnaloss might be of use in designing rna sequences for rapid folding . users may input an rna sequence consisting of upper or lower case nucleotides a , c , g , t , u , either by uploading a fasta - format file ( a ) or by pasting a nucleotide sequence into ( b ) . output of web server rnaloss on type iii hammerhead ribozyme af170517 from rfam ( 24 ) . the web server rnaloss outputs a table of number of k - locally optimal secondary structures , for each possible value of k 0 ( shown here ) . additionally , tables for the relative density of states and mfe values of sample k - locally optimal secondary structures are displayed in the browser ( data not shown ) . column graph of relative density of states is obtained by clicking a hot link from the previous screen shot . for each value of k 0 , the ratio of number of k - locally optimal secondary structures over the total number of locally optimal secondary structures is displayed .

rnaomics , analogous to proteomics , concerns aspects of the secondary and tertiary structure , folding pathway , kinetics , comparison , function and regulation of all rna in a living organism . given recently discovered roles played by micro rna , small interfering rna , riboswitches , ribozymes , etc . , it is important to gain insight into the folding process of rna sequences . we describe the web server rnaloss , which provides information about the distribution of locally optimal secondary structures , that possibly form kinetic traps in the folding process . the tool rnaloss may be useful in designing rna sequences which not only have low folding energy , but whose distribution of locally optimal secondary structures would suggest rapid and robust folding . website : .

INTRODUCTION METHODS Definition WEB SERVER DISCUSSION Figures and Tables

PMC3888764

the national diabetes epidemic continues to expand , with about 1.6 million new cases each year and an overall prevalence of 23.6 million people . additional 57 million american adults are at high risk for type 2 diabetes mellitus ( t2 dm ) . the annual diagnosed diabetes incidence is projected to almost double from 8 cases per 1,000 in 2008 to about 15 in 1,000 by 2050 , with a potential total prevalence projected to increase one - fifth of the us population by 2050 . currently , 10% of americans have t2 dm , and 20% to 25% are considered prediabetic with impaired glucose tolerance and elevated fasting glucose measurements . the long - term complications of uncontrolled hyperglycemia in diabetics include neuropathy , nephropathy , retinopathy , and other chronic issues . the in - hospital morbidity of diabetics and new - onset hyperglycemics is well documented . . showed that for inpatients with new hyperglycemia or known diabetes there was a higher in - hospital mortality , increased length of stay , increased intensive care unit ( icu ) stay , increased transitional or nursing home care , a higher rate of infections , and more neurologic events compared to a normoglycemic control group . in addition , diabetes and inpatient hyperglycemia increase mortality for patient with an acute myocardial infarction [ 5 , 6 ] . the role of diabetes in outcomes of the critically ill , intensive care unit patient continues to be debated [ 710 ] . diabetics are increasingly undergoing elective and emergent surgery ; however , whether the presence of diabetes compromises surgical outcomes remains unclear . it is accepted that diabetics have an increased risk of cardiovascular disease compared to nondiabetics . have shown that diabetics undergoing general and vascular surgery with postoperative hyperglycemia exceeding 150 mg / dl had increased rates of infection . moreover , bower et al . , in a study of prospective patient registries , showed increased morbidity in known diabetic patients with and without malignancy and that diabetes was related to degree of postoperative complications in a quantitative fashion . in addition , the detriment of uncontrolled hyperglycemia in surgical patients has been investigated [ 14 , 15 ] . in specific surgical populations , particularly cardiac and vascular patients , the negative effect of diabetes on postoperative outcomes is clear . patients undergoing surgery are subject to altered carbohydrate metabolism , increased production of glucose , and increased resistance to insulin , leading to a stress - induced hyperglycemia . in cardiac surgery patients , maintenance of perioperative blood glucose levels between 125 and 200 mg dl resulted in fewer episodes of atrial fibrillation and recurrent ischemia , as well as a shorter length of stay ( los ) [ 16 , 17 ] . similarly , in the vascular population suboptimal hgb a1c levels in diabetics and non - diabetics predicted poor postoperative outcomes . diabetics undergoing lumbar spine surgery fared worse than non - diabetics for spine - specific outcomes postoperatively . however , the neurosurgical literature reveals only modest benefits linked to strict control of glycemic indexes during acute periods of brain ischemia and in specific surgical situations such as spine and tumor surgery . a diagnosis of diabetes has not been shown to be an independent risk factor for poor outcomes in general surgery . studies investigating overall surgical outcomes of patients with a known diagnosis of diabetes , irrespective of perioperative hyperglycemia , are sparse , and most of these are fraught with small sample sizes , are specific to only one surgical specialty , are or guided towards one specific outcome measure ( e.g. , surgical site infection ) . we asked the question of whether a diagnosis of insulin dependent or non - insulin dependent diabetics impacted overall postoperative morbidity and mortality in a large administrative general and vascular surgical population . we sought to answer this question through query of a comprehensive statewide surgical outcomes database . this study was categorized as exempt from review at both central michigan university ( cmu ) college of medicine and covenant healthcare . we used data from the michigan surgical quality collaborative ( msqc ) for this study . briefly , the msqc is a statewide quality improvement effort representing 52 member hospitals in the state of michigan . it is modeled after the american college of surgeons ' national surgical quality improvement program . over 200 variables are collected about surgical interventions using reliable and validated systematic sampling techniques to provide a representative sample of cases from each participating hospital . the msqc prospectively collects data on ( 1 ) thirty - day morbidity , ( 2 ) preoperative laboratory values , ( 3 ) readmissions and unplanned return to the operating room , ( 4 ) preoperative risk factors , ( 5 ) postoperative laboratory values and discharge variables , and ( 6 ) 30-day postoperative mortality . at larger hospitals , in addition , quarterly and twice - a year reports are generated for participating hospitals to aid in quality improvement . we included subjects from the msqc database from the years 2007 through 2011 who had general or vascular surgery and diabetes status recorded and were over 18 years of age . in addition , we created a secondary outcome variable , any major morbidity , from six variables in the msqc database detailing postoperative occurrences : wound , respiratory , urinary tract , central nervous system , cardiac , and other occurrences . subjects experiencing one or more of these post - operative occurrences were classified as having any major morbidity . we also used a range of variables from the msqc database to model our outcome variables . preoperative risk variables included history of diabetes , smoking , dependence on ventilator within 48 hours , copd , dialysis , steroid use , 10% weight loss within six months , and sepsis within 48 hours . subjects were classified as having a cardiac preoperative risk if they had any of the following : congestive heart failure ( chf ) within 30 days , history of myocardial infarction ( mi ) , previous percutaneous coronary intervention ( pci ) or percutaneous transluminal coronary angioplasty ( ptca ) , previous cardiac surgery , history of angina within 30 days , or on hypertensive medications . finally , we included or time , wound classification , asa class , whether cases were emergent , and type of surgery : general or vascular . chf within 30 days is defined by the msqc as chf , congestive heart failure , or pulmonary edema . a history of mi within past 6 months is defined as a history of a non - q wave or a q wave myocardial infarct in the six months prior to surgery . a history of previous pci / ptca includes balloon dilatation and stent placement ; however , it does not include valvuloplasty . previous cardiac surgery includes procedures classified as off - pump repair or utilizing cardiopulmonary bypass , valve replacement or repair , great thoracic vessel repair , cardiac transplant , repair of atrial or ventricular septal defects , left ventricular aneurysmectomy , insertion of left ventricular assist devices , and so forth ; however , it does not include insertions of pacemakers or automatic implantable cardioverter defibrillators . hypertension requiring medication is defined as a persistent elevation of systolic pressure over 140 mmhg or a diastolic pressure over 90 mmhg or requires an antihypertensive treatment , at the time the patient is being considered for surgery . emergency cases are defined as being performed as soon as possible and no later than 12 hours after hospital admission or onset of appropriate symptoms . steroid use is defined as the regular administration of oral or intravenous corticosteroids 30 days prior to surgery for a chronic condition and does not include topical , rectal , or inhalation corticosteroids or a course of short course steroids ( 10 days or less ) . we conducted binary logistic regression models for 30-day mortality ( table 2 ) for the entire sample ( model 1 ) , a subgroup of diabetics with general surgery ( model 2 ) , and a subgroup of diabetics with vascular surgery ( model 3 ) . similarly , we conducted binary logistic regression models for any morbidity ( table 3 ) for the entire sample ( model 4 ) , a sub - group of diabetics with general surgery ( model 5 ) , and a subgroup of diabetics with vascular surgery ( model 6 ) . all models were saturated models , that is , using all independent variables within each dependent variable ( mortality and morbidity ) to see which independent variables had the highest contribution to the desired outcome . all analyses were done using statistical product and service solutions ( spss ) version 20 ( ibm , armonk , ny ) . a complete demographic and variable summary of our sample is presented in table 1 . of 211,436 subjects , there were 177,430 ( 83.9% ) general surgeries and 34,006 ( 16.1% ) vascular surgeries . there were 175,639 ( 83.1% ) nondiabetics , 15,050 ( 7.1% ) insulin dependent diabetics , and 20,747 ( 9.8% ) diabetics who were dependent on oral medications . overall thirty - day mortality was two percent ( 4,083 , 1.9% ) and 13.0% ( 27,533 ) of cases were observed to have any type of major morbidity . in addition , 24% of subjects were smokers with the past 12 months , 6% had chronic obstructive pulmonary disease ( copd ) , and 50.8% had some level of cardiac risk . 84% of patients were either asa class 2 or 3 , and 52.7% of wounds were classified as clean . our goal was to determine if specific patient characteristics influenced mortality and morbidity and so we began by running regression analyses on these variables . interestingly , our preliminary analyses indicated that smoking is not associated with an increased risk of mortality . while this is unexpected based on most other studies that associate smoking with increased mortality , in our sample , there is a negative correlation between smoking and diabetes . in addition , smokers were more likely to have anesthesia types other than general anesthesia . in both cases ( diabetes and anesthesia other than general ) the risk of mortality is decreased and the association of smoking with these characteristics likely impacts the influence of smoking . due to the high multicollinearity of smoking with several other characteristics , we removed smoking as a variable from the regression analyses reported in section 3.2 . we regressed the 30-day mortality variable using three binary logistic regression models ( table 2 ) to determine the ability of different variables to predict mortality in diabetic subgroups of general and vascular surgery patients . model 1 uses the entire sample . in this case , those subjects who died within 30 days were less likely to be oral ( or = 0.78 , 95% ci 0.700.86 ) or insulin ( or = 0.83 , 95% ci 0.750.92 ) dependent diabetics than not diabetic . in our sample , diabetes does not increase the risk of 30-day mortality . vascular surgery ( compared to general surgery ) is a significant predictor of mortality ( or = 1.57 , 95% ci 1.421.72 ) for the entire group . due to our interest in the diabetic population , we conducted further subgroup analyses for 30-day mortality using only diabetic patients . model 2 ( table 2 ) is a subgroup analysis of diabetic , general surgery subjects . in this case model 3 ( table 2 ) is a subgroup analysis of diabetic , vascular surgery subjects . as with the general surgery subgroup , there is no difference between oral and insulin dependent vascular surgery , diabetics in survival . moving from the consideration of diabetes as a variable , we also included our other variables in the analyses . in general and as we would expect , ventilator dependence , copd , cardiac risk factors , current dialysis , steroid use , weight loss , sepsis , emergent cases , increased asa class , and any wound classification increase the risk of 30-day mortality . in summary , our models for 30-day mortality show that for our entire sample , the presence of diabetes does increase the risk of 30-day mortality . there do not appear be significant differences in variables that contribute to mortality , when comparing models for diabetic subgroups of general and vascular surgery and , as expected , ventilator dependence , copd , current dialysis , weight loss , sepsis , emergent cases , increased asa and any wound classification increase the risk of 30 day mortality in diabetic patients . we also regressed the any morbidity variable using three binary logistic regression models ( table 3 ) to determine the ability of different variables to predict any type of morbidity in different subgroups of surgery patients . subjects with any type of morbidity were more likely to be insulin dependent ( or = 1.11 , 95% ci 1.061.16 ) diabetics than oral dependent diabetics , or not diabetic . vascular surgery ( compared to general surgery ) is also a significant predictor of morbidity ( or = 1.69 , 95% ci 1.621.75 ) . model 5 ( table 3 ) is a subgroup analysis of diabetic , general surgery subjects . in this case , insulin dependent diabetics are more likely to have any morbidity ( or = 1.20 , 95% ci 1.121.29 ) when compared to diabetics who are dependent on oral medications . model 6 ( table 3 ) is a subgroup analysis of diabetic , vascular surgery subjects . again , we found that insulin dependent diabetics are more likely to have any morbidity ( or = 1.24 , 95% ci 1.121.38 ) when compared to oral medication dependent . again , considering other variables across all models for any morbidity , we found that in general , ventilator dependence , copd , cardiac risk factors , current dialysis , steroid use , weight loss , sepsis , emergent cases , increased asa class , and any wound classification increase the risk of any morbidity . in summary , models for any morbidity show that for our entire sample , insulin dependent diabetics have an increased risk for any morbidity ( or = 1.11 , 95% ci 1.061.16 ) when compared to non - diabetics . this increased risk is also present in subgroup analyses comparing insulin dependent diabetics to oral medication dependent diabetics for general ( or = 1.20 , 95% ci 1.121.29 ) and vascular ( or = 1.24 , 95% ci 1.121.38 ) surgeries . , there do not appear be significant differences in variables that contribute to morbidity , when comparing models for diabetic subgroups of general and vascular surgical patients . as expected , ventilator dependence , copd , current dialysis , weight loss , sepsis , emergent cases , increased asa and any wound classification increase the risk of any morbidity in diabetic patients . our objective was to understand the postoperative risk for morbidity and mortality in diabetics versus non - diabetic patients undergoing general or vascular surgery . we found that , with regard to mortality , the presence of diabetes is not predictive of mortality ; however , vascular surgery itself is predictive of mortality . there are no significant differences in variables that contribute to mortality , when comparing diabetic and non - diabetic subgroups for both general and vascular surgery . in our morbidity analysis , insulin dependent diabetics have an increased risk for any morbidity compared to non - diabetics or non - insulin ( oral ) dependent diabetics in the general and vascular surgery subgroups . our multivariate analyses confirm findings in the literature of risk factors in general surgery that contribute to morbidity and mortality , such as ventilator dependence , copd , cardiac risk factors , dialysis , steroid use , significant weight loss , sepsis , emergent cases , and increasing asa class . interestingly , notable differences were detected when analyzing separate groups of diabetics and non - diabetics undergoing general surgery . for instance , male non - diabetics undergoing general surgery have an increased risk for any morbidity as compared male diabetics . it is possible that higher risk male diabetics were more likely denied elective surgical intervention , as opposed to higher risk non - diabetic males , resulting in our finding . as in the mortality analysis , there do not appear be significant differences in variables that contribute to morbidity for diabetics and non - diabetics specifically undergoing vascular surgery . this is the first study in the literature of overall post - operative outcomes ( morbidity and mortality ) of diabetics undergoing general surgery with such a large population of patients . examined 55,000 diabetic patients in the veterans heath administration national surgical quality improvement program database and analyzed the effect of different levels of preoperative hyperglycemia on the rate of wound infection . however , in their cohort , there was no comparison of diabetics to a non - diabetic control group , as that was not the purpose of their study . within their study , they showed that higher preoperative serum glucose levels correlated with worse wound - related outcomes ; however , preoperative hba1c ( long - term glucose control in diabetics ) had no association with infection rates . in a study of preoperative hba1c levels , o'sullivan found that , after multivariate analysis , elevated hba1c levels were predictive of morbidity in non - diabetic patients , but not in diabetics , undergoing vascular surgery . the o'sullivan study does highlight the disturbing problem of undiagnosed diabetics undergoing surgery without proper control of their glycemia . the lack of correlation between diabetic diagnosis and mortality in general surgery in our study is not unique in the literature , where a clear consensus on the issue can not be found . . showed in a study of over 600 patients undergoing hepatic resection that multivariate analysis did not identify diabetes as an independent variable having an impact on mortality and that overall complications were equally frequent between diabetic and non - diabetic patients . diabetes was also shown not to be a statistically significant predictor of overall mortality or cardiac morbidity in patients undergoing open aortic aneurysm repair . in a matched cohort retrospective review , diabetes did not increase the mortality rates of cardiac surgery , but increased the risk for renal and neurological complications , blood transfusion , reoperation , and length of icu stay . however , a smaller study than ours from finland , with a long 7-year followup , showed quite convincingly that short - term and long - term mortality was higher in diabetics versus non - diabetics undergoing similar noncardiac surgeries . described a high mortality , due mainly to cardiovascular complications , in cohort of diabetics undergoing noncardiac surgery ; however , there was no non - diabetic control group in the study . our study revealed that the presence of cardiac risk predicted overall morbidity in diabetic and non - diabetics undergoing general and vascular surgery equally . however , cardiac risk predicted mortality only in non - diabetic general and vascular surgery patients . conversely , hollenberg et al . showed that diabetes was one of five risk factors predicted of cardiac events in patients undergoing non - cardiac surgery . one difference that may account for the results is that the latter study began from the premise of a population of patients that were already high risk for a cardiac event occurring and underwent non - cardiac surgery . in contrast , our study evaluated all patients from the perspective of being diabetic or not and assessing factors predictive of morbidity and mortality in diabetic versus non - diabetic , that is , a difference of study design to test a different hypothesis . most studies regarding surgical outcomes of diabetics involve studying the presence of perioperative hyperglycemia in diabetics or non - diabetics and its effect on morbidity and mortality . currently , the literature regarding optimal target glucose ranges for the perioperative patient is unclear . the ada recommends screening for diabetes preoperatively in select populations , especially in obese patients . the united states preventive services task force ( uspstf ) recommends screening only hypertensive adults for diabetes . some of the first studies to evaluate the benefits of tight glucose control were the leuven study in 2001 and 2006 , which sparked widespread interest in intensive insulin therapy in critically ill patients [ 33 , 34 ] . some researchers relate the benefits identified in this study to our current understanding of the bodies stress response . in the leuven study , the majority of the critically ill patients studied had spent more than five days in the intensive care unit . the effects of an anabolic steroid , such as insulin , may be more beneficial as the response to the acute injury resolves . however , during the acute onset of stressful stimulus such as surgery or possibly traumatic events the effect of treatment with these hormones has not been fully explained and may be harmful . despite the fact , improvements in surgical outcome have been seen in patients with tight intraoperative glucose control . as previously mentioned , a growing body of evidence suggests that even small changes in glycemic ranges are associated with impaired outcomes . in one study , in patients undergoing cardiac surgery , a 20 mg / dl increase in mean intraoperative glucose was associated with increased risk of more than 30% of adverse outcome . however , it is important to make the distinction that these findings can not be generalized to general surgical patients . the broad array of patient demographics and options for surgical care encompass an extensive variety of settings , and as many operations performed by the modern general surgeon are now performed in the ambulatory setting , the assessment and management of the dysglycemia patient continue to be a looming challenge . the msqc is an excellent source of data for studying general and specific 30-day morbidity for various surgical procedures on a larger scale . the administrative database provides risk and reliability adjusted reports for over 52 participating hospitals in the state of michigan . the other option to answer our question would have been to use the national surgical quality improvement program ( nsqip ) database or the national inpatient sample ( nis ) hospital discharge data . the nis system , although widely available and currently the largest all - payer database of hospital discharge records , poses a number of limitations . first , inconsistencies across states and providers in the data element reporting may compromise data quality . second , other data elements that would be needed to analyze risk factors for certain preoperative patient populations , such as race / ethnicity , detailed test results , whether a condition was present on admission , functional status , severity of illness , and behavioral risk factors , would be absent in many cases . first , collaboration in the msqc is purely voluntary , and these hospitals may have different characteristics from those who have chosen not to participate . therefore , our results may not be generalizable to the entire cohort of statewide hospitals . moreover , michigan hospital practices may not be representative of us hospitals , although this database includes academic and community as well as various sizes and geographic locations within the state . secondly , data in the msqc is collected retrospectively and analyzed by the end - user retrospectively and therefore will have the biases inherent to such a study design . third , the present analysis does not address procedure specific outcomes for diabetes . for example , although a cholecystectomy may not have the level of morbidity or mortality as a pancreatic operation for a diabetic , the volume of cholecystectomies in the database outweighs that of complex pancreatic operations . although cases are easily distinguished between vascular and nonvascular surgeries , we did not separate out or group the 3000 cpt codes to identify procedure specific risk . in summary , we found that a diagnosis of diabetes ( insulin or oral dependent ) did not affect outcomes in general surgical patients . however , the presence of insulin - dependent diabetes was associated with increased morbidity in the vascular and general surgery subgroups greater than non - diabetics or non - insulin dependent diabetics . however , the reassuring fact that the presence of diabetes did not have an overt effect on mortality , along with morbidity risk of insulin - dependent diabetes , may greatly impact the surgeon - patient preoperative dialogue and our ability to appropriately risk - stratify and risk - optimize these patients .

aims . preoperative diabetic and glycemic screening may or may not be cost effective . although hyperglycemia is known to compromise surgical outcomes , the effect of a diabetic diagnosis on outcomes is poorly known . we examine the effect of diabetes on outcomes for general and vascular surgery patients . methods . data were collected from the michigan surgical quality collaborative for general or vascular surgery patients who had diabetes . primary and secondary outcomes were 30-day mortality and 30-day overall morbidity , respectively . binary logistic regression analysis was used to identify risk factors . results . we identified 177,430 ( 89.9% ) general surgery and 34,006 ( 16.1% ) vascular surgery patients . insulin and noninsulin diabetics accounted for 7.1% and 9.8% , respectively . insulin and noninsulin dependent diabetics were not at increased risk for mortality . diabetics are at a slight increased odds than non - diabetics for overall morbidity , and insulin dependent diabetics more so than non - insulin dependent . ventilator dependence , 10% weight loss , emergent case , and asa class were most predictive . conclusions . diabetics were not at increased risk for postoperative mortality . insulin - dependent diabetics undergoing general or vascular surgery were at increased risk of overall 30-day morbidity . these data provide insight towards mitigating poor surgical outcomes in diabetic patients and the cost effectiveness of preoperative diabetic screening .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

PMC5098515

the emergency care system in denmark has been reorganized during recent years , and a danish quality database on emergency care has been established to assess , monitor , and improve quality . the danish quality database for prehospital emergency medical services ( qems ) is one of the three national sub - databases . the aim of the qems is to assess , monitor , and improve the quality of prehospital emergency medical service ( ems ) care in the entire patient pathway and acts as basis for clinical and health - service research . provision of prehospital emergency care is subject to variations in organization among ems providers , with domestic and regional as well as international differences . there are few uniform quality indicators and standards based on high - level evidence , and most standards are based on expert panel opinions.14 some specific diagnostic groups benefit from quality indicators and standards , such as cardiac arrest,5,6 myocardial infarction ( mi),7,8 stroke,9,10 and severe injury . the major challenge and difficulty in emergency care , especially in the prehospital setting , is that the vast majority of patients present with symptoms and not diagnoses . thus , ems care has to be initiated and delivered on the basis of the symptoms with limited knowledge about the patient s objective condition and medical history . in 2013 , a working group was established by the danish regions with medical and administrative representatives from all five health regions , with the task of formulating recommendations on indicators and standards of ems quality . the working group consisted of medical directors and administrative professionals from prehospital organizations from each of the five regions . the selection of quality indicators was a consensus process , based on literature and the actual availability of prehospital data in our system and national registries on specific emergency conditions , cardiac arrest , mi , and stroke . the danish clinical registries a national improvement program was established in 2014 with medical representatives from the five public regional ems organizations , a professor in prehospital and emergency medicine and representatives from the danish society of cardiology , the danish society of neurology , and a clinical epidemiologist and a statistician from the danish clinical registries . an initial pilot test of indicators is currently underway in 2015 . due to lack of high evidence , we could not define all standards a priori . a process of defining the remaining standards will follow evaluation of the pilot test . ultimately , aggregated data at a regional level will be available monthly , and national and open - source annually , and provide stimulus for ems improvement . the aim of this review is to describe the design and the implementation of qems , a new danish clinical quality database . in denmark , access to health care is a tax - financed service , and access to ems and emergency hospital care is free of charge . during the last 7 years five regional health care regions were established and were responsible for the pre hospital ems in their own region . these regional ems organizations are responsible for the entire prehospital patient care pathway , from the 112 call made to the recently established emergency medical coordination centers ( emcc ) , through the care given on scene and during transport , and until the patient arrives at hospital , or is taken care of without being brought to hospital . at the emcc , the national emergency number 112 covers both ems , fire and police , and is answered by the police , except in the capital region . here all decisions regarding medical prehospital responses are made at the emcc , based upon a focused , structured interview of the person calling 112 . assessment is performed according to a criteria - based dispatch protocol , the danish index for emergency care ( danish index).11 the danish ems is a two- or three - tier system , with ambulances as the basic - level response , an intermediate level with paramedic- or nurse - manned cars , and an advanced level with dedicated prehospital doctors ( consultant anesthesiologists ) in cars ( mobile emergency care unit ) or helicopter ( helicopter emergency medical service [ hems ] ) in a rendezvous model . the danish hems has only recently been added to the system , established as national hems in 2014 . ambulances are run by municipal or private companies and staffed with emergency medical technicians or paramedics , educated and trained to national minimum standards according to national regulations.12 the regional ems organizations contract with ambulance providers after european union - regulated tenders . the regional ems is responsible for the quality of care delivered in the entire ems pathway , including the care delivered by private ambulance contractors . all ems providers nationwide are to use the same electronic prehospital patient record system , under implementation in 2015 . the study population consists of all 112 patient contacts defined as patients in need of emergency help , where access to the health care system is a 112 call forwarded to the emcc . . the corresponding number of 112 patient contacts will be lower , estimated to be 300,000350,000 as there might be more 112 calls on the same patient . the database includes data acquired from each of the five regional emcc and when fully implemented data from the prehospital electronic patient record . data from the danish database for acute hospital contacts will also be included in the future . emcc call - takers assess the level of emergency and respond in accordance with the danish index for emergency care , which consists of 37 main symptoms . the index defines five levels of urgencies : a : life- threatening or potentially life - threatening condition , immediate response required ; b : urgent , but not life - threatening condition ; c : nonurgent condition that needs an ambulance ; d : nonurgent supine patient transport ; and e : other service or advice / instruction including taxi transportation ( no ambulances dispatched ) . the emcc call - taker assigns the letter appropriate for the level of urgency coupling it with a specific number to the case according to the danish index criteria code that corresponds to the main symptom and specific subgroup symptom . the indicators aim at describing quality at the regional ems organizational level and the quality of the medical care . as the prehospital electronic patient record has not yet been fully implemented , only some of the indicators are monitored in the initial pilot test phase . indicator numbers 13 describe quality at an ems organizational level ; these include only responses of level of urgency a ( table 1 ) . time from arrival until the patient is handed over to the hospital personnel ( indicator 3 ) awaits the electronic patient record . concerning the three medical emergencies , cardiac arrest , st - elevation mi , and stroke , all these await full implementation in the electronic patient record . in the meantime , data will be obtained from the national research database , the danish out - of - hospital cardiac arrest registry , and regional and national databases.5 indicator number 4 , the proportion of 112 calls coded according to danish index , was included to assure registration of the initial symptom as this is the only patient information available at time of the decision on level of urgency . indicator number 5 was defined in order to monitor the proportion of patients calling 112 classified as having no need for an ambulance and/or no need for hospital care , who died or called 112 again within 24 hours . in denmark , each citizen is assigned a unique ten - digit civil registration ( cpr ) number , universally used in all patient records and danish registries , and enables unambiguous linkage between these registries . thus , the registration of this number , as indicator 9 , is essential as a key to patient care and for follow - up . table 3 describes the variables included in the primary data set . apart from being an identifier for an event and person , the variables comprise mainly date and time stamps identifying when the 112 call was received at the emcc , when the dispatcher sent a prehospital resource , and when the first resource arrived . the danish index , including urgency level , is also included in the data set . it is , however , a fact that the cpr number is missing in a substantial proportion of patients because it is not always possible to extract this information from the caller . patients with a valid danish cpr number are followed from the date of 112 call and until death for up to 2 days after the task has been completed using the danish civil registration system.13 the database is under establishment , and no studies have been performed yet , but there are previous studies using some of similar data from three regions comparing the danish index with diagnosis and outcome after hospitalization.11,14,15 data will be available to researchers after application to the steering committee , provided that permissions from the danish data protection agency and/or ethical committee were obtained . the qems is an approved clinical quality database ( the danish data protection agency record no 2013 - 58 - 0026 , the central denmark region record no 1 - 16 - 02 - 508 - 14 , and the state serum institute record no 14/18767 ) funded by the danish regions and receives administrative , epidemiological , and biostatistical support from the danish clinical registries ( danish regions ) . aggregated data at regional level will be reported annually in a published report , and data will be provided monthly to the danish regions for use in each regions information system . aggregated data at a regional level will be available monthly , and national and open - source annually , providing incentive for improvement of patient care pathways in the prehospital setting .

aim of databasethe aim of the danish quality database for prehospital emergency medical services ( qems ) is to assess , monitor , and improve the quality of prehospital emergency medical service care in the entire prehospital patient pathway . the aim of this review is to describe the design and the implementation of qems.study populationthe study population consists of all 112 patient contacts defined as emergency patients , where the entrance to health care is a 112 call forwarded to one of the five regional emergency medical coordination centers in denmark since january 1 , 2014 . estimated annual number of included 112 patients is 300,000350,000.main variableswe defined nine quality indicators and the following variables : time stamps for emergency calls received at one of the five regional emergency medical coordination centers , dispatch of prehospital unit(s ) , arrival of first prehospital unit , arrival of first supplemental prehospital unit , and mission completion . finally , professional level and type of the prehospital resource dispatched to an incident and end - of - mission status ( mission completed by phone , on scene , or admission to hospital ) are registered.descriptive datadescriptive data included age , region , and danish index for emergency care including urgency level.conclusionqems is a new database under establishment and is expected to provide the basis for quality improvement in the prehospital setting and in the entire patient care pathway , for example , by providing prehospital data for research and other quality databases .

Background and aim of database Setting Study population Quality indicators Main variables Follow-up Examples of research Administrative issues and funding Conclusion

PMC4248536

however , it has not been reported with fluoroscopically - guided endovascular procedures.the rapidly proliferating keratinocytes in the hair matrix are most sensitive to radiation . the insult damages the keratinocytes , which results in thin , weakened hair shaft that is susceptible to trauma.complete hair re - growth occurs in a time period between two to four months following the insult . the insult damages the keratinocytes , which results in thin , weakened hair shaft that is susceptible to trauma . complete hair re - growth occurs in a time period between two to four months following the insult . fluoroscopy - guided neuro - radiological endovascular procedures for treatment of arteriovenous malformations are getting commonplace in modern health care facilities of india . temporary alopecia of the scalp is an uncommon but documented side - effect of these procedures , especially when a certain threshold of radiation is exceeded . to the best of our knowledge , this is the first case of temporary alopecia after fluoroscopically - guided coiling procedure for a ruptured cerebral aneurysm being described in a dark haired woman from india . a 47-year - old apparently healthy woman was referred to this clinic ( sv ) for a dramatic non - scarring hair loss of occipito - vertical region since a month . it became known upon eliciting history that three weeks ago , she developed acute excruciating headache of the nape of the neck , profuse perspiration , vomiting , and lack of consciousness with incontinence . an urgent ct scan showed a ruptured anterior communicating artery aneurysm . a fluoroscopically - guided guglielmi detachable coil ( gdc ) coiling procedure as a non - invasive treatment was suggested . a preliminary angiography lasting for two and half hours was done followed by an attempt to perform coiling that lasted unsuccessfully for four hours . shoots had to be taken due to severe vasospasm . after waiting for two days to let the dye drain out , the procedure was attempted again , and coiling was done successfully in two and a half hours . a week later , the patient developed headache , for which another ct scan was done . the headache was attributed to cerbritis , and 24 mg of methylprednisolone was instituted on a slow taper . two weeks later , she noticed clumps of hair falling out initially during combing and then even while spontaneously running her fingers through her hair . there was a 6 4 inch - sized , asymptomatic , skin - colored alopecic patch in the occipital area with no erythema , scarring or scaling [ figure 1 ] . hair was sent for trichogram to an academic hospital in coimbatore where they demonstrated 80% anagen hair showing dystrophy , the so called bayonet hair. [ figure 2 ] . she had no exclamation mark hairs , poliosis , hair shaft disorders or positive history of similar affliction or atopy in her family . a vascular cause for the hair loss was ruled out by loud arterial pulse palpable in the temporal and occipital arteries . pressure alopecia due to prolonged supine position was ruled out by absence of fractured hair shafts . occipito - vertical alopecia after radiation exposure anagen hair with the pointed distal end to the left and telogen hair to the right diagnosis of fluoroscopic endovascular catheterization - induced alopecia was made . after a period of 14 weeks of observation and no active treatment , she showed significant re - growth of dark hair [ figure 3 ] . flouroscopically - guided endovascular procedures are becoming increasingly common all over the globe . while dermatologists have since long been consulted for hair loss following radiotherapy of tumors , there are only few reports of temporary hair loss following fluoroscopic endovascular procedures in dermatologic literature . other than radiation oncology , the maximum radiation exposure comes from equipment used for non - invasive interventions such as these . the two commonest temporary side - effects of such procedures , erythema , and temporary hair loss are termed deterministic and are indicative of a certain threshold of irradiation , below which the phenomenon would not be seen . the probability of encountering such a side - effect would increase as the dose exceeds the threshold . the time between the exposure and appearance of clinical symptoms depend on the cell turnover rate . in tissues such as hair follicle with its rapid turnover rate , it is thought that irradiation doses exceeding 3 gy are followed by such a hair loss during fluoroscopic procedures . an estimated 7% of such radiological interventions are said to reach the threshold set for temporary hair loss by the international commission on radiological protection . according to the interventionalist , it would have grossly exceeded 3 gy because the procedure turned out to be a very long one with prolonged exposure to the radiation owing to the difficulty in negotiating the loop through the site of pathology . it is not unusual for the patient to skip the history of radiation exposure because they are unaware of the fact that the procedure they have undergone involves radiation exposure and have not been forewarned by the interventionalist . severity of radiation - induced temporary alopecia is largely dependent on the dose , total duration of irradiation , interval between irradiations ( dose fractionation where applicable ) , the size of the skin area irradiated , the angle of irradiation , all affect the expression and severity of the injury . one of the commonest working positions during gdc coiling procedure is posteroanterior , which explains the occipito - vertical hair loss in this patient . loss of scalp hair can occur at relatively lower doses when compared with doses at which other body hair is lost . our patient with jet black hair developing this dramatic hair loss is suggestive of gross excess of radiation . the radiation insult damages these keratinocytes and diminishes the growth of the growing hair shaft . this results in thin , weakened hair shaft that is susceptible to trauma when it reaches the surface of scalp . hair breakage in anagen effluvium occurs within days to weeks ( 1 - 3 weeks ) following insult to the follicle . microscopic hair examination shows that the distal hair shaft is dystrophic with a rapidly tapering configuration called the bayonet hair. tapered hair can arise in any process that inhibits the cell division in the hair matrix . since 90% of hair on the scalp is in growing phase , smoking , malnutrition , pre - existing autoimmune and connective tissue disorders like diabetes , hyperthyroidism , scleroderma , rheumatoid arthritis , and systemic lupus erythematosus predispose patients to the development of severe radiation effects in an unpredictable manner . drugs that increase radiosensitivity include actinomycin d , doxorubicin , bleomycin , 5-fluorouracil , and methotrexate , mitoxantrone , 5-fluorourcil , cyclophosphamide , paclitaxel , docetaxel , and possibly tamoxifen . , it was literally adding insult to injury considering she had just recovered from a life - threatening situation . fortunately , unassisted re - population of hair in 2 - 4 months is the most likely outcome and , therefore , a gentle handholding and confidence building are all that is required . interventionalists should explain this possibility to patients in whom such a deterministic dose has been exceeded to avoid distress . fortunately , at such doses , there are very few chances of a serious or irreversible long - term consequence . however , all dermatologists should be aware of the association of hair loss with prolonged fluoroscopic endovascular procedures where the deterministic dose has been exceeded . the absorbed skin dose should be recorded on the patient 's charts or medical record . it is recommended that this dose be taken into account if the patient requires a future procedure involving exposure to ionizing radiation . minimizing the effective dose to achieve the therapeutic goal is the aim , and efforts are on to reach that goal . fluoroscopically - guided interventional procedures are now performed fairly commonly in advanced tertiary care centers in india . dermatologists who saw radiation - induced dermatoses until recently will now increasingly start seeing side - effects to the skin and hair due to these procedures , which is the second commonest cause of radiation exposure after radiotherapy.temporary alopecia of the scalp is a well - known early side - effect of ionizing radiation , which is emitted during the fluoroscopic - guided interventional procedures and is seen when a dose of 3 gy is exceeded . other factors that play a role include total duration of irradiation , interval between irradiations , size of the skin area irradiated , and the angle of irradiation.the resultant alopecia is due to anagen effluvium , and there is complete spontaneous re - growth in about 2 - 4 months . fluoroscopically - guided interventional procedures are now performed fairly commonly in advanced tertiary care centers in india . dermatologists who saw radiation - induced dermatoses until recently will now increasingly start seeing side - effects to the skin and hair due to these procedures , which is the second commonest cause of radiation exposure after radiotherapy . temporary alopecia of the scalp is a well - known early side - effect of ionizing radiation , which is emitted during the fluoroscopic - guided interventional procedures and is seen when a dose of 3 gy is exceeded . other factors that play a role include total duration of irradiation , interval between irradiations , size of the skin area irradiated , and the angle of irradiation . the resultant alopecia is due to anagen effluvium , and there is complete spontaneous re - growth in about 2 - 4 months .

a 47-year - old indian woman with dark hair developed dramatic alopecia of the occipito - vertical area two weeks after a fluoroscopically - guided endovascular procedure for treatment of cerebral aneurysm . there was spontaneous repopulation of hair in 14 weeks . neuro - radiological intervention procedures are becoming commoner by the day in india , and the rare but documented possibility of such reactions occurring in patients should be kept in mind by the treating surgeon as well as dermatologist . necessary counseling regarding this uncommon side - effect is of essence , especially when the radiation dose exceeds 3 gy . we believe this is the first case ever reported in indian dermatology literature .

Introduction Case Report Discussion

PMC4831500

the optimal time of catheter removal following radical prostatectomy ( rp ) is not defined . from a patient 's perspective , early removal of the urinary catheter is desirable due to catheter - related discomfort , penile pain , bladder irritation / spasm , pericatheter urine leaks , etc . , however , the surgeon 's concerns with early catheter removal are the possibility of anastomotic site leak and acute urinary retention . with experience and improvement in techniques , the duration of catheterization has decreased from an average of 21 days in open rp era to 710 days in cases where robot - assisted rp ( rarp ) is performed . in fact , there are numerous reports about the feasibility and safety of even earlier ( i.e. on the postoperative day 35 ) catheter removal after radical retropubic prostatectomy or rarp . with early catheter removal , some concerns have been raised about increased risk of acute urinary retention . therefore , in practice , the majority of the institutions perform catheter removal after 7 days of surgery . however , there is a variation in the practice of performing cystograms before catheter removal which is done primarily to rule out anastomotic site leak . with the improvement in anastomotic technique , it is rare to find anastomotic site leak after rarp , and it may not always be necessary to obtain a routine check cystogram . we , therefore , evaluated our 230 consecutive rarp patients and the outcomes of their cystogram findings to determine the indications for cystogram before catheter removal . we reviewed our prospectively collected rarp database of 230 consecutive patients who underwent robot - assisted rp by 2 surgeons from april 2010 to january 2015 . all the patients underwent rarp through transperitoneal approach using 4 arm da vinci surgical system . clinical characteristics included patient 's age , biopsy gleason score , clinical stage , and history of transurethral resection of prostate ( turp ) or other urethral / bladder neck surgery . intraoperative data recorded included : estimated blood loss , operative time , bladder neck reconstruction , visible leak upon bladder irrigation at completion of anastomosis , and placement of pelvic drains . bladder neck sparing was done whenever possible . in patients with a wide bladder neck , bladder neck reconstruction was performed by placing sutures at the 3 and 9 oclock positions . urethrovesical anastomosis was performed in 2 layers ( described earlier ) . at the end of anastomosis , a bladder fill test was performed ( using 120 ml of saline ) to assess intraoperative anastomotic leak . patients were called for office cystography and catheter removal , usually a week after surgery . cystography was performed at low pressure by gravity instillation of 150200 ml of diluted contrast through the catheter placed during rarp . patients were observed under fluoroscopy in lateral oblique position for any contrast leak at the site of anastomosis . in the absence of extravasation , the catheter was left in place until a subsequent cystogram revealed resolution , typically 1 week later . in patients with retention of urine after catheter removal , placement of a 16fr foley catheter all patients were followed for a minimum of 6 months , and the longest follow - up was 5 years . postoperative data included pathologic gleason score and stage , hospitalization time , and catheterization time . postoperative complications included urinary retention , urinary tract infection , bladder neck contracture , and urinary incontinence . continence status was defined as the use of either no pad or just a security liner . continence rate was assessed with the self - administered questionnaire during a visit in the outpatient department or by e - mail / postal mail at 6 week and at 3 , 6 , 9 , 12 , 18 , and 24 months and then once a year . continuous parametric variables were reported as the mean standard deviations or as the median values and interquartile range . a total of 207 patients ( 90% patients ) underwent catheter removal on the 7 postoperative day while 10% ( 23 patients ) had catheter removal on the 10 day or later . a decision about delay in catheter removal was usually made by the operating surgeon , based upon satisfaction with bladder neck reconstruction or observation of minimal leak on bladder fill test during surgery . two of these patients had prior bladder neck reconstruction for the wide neck , and other two underwent standard anastomosis . as a routine , patients with bladder neck reconstruction and/or intraoperative leak ( on bladder fill test ) had their catheter removed on the day 10 . only one patient out of four with an intraoperative leak on bladder fill test had leak on postoperative cystogram . demographic details of patients total of nine patients ( 3.9% ) had extravasation on initial cystogram . among the patient with anastomotic site leak , two patients had a history of turp and seven other had wide bladder neck ( resulting from wide margin due to basal tumor ) . out of the total nine patients with leak , 3 patients had a minimal amount of contrast leak ( which cleared on post void film ) ; therefore , catheter was still not replaced [ flow chart 1 ] . in rest of the 6 patient showing leak , continued catheter drainage was done for another week and repeat cystogram was done [ figure 1 ] . four out of nine patient showing leak needed catheter for a total of 14 days while the two patients needed the catheter for a total of 21 days . no significant difference in the intraoperative variables , blood loss , duration of drain , length of hospital stay , and continence outcomes was noted between the patients with anastomotic leak ( noted on cystogram ) when compared to rest of the patients . flow chart of outcome of cystogram and their management ( a ) cystogram showing minimal anastomotic leak on the postoperative day 7 . ( b ) resolution of leak after 7 days of indwelling catheter ( day 14 ) acute urinary retention after catheter removal was noted in 3 patients . only 1 out of 3 patients who had retention after catheter removal had bladder neck sparing surgery . none of the patient experienced immediate retention of urine . out of those who developed retention ( 3 cases ) , one occurred after a week of catheter removal , and the other two developed after 48 h. the catheter was easily replaced in an emergency without any problem . these patients were started on alpha - blocker and catheter removed after 3 days with successful voiding . no pad status at 3 months , 6 months , and 1 year was 76% , 86% , and 94% , respectively . all but one patient with urine leak was continent at 6 months ( one patient became continent by 9 months of follow - up ) . no significant difference in the intraoperative variables , blood loss , and duration of drain , length of hospital stay , prostate volume , grade or stage of the tumor , and continence outcomes was noted between the patients with leak compared to rest of the patients . cystogram at the time of catheter removal is an important tool that can help in documentation of healing at anastomotic site and identifying leak , if any . however , as noted in our study and also studied by other authors , the incidence of urine leak noted on cystogram done at day 7 is very low ( 25% ) . therefore , instead of using cystogram in all the patients , selective use of cystogram can prove to be cost - effective . compared to studies published from developed countries , our patients had a higher clinical stage and more cases of post - turp status , thus requiring wide bladder neck dissection and subsequent reconstruction . we found that there is specific utility of cystogram in the patients who have had a history of turp , wide bladder neck with intraoperative bladder neck reconstruction , or minimal leak noted on intraoperative bladder fill test . although not supported by any evidence , it is our practice to delay catheter removal to postoperative day 10 ( instead of day 7 ) , in patients who undergo bladder neck reconstruction . the optimal time for catheter removal ( or indwelling catheter drainage ) is still a point of scrutiny . understandably , catheter - related symptoms and discomfort are less with shorter duration of indwelling catheter . however , the risk of anastomotic leak , acute urinary retention , and the impact of urine leak on long - term continence and bladder neck contracture are some of the concerns with early catheter removal . most of the authors consider catheter removal after 7 days minimizes the risk of anastomotic leak as well as the chance of acute retention . interestingly , there are studies that have suggested even earlier catheter removal ( at 34 days ) without any significant increase in complication . have reported 1% cystogram leak rate after catheter removal at 24 days , but with a higher rate of acute retention . it is considered that anastomotic site urine leak can potentially delay the time to continence , and adversely impact the overall continence rates . several authors have studied the outcomes in their patients who had a varying degree of anastomotic site urine leak . all the studies based on robotic prostatectomy have concluded that urinary extravasation had no adverse impact on the long - term continence status . patil et al . reported that those patients who had a leak , 70% were dry at 3 months , compared to 90% in those with no leaks . at the 12-month follow - up , 95.2% of the patients with no leak were dry , compared with 94% of those with a leak , thus concluding that at 12 months there was no difference . in our study , the number of patient with anastomotic site leak was too small to make a comparative statement about the impact on recovery pattern of continence . however , all but one patient with urine leak had zero pad status at 6 months ( one patient became continent by 9 months ) . it is also postulated that urine leak ( especially prolonged duration ) will lead to a sustained inflammatory reaction , hampering wound healing , thus increasing the risk of delayed complication such as bladder neck contracture . in a large series reporting urine leak eight of 287 ( 2.8% ) patients it was further clarified that bladder neck contracture developed in patients with higher grade leaks . only three of our patient had retention of urine after few hour of catheter removal . it is likely that the cause of acute retention is mucosal edema or hematoma or increase bladder neck smooth muscle tone . the reported risk of acute retention is reportedly higher ( 6.721% ) with catheter removal at 4 days or less . therefore , results may not be generalizable . however , at the same time , constancy of anastomotic technique , operating surgeons and the follow - up methodology is the strength of this study . the results of our study suggest that in usual circumstances , urinary catheter can be safely removed on the 7 postoperative day after rarp without a routine cystography . selective use of check cystogram can be done in cases where bladder neck reconstruction is performed or those who have a history of turp with wide bladder neck and when an intraoperative leak noted on the bladder fill test .

introduction : with the improvement in anastomotic technique , it is rare to find anastomotic site leak after robot - assisted radical prostatectomy ( rarp ) . it may not always be necessary to do regular check cystogram before catheter removal . we evaluated our 230 consecutive rarp patients and their cystograms to determine the indications for selective use of cystogram before catheter removal.materials and methods : we reviewed our prospectively collected rarp database of 230 consecutive patients . cystography was performed at low pressure by gravity instillation of diluted contrast through the catheter . patients were observed under fluoroscopy in lateral oblique position for any contrast leak at the site of anastomosis . all patients were followed for a minimum of 6 months , and the longest follow - up was 5 years.results:a total of 207 patients ( 90% ) underwent catheter removal on postoperative day 7 . nine patients ( 3.9% ) had extravasation on initial cystogram . two patients with leak had a history of transurethral resection of prostate ( turp ) and seven other had bladder neck reconstruction for wide bladder neck . three patients with minimal leak did not require catheter replacement . in rest of the 6 patient with leak , continued catheter drainage was done . no significant difference in the intraoperative variables , blood loss , duration of drain , length of hospital stay , and continence outcomes was noted between the patients with leak compared to rest of the patients . none of the patient needed any procedure / intervention related to the surgery and none developed bladder neck stenosis.conclusion:in usual circumstances , catheter removal can be done safely on a postoperative day 7 without routine cystography . selective use of check cystogram can be done in the case where bladder neck reconstruction is performed or those had a prior turp and a wide bladder neck .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION None Financial support and sponsorship Conflicts of interest

PMC3337872

nutcracker syndrome is generally referred to as the compression of the left renal vein between the superior mesenteric artery and aorta ( 1 ) . this compression leads to lrv hypertension and results in direct communication of the renal vein with the calyceal cavity and the varicosities of the collateral pathway of the lrv ( 2 ) . renal varicosities or abnormal branching of the superior mesenteric artery from the aorta are the most common factors considered responsible for this condition ( 1 , 2 ) . the other causes , such as compression of the retroaortic lrv between the aorta and spine ( which is referred to as ' posterior ncs ' in contrast to the more common ' anterior ncs ' ) or aberrant right renal artery constricting the lrv , are uncommon ( 3 , 4 ) . however , three recently reported cases of nutcracker phenomenon associated with the left inferior vena cava ( livc ) suggest that some anomalies of the inferior vena cava ( ivc ) can also impair lrv blood outflow and result in ncs ( 4 - 6 ) . here we reported a case of complicated malformation of livc with interrupted ivc and azygos continuation and retroaortic right renal vein causing posterior ncs , which has been diagnosed by both color doppler ultrasound and computed tomographic angiography ( cta ) . to the best of our knowledge , this is the first report of an association between a complicated anomaly of the livc and ncs . a 42-year - old female patient was referred to our hospital for renal ultrasonographic scanning . she had a past medical history of intermittent microscopic hematuria or clots for nearly 10 years ' duration . the morphology of urinary red cells by phase - contrast microscopy revealed 53% dysmorphic cells . however , oblique transverse scanning of the upper abdomen showed two tubular structures on both sides of the abdominal aorta above the level of the renal vein . we traced the vessels in an effort to scrutinize every segment of these two tubular structures and found that the right common iliac vein crossed the vertebra and formed the left - sided infrarenal ivc ( l - ivc ) along with the left common iliac vein . the right renal vein ( rrv ) traveled retroaortically and joined the l - ivc ( fig . the post - anterior diameter of the hilar portion and that of the retroaortic stenotic portion of the rrv were 8.8 mm and 2.0 mm , respectively , revealed by grey - scale ultrasound , which , with peak velocities , was demonstrated by a spectral doppler of 26 cm / s and 87 cm / s , respectively . the diameter ratio and the peak velocity ratio between the dilated portion and the stenotic portion were 4.4 and 3.4 , respectively . after collecting blood from the right and left common iliac veins and the renal veins , the l - ivc crossed the midline retroaortically and flowed into the right - sided suprarenal ivc ( r - ivc ) with turbulent , high - velocity flow ( fig . the diameters of the l - ivc at the level of the lrv and the dilated portion of the lrv near the hilus and the diameter of the ivc at the stenotic portion were 18.6 mm , 14.3 mm and 2.3 mm , respectively , with the peak velocities of 30 cm / s , 27 cm / s and 149 cm / s , respectively . correspondingly , the diameter ratio between the dilated portion of lrv and the stenotic portion of l - ivc was 6.2 , accompanied by the peak velocity ratio of 5.5 . 1d ) . below the level of the rrv , no tubular structure was found at the right lateral portion of the aorta . the results demonstrated that the rrv traveled retroaortically to the l - ivc . after collecting blood from the right and left common iliac veins and the renal veins , the l - ivc traversed retroaortically to join the r - ivc above the level of the celiac artery . the r - ivc ascended with the azygos instead of joining to the hepatic vein . in addition , paravertebral collateral veins in the right lumbar region traveled upward tortuously and drained into the r - ivc ( fig . 1e - g ) . the findings of both color doppler ultrasound and cta revealed that the anomaly of the ivc in our patient was livc with continuation of the azygos , existence of a retroaortic rrv , and absence of the posthepatic segment of the ivc ( fig . cystoscopy was performed to evaluate otherwise asymptomatic episodes of microscopic hematuria , which demonstrated no positive findings . analysis the data obtained by image findings , together with the patient 's typical history and other laboratory findings , she was diagnosed as the posterior ncs associated with a complicated malformation of the livc . the normal ivc is composed of four segments : hepatic , suprarenal , renal , and infrarenal . its evolutionary process involves the development , regression , and anastomosis of three pairs of fetal veins : the posterior cardinal veins , subcardinal veins , and supracardinal veins , in the embryonic life during the sixth to eighth week of gestation ( 7 ) . the livc is the result of the persistence of the left supracardinal vein with regression of the right supracardinal vein . moreover , this case was more complicated because of the continuation with the azygos , the existence of the retroaortic rrv , and the absence of the posthepatic segment of the ivc . consequently , the pathology may be accompanied by the atrophy of the right subcardinal vein , the persistence of the dorsal limb as well as the regression of the ventral limb of the right renal collar , and the failure to form the right subcardinal - hepatic anastomosis ( 7 ) . furthermore , interruption of the ivc with azygos continuation is often associated with congenital heart disease , polysplenia , and less commonly , asplenia . in our patient no congenital heart diseases or other anomalies were found , so we have reason to believe that this condition is extremely rare . diagnosis of the anomalies of ivc may have significant clinical implications , especially during retroperitoneal surgery or in the treatment of thromboembolic diseases ( 5 - 7 ) . the majority of abnormal ivcs are clinically asymptomatic and only recognized incidentally during radiologic investigations performed for other reasons or during surgeries on the aorta or retroperitoneal structures or during postmortem examinations . in our literature review , we found only three cases of the anomalies of the ivc presenting as ncs . ( 5 ) first described data on an 80-year - old woman , who had a liver metastatic adenocarcinoma , and was incidentally found to have the anomaly of the livc . the authors believed that the compression of the livc between the aorta and superior mesenteric artery was responsible for her hematuria , a symptom of ncs . regardless of the various causes of ncs , an outlet obstruction of the lrv is the outcome of ncs . its typical symptoms , such as hematuria , left flank and abdominal pain , and varicoceles , are closely related to a backward venous renal hypertension due to the outflow impairment of the lrv and the development of compensating collateral circulation . in our present case , the aberrant vessels of the ivc resulted in two potential entrapments of the renal vein : 1 ) the pathway where the l - ivc drains into the r - ivc between the aorta and vertebra indirectly causing the hypertension of the lrv and 2 ) the location where the rrv runs to the l - ivc retroaortically . the data obtained by sonography revealed that the ratios of the diameter and of the peak velocity in rrv between the dilated portion and the stenotic portion were 4.4 and 3.4 , respectively , whereas those between the dilated segment of lrv and the narrowest segment of l - ivc were 6.2 and 5.5 , respectively . ct angiography also demonstrated the existence of obvious dilatation of lrv , which we believed to be the main factor of the occurrence of the hematuria . in addition , the distension of the lrv and tortuously paravertebral collateral veins in the right lumbar region could explain the patient 's feeling of a continuous dull lumbago for a long time . clinically , the diagnosis of ncs is based on the assessment of the pathologic anatomy and physiology ( 8) . the newer imaging modalities such as cta and magnetic resonance angiography have powerful abilities in providing accurate , three - dimensional reconstructive images in any desired plane and allowing better identification of these anomalies . spectral doppler ultrasound can give more details of the hemodynamics of the lrv at the site of compression . the case we presented here was first diagnosed by color doppler ultrasound and then identified by cta scanning with a high - quality delineated picture of the anomaly of the ivc . the dilated lrv as well as the l - ivc draining into the r - ivc in our patient were clearly visualized by ct and color doppler ultrasound . kim 's study showed that the cutoff values for ncs as the ratios of both antero - posterior diameter and peak velocity between the distended and narrowed segments of the lrv are greater than 5 , with sensitivity of 69% , 80% and specificity of 89% , 94% , respectively . but when combined with the cutoff value of more than 5 of the ratio of the antero - posterior diameter and the ratio of peak velocities divided by 2 , the sensitivity and the specificity can be elevated to 90% and 100% , respectively ( 9 ) . in our case , both the diameter ratio and the peak velocity ratio between the dilated segment of lrv and the narrowest segment of l - ivc were greater than 5 with a combined value of 5.9 . moreover , the morphology of urinary red cells is less than 75% , which is thought to be non - specific in nature ( 10 ) . in review of our patient 's typical history and clinical and radiologic findings , the diagnosis was most likely posterior ncs associated with complicated malformation of the livc . in conclusion , a congenital anomaly of the ivc , although rare , may be one of the contributing factors for the development of ncs . with the aid of ultrasonography and the newer cross - sectional imaging modalities , variant venous anatomy should be identified and its clinical significance , which had not been mentioned in previous reports , should be evaluated thoroughly .

various anatomic anomalies have been considered the causes of nutcracker syndrome ( ncs ) . posterior ncs refers to the condition , in which vascular narrowing was secondary to the compression of the retroaortic left renal vein while it is crossing between the aorta and the vertebral column . here , we report an unusual case of posterior ncs associated with a complicated malformation of the interrupted left inferior vena cava with azygos continuation and retroaortic right renal vein , diagnosed by both color doppler ultrasonography and ct angiography .

INTRODUCTION CASE REPORT DISCUSSION

PMC1538886

one important and interesting problem in genome research is the prediction of transcription factor binding sites ( tfbss ) . binding of transcription factors to their dna binding sites in the regulatory region of a gene is a prerequisite for its activation or repression . the combinatorial presence of tfbss controls gene regulation at least partially , and the prediction of tfbss is of importance for unraveling the underlying molecular mechanisms . wet - lab experiments allow the identification of tfbss , but these experiments are expensive and time consuming . computational methods , which are often less accurate but easy to conduct with available resources , are a welcome complementation to wet - lab experiments . many tfbs prediction algorithms employ statistical models for scoring a given sequence as tfbs or non - tfbs . for these algorithms , the family of markov models ( 13 ) is chosen in many tfbs prediction algorithms as well as for a variety of other classification problems . well - known examples are the position weight matrix ( pwm ) model ( 4 ) , which is an inhomogeneous markov model of order 0 , and the weight array matrix ( wam ) model ( 5 ) , which is an inhomogeneous markov model of order 1 . from a statistical point of view , markov models employ assumptions about the statistical independence of nucleotides at different positions of the binding site . the strongest independence assumption is realized by the pwm model , where each position is assumed to be statistically independent of all other positions . as indicated e.g. in ( 6 ) , it is an open question whether this strong independence assumption is reasonable in view of recent results indicating the presence of statistical dependences between positions ( 7,8 ) . markov models of higher order take into account statistical dependences from previous positions , called the context , where the length of the context is equal to the order of the markov model . despite the often unjustified independence assumption , pwm models are often found to outperform markov models of higher order in the prediction of tfbss . this is probably caused by the rather limited amount of training data available from experimentally verified tfbss . as the number of model parameters grows exponentially with the order of the markov model , markov models of higher order tend to be over - fitted , resulting in poor performance . one possibility to circumvent this problem is provided by variable order markov models , which do not require the contexts to be of a fixed length , but allow contexts with variable lengths ( 912 ) . the power of variable order markov models stems from the freedom to include only those contexts into the model for which there are strong statistical dependences . another possibility to capture statistical dependences between non - adjacent positions without the burden of increasing the number of model parameters exponentially are provided by bayesian trees ( bts ) ( 1316 ) . in bts the contexts are not restricted to the previous positions within the binding site , and it could be demonstrated that bts outperform pwm models and wam models in the prediction of splice sites ( 17,18 ) and tfbss ( 6,19 ) . as an extension of variable order markov models and bts , we introduce variable order bts ( 19 ) . the vombat web server is designed to train variable order markov models and variable order bts on user - supplied data and to apply the resulting models to the prediction of tfbss . in addition , the vombat web server provides a cross - validation platform , allowing advanced users to compare different model combinations with different parameter settings on problem - specific datasets . the vombat web server is primarily designed for the prediction of tfbss , but applicable to other types of fixed - length motifs as well . the prediction of tfbss is based on one statistical model for tfbss and one for non - tfbs sequences constituting the background . each statistical model assigns a probability p(x1xl ) to a given dna sequence x1xl of l nucleotides . for any value of m , 1 m l , p(x1xl ) markov models of order m assume that the conditional probabilities pl(xlx1xl1 ) do not depend on all previous nucleotides x1xl1 , but only on the m previous nucleotides xlmxl1 , which are called the context of position l. hence , a markov model of order m is defined by p(x1xl)=p0(x1xm)l = m+1lpl(xlxlmxl1 ) . if the conditional probabilities pl(xlxlmxl1 ) are identical at all positions , the markov model is called homogeneous , otherwise it is called inhomogeneous . in contrast to a markov model of order 1 , where the conditional probabilities at position l depend only on the previous nucleotide xl1 , a bt allows that the conditional probabilities at position l may depend on the nucleotide xpa(l ) at a possibly non - adjacent position pa(l ) , which is called the parent of position l. in a bt , arbitrarily remote positions may be chosen as parents as long as no cycles between positions are induced . this implies that there is one position , called the root position , which must not depend on any other position , and that the statistical dependences of a bt may be graphically represented by a rooted tree , where the positions are represented by nodes and the statistical dependences are represented by edges ( 1316 ) . the probability distribution defined by a bt with root position r decomposes as p(x1xl)=pr(xr)lrpl(xlxpa(l ) ) . in the vombat web server , the so - called motif model pmotif ( used for modeling tfbss ) and the so - called background model pbg ( used for modeling background dna sequences ) can be chosen by the user . an inhomogeneous markov model or a bt should be chosen if statistical dependences are assumed to vary from position to position within the sequence . a homogeneous markov model should be chosen if statistical dependences are assumed to be the same at all positions within the sequence . the appropriate order of a markov model depends on the expected range of statistical dependences and on the amount of available training data . for markov models of fixed order , the number of model parameters grows exponentially with the order , resulting in a sharp transition from under - fitted to over - fitted models . the idea of variable order markov models is to shorten the context in those cases where extending the context does not yield mathematically this is formulated by measuring to which degree the conditional probabilities change when extending or reducing the context . the kullback leibler divergence ( 20 ) is used as a measure of change of the conditional probability distributions . it measures the degree of dissimilarity of the conditional probability distribution given the extended context and the conditional probability distribution given the reduced context . leibler divergence is always non - negative , but small positive values of the kullback the threshold above which the kullback leibler divergences are considered important can be set by an external parameter , c , called pruning constant . for c = 0 , any extension of the context is considered important , and the resulting variable order markov model becomes a traditional markov model of order m. for c , any extensions of the context is considered unimportant , and the resulting variable order markov model becomes a traditional markov model of order 0 , i.e. a pwm model . as c grows from 0 to , more and more contexts are considered unimportant , and the resulting variable order markov models become smaller and smaller , interpolating between a markov model of order m and a pwm model . vombat uses a maximum likelihood estimator with optional pseudo counts , which can be specified by the user , for smoothing the conditional probability distributions and to compensate for zero occurrences of nucleotides and contexts . the motif model pmotif is learned from a user - supplied training set of tfbss , and the background model pbg is learned from a user - supplied training set of background sequences . after training pmotif and pbg , vombat can be used to compute genome - wide predictions of the learned motif in a set of user - supplied sequences . for each position of a sliding window of length l , the log - likelihood ratio of the oligonucleotide y1yl occurring at that position is computed , and y1yl is predicted as tfbs if the log - likelihood ratio is greater than a given threshold t , i.e. if log2pmotif(y1yl)pbg(y1yl)t . specific knowledge , such as the number of tfbss expected in the genome , can be taken into account to adjust the threshold t to achieve tfbs predictions with the desired balance of sensitivity and specificity . for advanced users , vombat allows a cross - validation of the prediction accuracy of a user - defined combination of pmotif and pbg based on a user - supplied set of tfbss and a user - supplied set of background sequences . from each of the datasets , 10% of the sequences are randomly excluded , and the models pmotif and pbg are trained on the remaining 90% . subsequently , both models are used for the prediction of the withheld data , and the sensitivity given a user - defined value of the specificity is computed . this procedure is repeated k times , where k can be specified by the user , and the mean sensitivity and its standard error are reported . vombat is based on a variant of the three - tier architecture , where the presentation layer , the management layer and the execution framework are logically separated and physically located on different servers . the web front - end of vombat is based on standard technologies like javaserver faces for the web forms and servlets for displaying images and providing downloads of results . the user - supplied parameters and input files are stored in a mysql - database . this database is queried by the execution framework . if the execution framework detects a job to be executed , it requests the necessary parameters and input files and submits the corresponding job to the scheduler of the attached linux cluster . after the job is finished , the results are written to the database and can be displayed by the web front - end , which loads numerical results , model descriptions and images from the result - tables of the mysql - database . imagine a user interested in a genome - wide prediction of binding sites of the transcription factor sp1 in gc - poor upstream regions of human refseq ( 21 ) genes . if the user were interested in a prediction based on pre - trained pwm models stored in the transfac database ( 22 ) , he could use the tfbs prediction program match ( 3 ) . if the user were interested in a prediction based on variable order markov models or variable order bts , he could use the vombat web server . the two main functions of the vombat web server are training variable order markov models or variable order bts based on user - supplied sequences and predicting putative tfbss based on user - supplied variable order markov models or variable order bts . the training function allows the user to choose an optimal combination of a motif model and a background model and train these models on problem - specific datasets . for example , the user might choose a variable order bt as motif model and a variable order markov model of order 5 as background model , he might choose that subset of sp1 binding sites available from transfac that are located in gc - poor promoters as motif dataset , and he might choose a representative set of gc - poor promoter regions as background dataset . for training a model from user - supplied input data , the user first selects train a model from data from the selection of tasks . the user can enter a comment on the model to be trained in order to make it easier to identify the corresponding results in a list of all results . the rest of the parameters defines the type of model to be trained as well as the pruning constant and the pseudo count . the default value of the pseudo count is 1 , and we recommend the user to always specify a pseudo count greater than 0 to compensate for zero occurrences . if the model is defined to be inhomogeneous , the user can select between a markov model and a bt as a third parameter . based on ( 6,19 ) and based on systematic cross - validation analyses of different model combinations applied to different sets of tfbss and background sequences , we generally recommend to use a bt as motif model and a homogeneous markov model with an initial order of at least 3 as background model . the user - supplied input file to train an inhomogeneous model must consist of aligned sequences of identical length , separated by line breaks . the vombat web server is designed for , but not restricted to , the prediction of motifs in dna sequences . hence , it is possible to specify the alphabet of the sequences . in case of dna sequences the alphabet is acgt. as a last option the user may select a checkbox that determines if a graphical representation of the trained model shall be displayed as supplementary data . if the job of training a model could be successfully submitted to the scheduler of the attached linux cluster , the subsequently displayed page reports jobs success, and vombat gives the user the possibility to go either to the list of tasks for starting another job or to the job overview . the job overview displays a table of all jobs with the following columns : the time at which the job was started , the comment that was entered by the user , the type of the job ( train , classify , crossval ) , the current state of the job , a button for the already available results and a button to stop a running job . the possible states of a job are view results in the same row to inspect the results of this job . the results of training a model are a link to an xml representation of the trained model , which can be saved and used as input for one of the other tasks presented in the following . if the user selected to obtain a graphical representation of the trained model , this representation is also displayed as a series of images , which may be saved using the corresponding browser command . after a motif model and a background model have been trained and saved , they can be combined to predict putative tfbss in a user - supplied set of input sequences . for this purpose , the first input field of the subsequently displayed form , which is shown in figure 1 , allows to enter a user - defined comment on the job . next , the user uploads two files containing the xml representations of the desired motif and background models , which have been trained using the train a model from data task described above . the classification threshold must be specified , and the file containing the set of sequences in which the motif is to be predicted must be uploaded . after a classification job has been started , the user can monitor the current state of the job in the job overview of vombat . if the job is finished , the predictions can be inspected by clicking on the corresponding view results button . the html file contains a textual description of the putative motifs , which contains for each predicted motif its position , its sequence , its probabilities returned by the two models , and the corresponding log - likelihood ratio . additionally , a profile of the log - likelihoods and the log - likelihood ratio is plotted for each sequence of the input file . the chosen classification threshold is also plotted as a reference for the log - likelihood ratio , allowing the user to judge the influence of the classification threshold on the prediction results at a glance . as an auxiliary function for advanced users , vombat provides a platform for cross - validation analyses of different combinations of user - defined models based on problem - specific data . selecting cross validation from the list of tasks brings up the cross - validation input form . the first input field allows to enter a user - defined comment on the cross - validation job . next , the number of iterations of the cross - validation job can be entered . two files containing xml representations of the motif model and the background model must be uploaded by the user . these files can be obtained by a standard training on the datasets to be used for cross - validation . next , the user must specify the desired specificity for which the mean sensitivity is to be computed . the last option in the form allows to plot a histogram of the log - likelihood ratios for the motif sequences and background sequences , which allows further analyses of the cross - validation results , such as judging the effect of a shifted threshold or inspecting the separation of the samples of both classes . the presented results are the mean ( and standard error ) of the sensitivity for the fixed specificity entered in the cross - validation form , the mean ( and standard error ) of the maximum correlation coefficient , and the corresponding thresholds and optionally the histogram of the log - likelihood ratios as shown in figure 3 . the following prototypical example illustrates how the three functions of vombat may be utilized for the prediction of putative tfbss . typically , the user has no a priori knowledge of which model combination and which model parameters could provide an accurate prediction of these binding sites in gc - poor upstream regions . hence , he would start a series of different cross - validation experiments for different model combinations and different parameter settings using the cross - validation function of vombat . after having established an optimal model combination ( including optimal parameters ) for his specificity requirements and based on his problem - specific datasets , e.g. based on already annotated sp1 binding sites from transfac as motif dataset and representative gc - poor upstream regions as background dataset , he would then use these models to run the training and prediction functions of vombat as outlined above . for training a model from user - supplied input data , the user first selects train a model from data from the selection of tasks . the user can enter a comment on the model to be trained in order to make it easier to identify the corresponding results in a list of all results . the rest of the parameters defines the type of model to be trained as well as the pruning constant and the pseudo count . the default value of the pseudo count is 1 , and we recommend the user to always specify a pseudo count greater than 0 to compensate for zero occurrences . if the model is defined to be inhomogeneous , the user can select between a markov model and a bt as a third parameter . based on ( 6,19 ) and based on systematic cross - validation analyses of different model combinations applied to different sets of tfbss and background sequences , we generally recommend to use a bt as motif model and a homogeneous markov model with an initial order of at least 3 as background model . the user - supplied input file to train an inhomogeneous model must consist of aligned sequences of identical length , separated by line breaks . for homogeneous models the length of the sequences may differ . the vombat web server is designed for , but not restricted to , the prediction of motifs in dna sequences . hence , it is possible to specify the alphabet of the sequences . in case of dna sequences the alphabet is acgt. as a last option the user may select a checkbox that determines if a graphical representation of the trained model shall be displayed as supplementary data . if the job of training a model could be successfully submitted to the scheduler of the attached linux cluster , the subsequently displayed page reports jobs success, and vombat gives the user the possibility to go either to the list of tasks for starting another job or to the job overview . the job overview displays a table of all jobs with the following columns : the time at which the job was started , the comment that was entered by the user , the type of the job ( train , crossval ) , the current state of the job , a button for the already available results and a button to stop a running job . the possible states of a job are if the state of a job is finished, the user can click on the button view results in the same row to inspect the results of this job . the results of training a model are a link to an xml representation of the trained model , which can be saved and used as input for one of the other tasks presented in the following . if the user selected to obtain a graphical representation of the trained model , this representation is also displayed as a series of images , which may be saved using the corresponding browser command . after a motif model and a background model have been trained and saved , they can be combined to predict putative tfbss in a user - supplied set of input sequences . for this purpose , the first input field of the subsequently displayed form , which is shown in figure 1 , allows to enter a user - defined comment on the job . next , the user uploads two files containing the xml representations of the desired motif and background models , which have been trained using the train a model from data task described above . the classification threshold must be specified , and the file containing the set of sequences in which the motif is to be predicted must be uploaded . after a classification job has been started , the user can monitor the current state of the job in the job overview of vombat . if the job is finished , the predictions can be inspected by clicking on the corresponding view results button . the html file contains a textual description of the putative motifs , which contains for each predicted motif its position , its sequence , its probabilities returned by the two models , and the corresponding log - likelihood ratio . additionally , a profile of the log - likelihoods and the log - likelihood ratio is plotted for each sequence of the input file . the chosen classification threshold is also plotted as a reference for the log - likelihood ratio , allowing the user to judge the influence of the classification threshold on the prediction results at a glance . as an auxiliary function for advanced users , vombat provides a platform for cross - validation analyses of different combinations of user - defined models based on problem - specific data . selecting cross validation from the list of tasks brings up the cross - validation input form . the first input field allows to enter a user - defined comment on the cross - validation job . next , the number of iterations of the cross - validation job can be entered . a higher number of iterations produces more reliable results . on the other hand , two files containing xml representations of the motif model and the background model must be uploaded by the user . these files can be obtained by a standard training on the datasets to be used for cross - validation . next , the user must specify the desired specificity for which the mean sensitivity is to be computed . the last option in the form allows to plot a histogram of the log - likelihood ratios for the motif sequences and background sequences , which allows further analyses of the cross - validation results , such as judging the effect of a shifted threshold or inspecting the separation of the samples of both classes . the presented results are the mean ( and standard error ) of the sensitivity for the fixed specificity entered in the cross - validation form , the mean ( and standard error ) of the maximum correlation coefficient , and the corresponding thresholds and optionally the histogram of the log - likelihood ratios as shown in figure 3 . the following prototypical example illustrates how the three functions of vombat may be utilized for the prediction of putative tfbss . typically , the user has no a priori knowledge of which model combination and which model parameters could provide an accurate prediction of these binding sites in gc - poor upstream regions . hence , he would start a series of different cross - validation experiments for different model combinations and different parameter settings using the cross - validation function of vombat . after having established an optimal model combination ( including optimal parameters ) for his specificity requirements and based on his problem - specific datasets , e.g. based on already annotated sp1 binding sites from transfac as motif dataset and representative gc - poor upstream regions as background dataset , he would then use these models to run the training and prediction functions of vombat as outlined above . the form requesting the parameters for a tfbs prediction . the results of a tfbs prediction . the list of putative tfbss are displayed as a link to sites.html, and the profiles for the sequences are plotted . the results of a cross - validation .

variable order markov models and variable order bayesian trees have been proposed for the recognition of transcription factor binding sites , and it could be demonstrated that they outperform traditional models , such as position weight matrices , markov models and bayesian trees . we develop a web server for the recognition of dna binding sites based on variable order markov models and variable order bayesian trees offering the following functionality : ( i ) given datasets with annotated binding sites and genomic background sequences , variable order markov models and variable order bayesian trees can be trained ; ( ii ) given a set of trained models , putative dna binding sites can be predicted in a given set of genomic sequences and ( iii ) given a dataset with annotated binding sites and a dataset with genomic background sequences , cross - validation experiments for different model combinations with different parameter settings can be performed . several of the offered services are computationally demanding , such as genome - wide predictions of dna binding sites in mammalian genomes or sets of 104-fold cross - validation experiments for different model combinations based on problem - specific data sets . in order to execute these jobs , and in order to serve multiple users at the same time , the web server is attached to a linux cluster with 150 processors . vombat is available at .

INTRODUCTION ALGORITHM INPUT AND OUTPUT Training a model Predicting putative transcription factor binding sites Cross-validation SUPPLEMENTARY DATA Figures and Tables

PMC4770306

bromodomains are epigenetic readers that specifically recognize acetyl - lysine ( kac ) marks on proteins . targeting this protein interaction module with small molecules has recently emerged as a potential therapeutic strategy for the treatment of several diseases including cancer and inflammation . to date , most inhibitor development efforts have been focused on the bet family of bromodomain proteins for which several inhibitors have now entered clinical testing . bromodomains have good predicted druggability , and selective chemical tool compounds have been developed even for less attractive binding sites that possess open or highly charged acetyl - lysine binding pockets such as baz2 and atad2 . in addition , potent inhibitors have been developed for highly druggable bromodomains present in brpf , cbp , and brd9 . interestingly , several recent inhibitor development projects have highlighted the success of fragment - based approaches identifying inhibitors , in particular for poorly druggable bromodomains . p300/cbp - associated factor ( pcaf , also known as histone acetyltransferase kat2b ) is a multidomain protein that harbors an acetyltransferase ( hat ) and e3 ubiquitin ligase domains as well as a c - terminal bromodomain that may associate with the hats p300 and cbp . while the roles of the acetyltransferase and the e3 ubiquitin ligase activities have been shown to be required for cell proliferation and apoptosis , little is known about the regulatory function of the pcaf bromodomain in cellular processes . selective inhibitors , so - called chemical probes , would therefore be interesting reagents to unravel the functions of the pcaf bromodomain and to assess its therapeutic potential as a targeting site for drug development . an important role of the pcaf acetyl - lysine recognition module has already been demonstrated for the replication of aids viruses . the pcaf bromodomain targets the hiv tat protein acetylated at k50 , an essential association that activates hiv-1 transcription and promotes the integrated proviral replication . development of pcaf bromo - domain inhibitors has therefore been proposed as a potential strategy for the treatment of aids , and this strategy has been confirmed by early n1-aryl - propane-1,3-diamine - based compounds that have been shown to prevent pcaf / tat association . this compound class has recently been followed up by the development of a series of 2-(3-aminopropylamino ) pyridine 1-oxide derivatives that however did not lead to improvement of the micromolar potency of current pcaf bromodomain inhibitors . this prompted us to identify more diverse chemical starting points using fragment screening by thermal shifts assays and nmr as well as structure - based approaches . to extend structural knowledge from our previous apo crystal structure , we initially determined the crystal structure of the pcaf - acetyl - lysine complex to provide insights into acetyl - lysine recognition . the binding mode of the acetyl - lysine in pcaf strongly resembled that observed in other bromodomains with the kac carbonyl forming two canonical hydrogen bonds to a conserved n803 and a conserved water molecule . this water is part of a conserved intricate network of structural waters located at the bottom of the pocket that bridge interaction with the za loop y760 and the backbone carbonyl of e750 ( figure 1a ) . despite sharing the conserved interactions , the kac binding groove in pcaf was fenced on one side by the bulky residue y809 , resulting in a tight and restricted pocket ( figure 1b ) . large aromatic residues in this position are not common for human bromodomains , present in less than a third of proteins in the family . ( a ) detailed interactions between the bound acetyl - lysine ( kac , shown in yellow stick ) and the pcaf bromodomain . ( b ) electrostatic surface representation reveals a tight central kac pocket that is extended by wide cavities lined by the za and bc loop regions . another interesting structural feature was the extension of the binding pocket by a shelf created by the za loop harboring a mixture of hydrophobic and polar surface residues ( figure 1b ) . however , recently the development of baz2 inhibitors demonstrated that such large binding pockets can be efficiently occupied for instance by aromatic ring systems of the baz2-icr probe that are aligned by intramolecular aromatic -stacking . the low affinities of the previously developed compounds prompted us to search for other kac mimetics that would be compatible with the constraint nature of the central pcaf pocket . we employed different techniques to screen two fragment libraries . in one set of experiments , we used a thermal stability shift assay to screen a small fragment set at 1 mm and identified several fragments that resulted in positive melting temperature ( tm ) shifts in the range of 12 c ( figure 2 ) . the highest tm shifts were recorded for 1-methylquinolin-2(1h)-one ) ( 1 , br004 ) , 1h - indole-5-carboxamide ( 2 , br005 ) , 2-methylisoindolin-1-one ( 3 , br013 ) , 4-methoxybenzo[d]isoxazol-3-amine ( 4 , mb360 ) , 2,3-dihydrobenzo[b]dioxine-5-carboxamide ( 5 , mb364 ) , and ( 4-(1,2,3-thiadiazol-4-yl)phenyl)methanamine ( 7 , mb093 ) , most of which comprised known kac mimetic scaffolds . in parallel , we used target immobilized nmr screening ( tins ) to screen a diverse collection of approximately 950 commercially available fragments . we first confirmed that binding of kac to immobilized pcaf could be observed in 2% dmso ( supporting information , figure s1 ) . subsequently mixtures of fragments were screened with a maximum of four compounds per mix such that the dmso concentration was never more than 2% . consistent with earlier predictions , we observed a tins profile indicating that pcaf is highly ligandable ( figure 2c ) . on the basis of the profile , 63 hits were selected , a 6.6% hit rate . in contrast to tins , binding of kac to immobilized pcaf in the presence of dmso could not be detected by spr . consequently , only those hits that were readily soluble in an aqueous buffer were orthogonally validated by spr . the binding of 17 hits could be fully characterized by spr , and three were selected for structural studies based on novel features ( supporting information , figure s2 and table s1 ) . these fragments ( 9 , 10 , and 11 ) also resulted in substantial tm shifts ( figure 2 ) . fragments containing kac mimetics identified as potential binders for pcaf bromodomain . chemical structures of the identified fragments are shown in ( a ) together with average tm shifts ( sem ) using triplicate experimental data listed in ( b ) . the t / r ratio of each compound screened was calculated as a height weighted average of the ratio of the peak amplitude of each nmr resonance in the presence of pcaf over that in the presence of the reference protein . the t / r ratios were then binned , and the frequency is plotted above . the asymmetry and tailing to the left ( i.e. , a large number of compounds displaying preferential binding to pcaf ) are indicative of high ligandability of pcaf . we next attempted to verify the binding modes of the identified fragments and successfully determined the complex crystal structures for seven kac mimetic fragments . as expected from their chemical structure , all of these fragments occupied the kac binding site through groups that mimicked the hydrogen bond interaction of acetyl - lysine . because the co - crystallized compounds were small , the contacts with the bromodomain were limited only to the canonical hydrogen bond with n803 and the typical water - mediated contact with y760 ( figure 3a ) . however , additional interactions were also observed for fr11 , of which the 1-ethanol decoration was oriented toward the open za cavity and formed both direct and water - mediated hydrogen bonds to the backbones of the za loop v752 and p751 . superimposition of all structures revealed that the co - crystallized fragments fit tightly into the narrow kac pocket , and most fragments formed aromatic interactions with y809 that lines the central acetyl - lysine binding groove of the pcaf bromomdomain ( figure 3b ) . no significant structural alterations were observed when comparing all complexes , suggesting that the pcaf bromodomain contains a rigid acetyl - lysine binding pocket . some kac mimetic groups of the identified fragments were not specific for pcaf and have been previously shown to bind to other bromodomains , for example , 1 also interacts with atad2 and the isoxazole 8 with brd4 and crebbp with highly conserved binding modes . ( a ) detailed interactions between the bound fragments ( yellow stick ) within the pcaf kac binding site . the conserved water molecules at the bottom of the pocket are shown in pink spheres , and an additional water molecule involving in additional water - mediated interactions observed in the complex with 11 is highlighted by magenta sphere . ( b ) superimposition of the bound fragments and kac revealed canonical acetyl - lysine mimetic binding modes and no significant conformational changes upon inhibitor binding within the pocket . coordinates have been deposited under accession codes : 5fe1 , 5fe2 , 5fe3 , 5fe4 , 5fe6 , 5fe7 , and 5fe5 . with the success in identifying the kac mimetic fragments and obtaining the complex structures , we next expanded the ligand series to more decorated compounds by choosing two diverse scaffolds ( figure 4a ) . two compounds , cpd1 ( 12 ) ( n-(1,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)acetamide ) and cpd2 ( 13 ) ( n-(1,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)methanesulfonam ) , were derivatives based on fragment hit 1 containing an additional 4-methyl group and two different decorations at the seventh position , either with n - linked acetamide or sulfonamide . a third inhibitor , cpd3 ( 14 ) ( n - methyl-5-(2-oxo-2-(phenylamino)ethoxy)-2-((tetrahydro-2h - pyran-4-yl)oxy)benzamide ) , contained instead a benzene core harboring an acetamide extension , a kac mimetic group resembling fragment 1 . in addition , this latter compound was also further decorated by tetrahydropyran and n - phenylacetamide linked to the benzene ring via an ethoxy linkage at the ortho and meta positions relative to the acetamide group , respectively . ( a ) chemical structures of three in - house available compounds that contain the fragment - related kac mimetic scaffolds . ( b ) average tm shift values from triplicate experiments for the selected compounds . ( c ) itc binding data for the interactions between 13 and 14 with pcaf with the isotherms of raw titration heat shown on the top and the normalized binding heat with the single - site fitting ( red line ) shown at the bottom . the binding of these three compounds was initially confirmed by dsf with 12 and 13 , showing a similar degree of stabilization with tm shifts of 2.62.9 c at 1 mm concentration ( figure 4b ) . because of signal interference , 14 was used at half the concentration of the others but nonetheless produced a similar tm shift of 3.3 c . as expected , the increase of the tm shift values of these three ligands suggested stronger binding to pcaf bromodomain . we used isothermal titration calorimetry ( itc ) to determine binding affinities in solution . in agreement with the dsf results , the measured kd of 13 was observed to be 21 m , which was 3-fold higher than that of the more decorated 14 ( kd of 7 m ) ( figure 4c ) . the increase in the affinity of the latter was due to significantly larger negative binding enthalpy , suggesting more favorable polar interaction of this inhibitor . however , the ligand efficiency ( le ) decreased to 0.24 for 14 compared to le of 0.34 for 13 . to gain insight into the selectivity of the most potent fragment hits , we performed a temperature shift screen using a panel of 48 recombinant bromodomains ( supporting information , figure s3 ) . this selectivity screen revealed strong interaction of 14 to bromodomains present in cecr2 ( 7.1 c ) , brd7 ( 6.5 c ) , suggesting that this fragment can also be optimized targeting other bromodomains . , both inhibitors showed strong inhibition for brd9 ( supporting information , table s2 ) . this is not surprising because 1-methylquinoline-2-ones have been developed into highly specific brd9 inhibitors . the crystal structures of the ligand pcaf complex allowed us to evaluate the interactions of the introduced decorations . all compounds could be unambiguously positioned based on the well - defined electron density map ( figure 5 ) . as expected in complexes with 12 and 13 , the 1-methylquinoline-2-one group occupied the kac binding groove and formed similar canonical interactions as observed in the pcaf complex with 1 , while the decoration of both compounds oriented toward the za pockets . in contrast , the 13 n - linked sulfonamide was observed to replace a water molecule that bridged interactions between e756 and k753 in the kac complex ( figure 1a ) , enabling the formation of two direct hydrogen bonds to these two residues as well as a water - mediated contact to the carbonyl backbone of w746 ( figure 5 ) . interestingly , a sulfonamide at this analogous position also engaged within the kac site of baz2b in the gsk2801 inhibitor complex . crystal structures of pcaf in complexes with 12 , 13 , and 14 . detailed interactions between the bound compounds with the pcaf bromodomain are shown . the conserved water at the bottom of the pocket is displayed in pink spheres , while an additional water molecule involving in contact between the 13 and protein is shown in magenta sphere . coordinates have been deposited under accession codes : 5fe8 , 5fe9 , and 5fdz , respectively . the binding mode of 14 was slightly different potentially due to its larger decorations and its different acetyl - lysine mimetic moiety ( figure 5 ) . the acetamide interacted with pcaf in a typical kac mimetic manner , with the benzene core stacking against the y809 in a similar fashion observed also for the other fragments . the tetrahydropyran expanded into the open za pocket , while the n - phenylacetamide extension occupied the bc pocket with the phenyl ring located above n803 and oriented perpendicularly to y802 and p804 within a distance for -stacking . considering its affinity , such limited interactions were rather surprising when compared to the binding mode of the weaker 13 , of which the extended group engaged in more hydrogen bond interactions . we hypothesize therefore that 14 may gain its increased binding potency through a larger contact surface . hence , an extension of the kac - mimetic fragments resulted in improved binding affinities despite the lack of direct polar interaction with the protein . in addition , the large surrounding space adjacent to the za and bc loops provides few spatial constraints , enabling the binding of diverse chemical moieties , albeit with little shape complementarity . it is likely that elongated , flexible , and uncharged substituents that could span the interface as well as engage a few interactions may be beneficial for increasing ligand potency through targeting the za and bc pockets in the pcaf bromodomain . the fragment - based approach coupled with structural information presented here offers a platform expanding the knowledge on pcaf kac - mimetic scaffolds and binding modes to aid development of potent and selective pcaf bromodomain inhibitors . the structural models provide valuable data for the rational design of inhibitors to the rigid and poorly enclosed pcaf kac binding pocket . comparison of inhibitor binding modes highlights the importance of aromatic interactions with y809 and provides several diverse acetyl - lysine mimetic chemotypes . these chemotypes may serve as attractive starting points for inhibitor development efforts targeting this interesting bromodomain . all fragments that were cocrystallized formed an acetyl - lysine mimetic hydrogen bond to n803 . thus , for future development of these fragments into more potent hits the fragment need to be grown rather than linked in order to increase potency . the pcaf bromodomain ( aa 715831 ) containing an n - terminal his6-tag was recombinantly expressed in escherichia coli rosetta . the protein was initially purified by ni - affinity chromatography and subsequently cleaved the tag with tev protease . the cleaved protein was further purified using size exclusion chromatography and stored in 25 mm hepes , ph 7.5 , 150 mm nacl , and 0.5 mm tcep . fragments were purchased from chemical vendors and were at least 95% pure according vendor specifications . the purity of the synthesized compounds 12 , 13 , and 14 was assessed by analytical hplc using an agilent 1100 equipped with photodiode array detector ( dad ) , quaternary gradient pump , and micro plate sampler ( agilent 220 ) . separation was performed upon centurysil c18-aq + 5 m , 50 mm 4.6 mm ( johnson ) . the flow rate of the mobile phase was kept at 3.5 ml / min . mobile phases b and c were acetonitrile with 0.35% cf3co2h and water with 0.35% cf3co2h , respectively . the gradient conditions were as follows : 00.5 min 1% b and 99% c , 3.7 min 90% b and 10% c , 5 min 99% b and 1% c. the injection volume was 10 l . 12 and 13 were 90% pure by hplc at 254 nm and 99% by evaporative light scattering detection ( elsd ) . the protein at 2 m in 10 mm hepes , ph 7.5 and 250 mm nacl was mixed with the fragments / compounds at 1 mm concentration ( unless stated ) . the assays , data evaluation , and melting temperature ( tm ) calculation were performed using a real - time pcr mx3005p machine ( stratagene ) with the protocols described previously . all isothermal calorimetry ( itc ) experiments were carried out on nanoitc ( ta instruments ) at 15 c in the buffer containing 25 mm hepes , ph 7.5 , 150 mm nacl , and 0.5 mm tcep . titration was performed by injecting pcaf ( 250 m ) into the reaction cell containing the compounds ( 1520 m ) . the corrected data of the integrated heat of titration were fitted to an independent single binding site model based on the manufactor protocol , from which the thermodynamics parameters ( h and ts ) , equilibrium association and dissociation constants ( ka and kd ) , and stoichiometry ( n ) were calculated . purified , biotinylated pcaf was captured onto a neutraavidin - coated cm5 sensor chip surface in 20 mm sodium phosphate ph7.5 , 150 mm nacl , 2 mm dtt , and 0.05% p20 tween . an acetyl - lys 16 peptide derived from h4 was used to monitor the ligand binding capacity of the immobilized pcaf bromodomain at regular intervals . apo crystals of pcaf ( 1418 mg / ml ) were obtained using the vapor diffusion method at 4 c and either single - peg - based ( 2135% peg 3350 , 0.1 m bis - tris ph 5.57.0 ) or peg - smear - based ( 2140% medium - molecular - weight peg smears buffered either with 0.1 m bis - tris ph 6.07.5 or 0.1 m tris ph 7.58.8 ) reservoir solutions . ligand complex crystals , soaking was performed using the mother liquor supplemented with the fragments / compounds at 1020 mm concentration and 20% ethylene glycol . diffraction data were collected either in - house or at the diamond light source . initial structure solution was achieved using molecular replacement with phaser from ccp4 suite and the published coordinates of pcaf . manual model building alternated with refinement were performed in coot and refmac , respectively .

the p300/cbp - associated factor plays a central role in retroviral infection and cancer development , and the c - terminal bromodomain provides an opportunity for selective targeting . here , we report several new classes of acetyl - lysine mimetic ligands ranging from mm to low micromolar affinity that were identified using fragment screening approaches . the binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective pcaf inhibitors .

Introduction Results and Discussion Conclusion Experimental Section

PMC3157863

in order to select dna fragments of interest to be purified , these should be resolved in agarose or acrylamide gels gel stained with ethidium bromide by electrophoresis using 0.5x tbe buffer ( tris borate , edta ) at 120 volts for 1 hour . for this , ~ 700 ng of plasmid pproex - gdmre11s ( 6000-bp ) previously digested with ndei and hindiii was used to release a 900-bp fragment ( 560 ng of plasmid and 84 ng of fragment ) . the electroeluter tank ( figure 1 ) should be filled with 0.5x tbe equally distributed in each side of the mid plateau taking care of not spill it onto the mid plateau . the selected band ( or bands ) is cut out of the gel , and placed in sample chamber as close as possible to the v channel ( one slice per well ) . running buffer should be added to each chamber ; just enough to cover the gel slice . each v channel used must be flushed with a pasteur pipette to eliminate any air bubble trapped . then 100 ul of 10 m nh4 acetate ( to facilitate visualization a small amount of bromophenol blue is added , just enough to color it ) are gently added inside the v channel . the lid should be closed gently to prevent any resuspension of the high salt cushion . when electrolution is completed , 400 ul are removed from v - channel , and placed in a microfuge tube to be precipitated with 2 volumes of cold ethanol and glycogen ( recommended to improve dna recovery ) at 4 c for 1 hour or overnight . the recovery obtained was 75% ( 63 ng recovered when 84 ng were placed on the v - channel ) . anod an cathode are indicated on each side of tank , dna contained in gel sliced ( illustrated as a black square ) will migrate towards cathode due to its negative charge . then it will be trapped in the salt cushion ( represented as an inverted black triangle ) located in the v - channel . the duration of the run will depend highly on the size of the fragments , normally for fragments up to 20 kb 50 minutes to 1 hour is enough , while for small fragments uv light monitoring every 10 minutes is required to prevent the fragment to go through the salt cushion and consequently to the anodal buffer chamber decreasing recovery . it is important to make sure that when you set your power supply at 100 volts , there is a current at least of 10 mamp . the dna band can be monitored by using a uv - hand lamp and detect when this abandons the gel slice . this procedure allows the purification of dna or rna fragments of different sizes with good recovery ( ~80% ) to be radiolabeled , digested by restriction enzymes , processed by modifying enzymes , etc .

purified dna fragments are used for different purposes in molecular biology and they can be prepared by several procedures . most of them require a previous electrophoresis of the dna fragments in order to separate the band of interest . then , this band is excised out from an agarose or acrylamide gel and purified by using either : binding and elution from glass or silica particles , deae - cellulose membranes , " crush and soak method " , electroelution or very often expensive commercial purification kits . thus , selecting a method will depend mostly of what is available in the laboratory . the electroelution procedure allows one to purify very clean dna to be used in a large number of applications ( sequencing , radiolabeling , enzymatic restriction , enzymatic modification , cloning etc ) . this procedure consists in placing dna band - containing agarose or acrylamide slices into sample wells of the electroeluter , then applying current will make the dna fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation .

Protocol Discussion Disclosures

PMC3609559

if mitochondrial deficiency causes insulin resistance it must occur before the onset of the insulin resistance . it does not seem possible to answer the question of whether or not mitochondrial deficiency precedes insulin resistance in humans , because t2 dm patients and obese insulin resistant individuals are insulin resistant for many years before they are diagnosed therefore , available evidence comes from studies on laboratory rodents , which develop muscle insulin resistance within a few weeks after being started on a high - fat diet . if the high - fat diet is continued , the rodents become obese and develop the rodent equivalent of the visceral obesity / metabolic syndrome and/or t2 dm ( 17,18 ) . in a number of early studies , high - fat diets were found to induce an increase in the levels of mitochondrial marker enzymes , such as betahydroxybutyrate dehydrogenase and citrate synthase in muscle ( 19,20 ) . more recently , it was found that feeding rats high - fat diets ( 21,22 ) or intermittently increasing plasma fatty acids to high levels ( 23 ) induces an increase in mitochondrial biogenesis in skeletal muscle . this is evidenced by increases in mitochondrial enzyme proteins , in the capacity to oxidize fatty acids , and in mitochondrial dna copy number . the increase in mitochondria appears to be an early , transient event that is lost as the insulin resistance and obesity progress ( 24 ) . the fatty acid - induced increase in muscle mitochondria appears to be mediated by the nuclear receptor peroxisome proliferator activated receptor- ( ppar- ; also referred to as ppar- ) . ppar- is a transcription factor for the genes encoding the enzymes of the mitochondrial fatty acid oxidation pathway . previous studies had shown that overexpression or activation of ppar- in muscle induces an increase in mitochondrial biogenesis ( 25,26 ) . the investigators who performed these studies concluded that the increase in mitochondrial biogenesis was mediated directly by ppar- , because overexpression of ppar- in muscle did not result in an increase in peroxisome proliferator activated receptor coactivator-1 ( pgc-1 ) mrna . however , this did not seem possible , because ppar- regulates expression of only a subset of mitochondrial proteins and , therefore , can not mediate mitochondrial biogenesis , which requires the coordinated activation of numerous transcription factors , a process that is mediated by the transcription coactivator pgc-1 ( 27 ) . it seemed probable , therefore , that an increase in ppar- induces an increase in mitochondria by mediating a posttranscriptional increase in pgc-1 protein . this turned out to be the case , as both overexpression of ppar- or activation of ppar- by raising plasma fatty acids resulted in an increase in pgc-1 ( 22 ) . in light of these findings , it seems clear that mitochondrial deficiency is not necessary for the development of insulin resistance and that , at least in the fat - fed rat model , insulin resistance develops despite an increase in muscle mitochondria . that insulin resistance is also not due to mitochondrial deficiency in humans is evidenced by the finding that insulin - resistant asian indians with t2 dm have a muscle mitochondrial capacity for oxidative metabolism similar to that of nondiabetic indians and higher than that of healthy north americans of northern european ancestry ( 28 ) . various transgenic models have been used to test the hypothesis that a deficiency of mitochondria causes muscle insulin resistance . ( 29 ) studied transgenic mice in which mitochondrial transcription factor a ( tfam ) was knocked out in skeletal muscle . tfam is a transcription factor that mediates transcription of genes encoded in the mitochondrial genome , which includes a number of key mitochondrial respiratory chain proteins . there was a progressive , severe deterioration of respiratory chain function in the muscles of the tfam knockout mice . however , rather than causing insulin resistance , the marked decrease in mitochondrial oxidative capacity resulted in increases in insulin action and glucose tolerance , as well as enhanced basal and insulin - stimulated 2-deoxyglucose uptake in isolated muscles . in another study , pospisilik et al . ( 30 ) examined the effect of deletion of mitochondrial apoptosis - inducing factor , which is required for maintenance of a functional mitochondrial respiratory chain . skeletal muscle apoptosis - inducing factor knockout mice had reduced levels of mitochondrial respiratory chain protein complexes i and iv with an associated decrease in the capacity for substrate oxidation . this reduction in mitochondrial respiratory capacity resulted in improved insulin sensitivity and glucose tolerance , and enhanced insulin - stimulated muscle glucose transport activity . in a third study , kelly and colleagues ( 31 ) generated mice that had combined deficiency of pgc-1 and pgc-1 in skeletal muscle . these totally pgc-1deficient mice had markedly reduced levels of mitochondrial enzymes and 90% decrease in the capacity of muscle to oxidize fat ( 31 ) . this severe mitochondrial deficiency resulted in an improvement in glucose tolerance . in an earlier study by our group , we generated transgenic mice that expressed uncoupling protein-1 ( ucp-1 ) in their skeletal muscles ( 32 ) . the original purpose of this study was to determine the effect of a modest reduction of steady - state atp concentration on mitochondrial biogenesis . however , the amount of ucp-1 expressed in the muscles of the transgenic mice was too high and resulted in disruption of mitochondrial structure ( fig . 1 ) , and caused large decreases in the levels of key mitochondrial enzyme proteins . the mechanism by which a high content of ucp-1 damages muscle mitochondria is not known . despite the severe reduction in mitochondrial function , and the associated accumulation of large amounts of intramuscular lipid ( fig . 1 ) , there were large increases in basal and insulin - stimulated muscle glucose transport activity ( 32 ) . electron microscopic images of muscle from wild - type mice ( a and c ) and from muscles of mice with severely disrupted mitochondria caused by high overexpression of uncoupling protein 1 ( ucp - h ) ( b , low magnification ; d , high magnification ) . * large lipid droplets surrounding abnormal mitochondria . reprinted with permission from han et al . , kelley and colleagues ( 33 ) published an article in which they concluded that insulin resistance in t2 dm is mediated by a deficiency of the electron transport chain ( etc ) enzymes with no deficiency of the citrate cycle or fatty acid oxidation pathway enzymes . they postulated that this results in an imbalance between the etc and the citrate cycle and fatty acid oxidation pathways , and that this imbalance causes insulin resistance . this claim is puzzling , both because their initial reports that t2 dm patients have mitochondrial dysfunction were largely based on the finding of decreases in citrate synthase ( 1,3 ) , and carnitine palmityl transferase ( 2 ) , and because the studies by pospisilik et al . ( 29 ) had shown that severe etc enzyme deficiency results in improved insulin action . to further test the hypothesis that a selective decrease in components of the mitochondrial etc , that results in an imbalance between the etc and the fatty acid oxidation pathway and citric acid cycle is responsible for insulin resistance , rats were made severely iron deficient by means of an iron - deficient diet ( 34 ) . iron deficiency results in decreases in the iron - containing mitochondrial respiratory chain proteins without affecting the noniron - containing enzymes of the fatty acid oxidation pathway and citrate cycle ( 35 ) . some of the iron - deficient rats were fed a high - fat diet . iron deficiency resulted in large decreases in iron - containing mitochondrial respiratory chain proteins in muscle ( 34 ) . citrate synthase and long - chain fatty acyl - coa dehydrogenase , used as markers for the citrate cycle and fatty acid oxidation pathways , were unaffected by the iron deficiency . oleate oxidation by muscle homogenates was increased by high - fat feeding and markedly decreased by iron deficiency despite a high - fat diet . the high - fat diet also increased long - chain acyl - coa dehydrogenase expression . as a result , there was a fivefold increase in the ratio of long - chain acyl - coa dehydrogenase to cytochrome c , indicating a large imbalance between the fatty acid oxidation pathway and respiratory chain . the iron deficiency completely protected against the high - fat diet induced insulin resistance , and also resulted in a higher insulin - stimulated muscle glucose transport activity compared with animals in the low - fat diet , normal iron group ( 34 ) . these studies on rodents showing that mitochondrial deficiency / dysfunction causes improvements in insulin action and glucose tolerance raise the question , why are t2 dm patients insulin resistant despite a reduction in muscle mitochondria ? the hypothesis that was tested in the studies on rodents reviewed above was , does mitochondrial deficiency / dysfunction severe enough to limit fat oxidation in resting muscle cause insulin resistance ? to test this hypothesis , an extremely severe reduction in functional mitochondria is necessary . the rate of substrate oxidation is determined by the demand for energy and is regulated by the rate of atp breakdown / adp production . the energy requirement of resting muscle is determined by housekeeping functions such as maintenance of electrochemical gradients and protein synthesis and is very low relative to the maximum capacity for substrate oxidation . therefore , a very severe reduction in functional mitochondria is necessary in order to result in limitation of resting substrate oxidation in resting muscle . in the various animal models in which mitochondrial deficiency / dysfunction resulted in improved insulin action and glucose tolerance , substrate oxidative capacity was so low that it resulted in a decrease in steady - state atp concentration and an increase in amp ( fig . the increase in amp resulted in activation of amp kinase , which directly stimulates glucose transport and also induces an increase in the glut4 ( fig . the increase in amp kinase activity explains the improvement in insulin action , glucose tolerance , and muscle glucose uptake in rodent muscle with severe mitochondrial deficiency / dysfunction . atp ( a ) , amp ( b ) , amp kinase activity ( c ) , glut4 protein ( d ) , and basal and insulin - stimulated 2-deoxyglucose transport rates ( e ) in muscle of control mice and mice with high overexpression of uncoupling protein 1 ( ucp - h ) and severe disruption of mitochondria . * in contrast to the rodent models of mitochondrial deficiency / dysfunction and improved insulin action in which the decrease in muscle respiratory capacity is so severe they ca nt exercise , t2 dm patients , despite a low vo2max of 26 ml o2 kg min ( 6 ) , are able to exercise sufficiently vigorously to increase their whole body rates of o2 uptake / substrate oxidation 89-fold . since the muscle mass involved in the exercise makes up 20% of body weight , this means muscles of t2 dm patients have a sufficiently high respiratory capacity to increase substrate oxidation 40-fold . in light of this huge reserve capacity , it is clear that a 30% lower than normal content of mitochondria is not sufficient to limit resting substrate oxidation in muscles of t2 dm patients or , therefore , to cause a decrease in atp or an increase in amp and amp kinase activity . it is clear from the many studies showing that mitochondrial deficiency / dysfunction severe enough to limit fat oxidation in resting muscle increases insulin responsiveness , that mitochondrial deficiency plays no role in mediating insulin resistance . studies by kelley and colleagues have supported the concept that fat oxidation is reduced in insulin - resistant individuals . in one of these studies , fat oxidation was decreased and glucose oxidation was increased in the resting , postabsorptive state in 11 t2 dm patients compared with 9 nondiabetic control subjects ( 36 ) . in another , fat oxidation by muscle was lower in 40 obese men than in lean control subjects ( 2 ) . however , in a subsequent study on obese men , kelley and colleagues in collaboration with wolfe ( 37 ) found that obese men derive a greater proportion of energy from fat oxidation than lean men ( 43 vs. 31% ) during mild exercise . in another study , kelley and colleagues found that fat oxidation was not reduced in obese insulin - resistant individuals or t2 dm patients , with fat providing 37% of total energy in the lean control subjects , 47% of energy in the obese insulin - resistant individuals , and 43% in the t2 dm patients ( 38 ) . there have also been reports that plasma fatty acid oxidation was reduced in insulin - resistant individuals , giving the impression that fat oxidation was impaired ( 39,40 ) . however , in these studies , nonplasma fatty acid oxidation ( probably from intramuscular fat ) was increased , so that total fat oxidation was not significantly different between the control and insulin - resistant groups . ( 40 ) , total fat oxidation averaged 0.92 0.08 mol kg min in the control subjects and 1.07 0.18 mol kg min in the obese group . most of the studies by other investigators comparing obese insulin - resistant individuals and/or patients with t2 dm have , however , found that insulin resistant individuals have increased fat oxidation ( 7,37,4147 ) . a hypothesis that used to be rather widely accepted is that insulin resistance is mediated by increased fat oxidation . ( 48 ) that fat oxidation inhibits glucose uptake and metabolism and is , therefore , referred to as the evidence for the randle effect in insulin resistant humans was provided by defronzo and colleagues ( 41 ) , who found that fat oxidation is increased in patients with t2 dm and in obese insulin - resistant individuals compared with normal control subjects . most of the other studies comparing fat oxidation in normal controls with t2 dm patients and/or obese insulin - resistant individuals have also shown that fat oxidation is increased in insulin - resistant obese individuals and patients with t2 dm ( 7,37,4147 ) . in conclusion , the answer to the three questions addressed in this counterpoint article is clearly no . rather than a decrease in mitochondria , an increase in mitochondrial biogenesis occurs in skeletal muscle concomitantly with the development of muscle insulin resistance ( 21,22 ) . rather than causing insulin resistance , a decrease in mitochondria or disruption of mitochondrial function sufficiently severe to limit fat oxidation in resting muscle results in increases in basal and insulin stimulated glucose transport into skeletal muscle and an improvement glucose tolerance ( 2932,34 ) . rather than a reduction in fat oxidation in muscle , the majority of studies show that fat oxidation is increased in obese , insulin resistant individuals and patients with t2 dm ( 7,37,4147 ) .

based on evidence that patients with type 2 diabetes ( t2 dm ) , obese insulin - resistant individuals , and lean insulin - resistant offspring of parents with t2 dm have 30% less mitochondria in their muscles than lean control subjects , it appears to be widely accepted that mitochondrial deficiency is responsible for insulin resistance . the proposed mechanism for this effect is an impaired ability to oxidize fat , resulting in lipid accumulation in muscle . the purpose of this counterpoint article is to review the evidence against the mitochondrial deficiency concept . this evidence includes the findings that 1 ) development of insulin resistance in laboratory rodents fed high - fat diets occurs despite a concomitant increase in muscle mitochondria ; 2 ) mitochondrial deficiency severe enough to impair fat oxidation in resting muscle causes an increase , not a decrease , in insulin action ; and 3 ) most of the studies comparing fat oxidation in insulin - sensitive and insulin - resistant individuals have shown that fat oxidation is higher in t2 dm patients and obese insulin - resistant individuals than in insulin - sensitive control subjects . in conclusion , it seems clear , based on this evidence , that the 30% reduction in muscle content of mitochondria in patients with t2 dm is not responsible for insulin resistance .

DOES A REDUCTION IN MITOCHONDRIA PRECEDE THE DEVELOPMENT OF INSULIN RESISTANCE? DOES A REDUCTION IN MUSCLE MITOCHONDRIA CAUSE INSULIN RESISTANCE? DOES MITOCHONDRIAL DEFICIENCY DECREASE FAT OXIDATION IN T2DM PATIENTS AND INSULIN-RESISTANT OBESE INDIVIDUALS?

PMC2989867

the term parkinson 's disease refers to a neurodegenerative disease that affects several regions of the brain , including the pigmented nuclei in midbrain and brainstem , the olfactory tubercle , the cerebral cortex , and elements of the peripheral nervous system . parkinson 's disease was first described in a medical context in 1817 by james parkinson , a general practitioner in london in his essay on the shaking palsy . even earlier many physicians have picked up some of the features of parkinson 's disease and described them in their writings , for example , franciscus de le be ( 16141672 ) who described tremors and franois boissier de sauvages de la croix ( 17061767 ) who described patients with running disturbances of the limbs . wilhelm von humboldt ( 17671835 ) , the celebrated academic reformer and writer who lived in the era of parkinson , has described his own neurological condition in a series of letters . reviewing the multiple clinical findings in pd will show us the need of more researches to reach a possible curative therapy . a group of neurobehavioral abnormalities can be found in pd such as apathy , fearfulness , anxiety , emotional liability , social withdrawal , increasing dependency , depression , dementia , bradyphrenia , a type of anomia termed the tip - of - the - tongue phenomenon , visual - spatial impairment , sleep disturbance , psychosis , and other psychiatric problems . the variable presentations of pd often cause diagnostic confusion and a delay in treatment . in the early stages , parkinsonian symptoms are often mistaken for simple arthritis or bursitis , depression , normal aging , alzheimer 's disease , or stroke . characterized a prodromal phase 46 years before the main manifestation in pd patients . during this period , pd patients , compared with normal controls , had a higher frequency of mood disorder , fibromyalgia , and shoulder pain . a sum of possible pd manifestations would be tremors at rest , rigidity , bradykinesia , and loss of postural reflexes which represents the cardinal manifestations besides possibility of the following . the basal ganglia include the neostriatum ( caudate nucleus and putamen ) the external and internal pallidal segments ( gpe , gpi ) , the subthalamic nucleus ( stn ) , and the substantia nigra with its pars reticulata ( snr ) and pars compacta ( snc ) . they participate in anatomically and functionally segregated loops that involve specific thalamic and cortical areas . striatum and stn receive glutamatergic afferents from specific areas of the cerebral cortex or thalamus , and transfer the information to the basal ganglia output nuclei , gpi and snr . the nigrostriatal projection terminates predominately at the necks of dendritic spines of the striatal medium spiny output neurons ( msns ) . this anatomic arrangement places the dopaminergic inputs in a position to regulate or gate the corticostriatal transmission . in parkinson 's disease , the degeneration of dopaminergic snc neurons and their projections to the striatum is a slowly evolving process that may take decades to develop . snc projections to the putamen degenerate earlier than projections to associative or limbic portions of the striatum . corresponding to this time course of degeneration , the motor symptoms and signs of parkinson 's disease develop before the nonmotor signs . recognizable motor or nonmotor signs appear only after substantial degeneration of the nigrostriatal neurons ( affecting at least 70% ) , this is due to the remarkable compensatory capacity within the dopaminergic system , or in the circuits it modulates . there may be mild frontal atrophy is some cases , but this is variable . histologically , there is neuronal loss in the substantia nigra pars compacta along with compensatory astrocytic and microglial proliferation . although biochemically there is loss of dopaminergic termini in the striatum , the striatum is histologically unremarkable . hyaline cytoplasmic inclusions or lewy bodies and less well - defined pale bodies are found in some of the residual neurons in the substantia nigra . lewy bodies are proteinaceous neuronal cytoplasmic inclusions . in some regions of the brain , such as the dorsal motor nucleus of the vagus , lewy bodies tend to form within neuronal processes and lewy bodies are accompanied by a variable number of abnormal neuritic profiles , referred to as lewy neurites . lewy neurites were first described in the hippocampus , but are also found in other regions of the brain , including the amygdala , cingulate gyrus , and temporal cortex . at the electron microscopic level , lewy bodies are composed of densely aggregated filaments and lewy neurites also are filamentous , but they are usually not as densely packed . some authors suggested that pre - parkinsonian manifestations would be nonmotor ( e.g. , autonomic dysfunction and anosmia ) and that the late stage would be associated with cortical lewy body dementia . synuclein has gain interest for its role in pd neurodegeneration since the demonstration that -synuclein is the major component of lewy bodies and lewy neuritis in idiopathic pd . the exact mechanisms of how conformational changes of -synuclein induce cell death are currently the subject of intense investigation , but are still poorly understood . it has been suggested that -synuclein oligomers might form pores on intracellular membranes such as the plasma membrane , and may increase cation permeability . -synuclein has been reported to negatively regulate the activity of tyrosine hydroxylase by maintaining the protein in the dephosphorylated inactive form , thus modulating dopamine biosynthesis in catecholaminergic neurons . in addition , -synuclein was proposed to negatively regulate the function of the dopamine transporter in dopaminergic nerve terminals by controlling the amount of the transporter at the plasma membrane . tau is a microtubule - binding protein that stabilizes microtubules and promotes their polymerization in neurons . while pd is the best known of the -synucleinopathies , tau pathology is also seen in many pd cases . staining of lewy bodies with multiple tau antibodies has been reported , suggesting cellular colocalization of these two pathologies . both of the proteins ( uchl1 and parkin ) are involved in the ubiquitin - proteasome pathway of abnormal protein degradation which is considered to be a cellular quality control . thus , it is possible that mutations in the parkin and uchl1 genes may lead to malfunction of the pathway and damaged proteins are not degraded . instead , they form aggregates that ultimately lead to cell degeneration with an unknown mechanism . the direct relation between mitochondrial dysfunction and pd came from the postmortem description of complex i deficiency in the substantia nigra of patients with pd . subsequently , the deficiency was also seen in the skeletal muscle and platelets and there was a decrease in complex i proteins in the substantia nigra of patients with pd . . da metabolism and auto - oxidation combined with increased iron , decreased total glutathione levels , and mitochondrial complex i inhibition can lead to cell death by exceeding the oxidative capacity of da - containing cells in the region . it has been an attractive hypothesis for decades that excitotoxicity and disturbance in calcium homeostasis are mechanisms leading to neurodegeneration ; disturbed calcium homeostasis is done through the activation of calpains , a family of cysteine proteases . these are elevated in the mesencephalon of patients with pd but not in other neurodegenerative disorders involving the mesencephalon . david park and colleagues have shown that calpains are activated and required for mptp - induced neuronal death in mice . in one of his dialogues , the greek philosopher plato speculated that our minds contain a block of wax , the size , hardness , and consistency of which varies from person to person . everything we think or perceive leaves an impression on the wax , and the quality and duration of this impression is the basis of our memory and knowledge . this metaphor was used by di monte , to describe the relation between genetics and environmental risk factors in developing pd . pd is considered a multifactorial disease resulting from the effect of environmental factors and genetic susceptibility . despite the mysterious cause of pd , the pathologic and neurochemical basis of parkinsonian signs and symptoms have been in part unraveled as discussed earlier . toxic insults could both modify the structure of -synuclein and interfere with the ubiquitin - proteasomal pathway , thus , promoting -synuclein aggregation and impairing the process of degradation of the abnormal protein . barlow et al . found that even in utero exposure with subsequent genetic damage can be a risk factor for neurodegenerative diseases , for example , alzheimer and pd . this can explain the finding of tsai et al . who described what he has called a young onset parkinsonian disease ( yopd ) which is a peculiar group of patients developing pd before 40 years on contrary to normal age group of more than 50 years . the reason for trying to identify toxic causes of pd ( besides future protection ) is the search for a perfect pd model . scientists are trying to unravel the exact cause of pd ; also we are trying to find out a perfect therapy and both the therapy and cause need a perfect model to act on . finding a toxin that can be a true cause of pd would solve the problem . since the discovery of mptp , parkinsonian inducing effects this arouse the possibility of other similar compounds relevant to mptp , for example , paraquat to induce pd . although pd existed long before the introduction of these pesticides , the thought is that pesticide exposure has contributed to the increased incidence of the disease . fumigantsthe fumigant class encompasses a variety of agents most commonly used to control insects or fungi in grains , soil , or other various consumables . fumigants , such as ethylene dibromide , are highly toxic to humans but most adverse actions are nonneuronal . although there is some evidence that carbon disulfide - based fumigants can induce parkinsonian - like neurotoxicity . this chemical class is not suitable for induction of pd in an animal model . the fumigant class encompasses a variety of agents most commonly used to control insects or fungi in grains , soil , or other various consumables . fumigants , such as ethylene dibromide , are highly toxic to humans but most adverse actions are nonneuronal . although there is some evidence that carbon disulfide - based fumigants can induce parkinsonian - like neurotoxicity . this chemical class is not suitable for induction of pd in an animal model . maneb , or manganese ethylene bis - dithiocarbamate , is one type with possible parkinsonian symptoms which may be secondary to exposure to the manganese metal core .in fact , maneb is one of the toxins used to induce pd in animal models but usually in combination with other agents specially paraquat . the maneb effect has been undoubtedly documented to increase the severity of pd models which make it one of the good candidates in pd research . maneb , or manganese ethylene bis - dithiocarbamate , is one type with possible parkinsonian symptoms which may be secondary to exposure to the manganese metal core . in fact , maneb is one of the toxins used to induce pd in animal models but usually in combination with other agents specially paraquat . the maneb effect has been undoubtedly documented to increase the severity of pd models which make it one of the good candidates in pd research . herbicides ( paraquat)the possibility that paraquat ( 1,1_-dimethyl-4,4_-bipyridinium ) may damage the nigrostriatal dopaminergic system and therefore contribute to the neuropathology of parkinson 's disease ( pd ) was first proposed in the mid 1980s following the observation that its chemical structure closely resembles that of mpp(1-methyl-4-phenylpyridinium ion ) .animal studies confirmed the ability of paraquat to induce selective dopaminergic nigrostriatal degeneration [ 3941].li et al . have developed subacute model for inducing pd in mice through using intraperitoneal injection of paraquat ( 10 mg / kg ) in old c57/bl mice . similar results were obtained 2 years later by kuter et al . using a similar approach.as described earlier , an improvement in modeling of paraquat induced pd was through adding maneb . a combination which improved the results . it also supported the multiple hit theory of pd .the presence of differences between paraquat and mptp has been questioned and the answer would come from a group of articles in toxicological sciences journal regarding mechanism of paraquat toxicity . richardson et al . proposed that paraquat would have different mechanisms for toxicity than mptp . later , ramachandiran et al . have shown that paraquat and mptp have divergent mechanisms of toxicity . another hot debate emerged on the same journal regarding the possibility of different mechanisms of neurotoxicity between paraquat and mptp [ 4548].despite the debate , there are certainly toxicokinetic and toxicodynamic differences which would give paraquat a unique pattern of neurotoxicity . despite the fact that paraquat models are still less validated than the previous well - established mptp models , combining maneb with paraquat would introduce a promising modeling technique that can be ( in our opinion ) a better model specially with the limitation in the other available toxic models . the possibility that paraquat ( 1,1_-dimethyl-4,4_-bipyridinium ) may damage the nigrostriatal dopaminergic system and therefore contribute to the neuropathology of parkinson 's disease ( pd ) was first proposed in the mid 1980s following the observation that its chemical structure closely resembles that of mpp(1-methyl-4-phenylpyridinium ion ) . animal studies confirmed the ability of paraquat to induce selective dopaminergic nigrostriatal degeneration [ 3941 ] . have developed subacute model for inducing pd in mice through using intraperitoneal injection of paraquat ( 10 mg / kg ) in old c57/bl mice . similar results were obtained 2 years later by kuter et al . using a similar approach . as described earlier , an improvement in modeling of paraquat induced pd was through adding maneb . a combination which improved the results . the presence of differences between paraquat and mptp has been questioned and the answer would come from a group of articles in toxicological sciences journal regarding mechanism of paraquat toxicity . richardson et al . proposed that paraquat would have different mechanisms for toxicity than mptp . later , ramachandiran et al . have shown that paraquat and mptp have divergent mechanisms of toxicity . another hot debate emerged on the same journal regarding the possibility of different mechanisms of neurotoxicity between paraquat and mptp [ 4548 ] . despite the debate , there are certainly toxicokinetic and toxicodynamic differences which would give paraquat a unique pattern of neurotoxicity . despite the fact that paraquat models are still less validated than the previous well - established mptp models , combining maneb with paraquat would introduce a promising modeling technique that can be ( in our opinion ) a better model specially with the limitation in the other available toxic models . many of the compounds in this class are , by design , neurotoxic . similarities between the insect and human nervous systems can lead to cross - toxicity of these compounds . organophosphatesalthough evidence supporting a role for ops in pd pathogenesis is scant , there are case reports of people with severe parkinsonism after op exposure ; however , the reversibility of the symptoms and lack of responsiveness to l - dopa are not consistent with pd . a recent family - based case - control study did implicate organophosphates and other insecticides in pd .however , it might be that the effects of op exposure probably involve disturbance of the balance between the dopamine and acetylcholine systems and not specific pathological changes in the dopamine system which makes them unsuitable in toxic modeling of pd rotenonedue to its ability of inhibiting mitochondrial complex i ( nadh dehydrogenase ) , rotenone has become one of the toxic models used to study pd in animals [ 33 , 53].chronic systemic exposure to rotenone reproduced many features of pd , including nigrostriatal dopaminergic degeneration and the formation of cytoplasmic inclusions in these neurons . rats exposed to rotenone showed bradykinesia , postural instability , unsteady gait , and some evidence of tremors [ 54 , 55 ] . reported an improved rotenone model through giving 3 mg / kg daily with observation of behavioral changes including bradykinesia , postural instability / unsteady gait , and rigidity , which occurred maximally after 6 days of treatment where postmortem analysis ( immunohistochemistry of the th+ve neurons in substantia nigra ) confirmed the behavioral observations . others have shown the efficacy of rotenone as a model of pd in mice and suggested its use for assessing candidate anti - parkinson drugs .rotenone model can be used as an alternative to other classical pd models , for example , mptp and 6-ohda , especially when testing the neuroprotective effects of novel therapeutic modalities . a limiting factor of rotenone models would be the high mortality rate of the examined animals . another limitation would be the less - accurate results that can be obtained usually in cases of oral administered rotenone . organochlorinesthe first clue of a relation between organochlorines and pd was the presence of them in postmortem specimens of pd patients . kanthasamy et al . have postulated possible mechanisms for the influence of organochlorines on pd patients . effects on dopamine and dopamine transportersthe assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum).sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin - induced oxidative stressdieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64].chun et al . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin and mitochondrial dysfunctiondieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line .neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons .pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669].dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . 2.4.1.4c.v . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation.these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . pyrethroidsalthough pyrethroids are often considered environmentally labile , exposure of mice to the pyrethroid pesticides deltamethrin and permethrin has been demonstrated to increase dat - mediated dopamine uptake [ 7375 ] . acute toxicity of pyrethroids is primarily mediated through interaction with sodium channels , leading to prolonged depolarization and hyperexcitation of the nervous system .pyrethroids have also been shown to be potent releasers of neurotransmitters , including dopamine .although the mechanism by which pyrethroids are capable of increasing dat - mediated dopamine uptake is not clear , elwan et al . suggest that the effects of pyrethroids on dat are indirect and that longer - term exposures may be capable of damaging cells through an apoptotic mechanism.the use of pyrethroids in modeling of pd is still far from validation as frequent researches are needed to reach a final exposure protocol . the biometals , iron , copper , zinc , and manganese participate in many essential activities , and indeed deficiencies can be lethal . zinc and iron are increased and copper is decreased in the substantia nigra ( sn ) in parkinson 's disease ( pd ) . iron and pdof great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals .searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese : nontypical pd manifestationsmanganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . metals and -synucleinuversky et al . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation.the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice .however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . mptpthe potent parkinsonian neurotoxin , 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) causes pd in animals and is one of the most accepted models . this may be due to its capacity to show some of pd pathological findings ( sn neuron loss and striatal da depletion similar to pd ) .mptp - treated animals ( including humans ) do not exhibit lewy bodies ( lb ) , a hallmark neuronal inclusion of pd , but old treated primates have inclusions that are somewhat similar . humans and many other animal species including nonhuman primates , guinea pigs , mice , and cats are susceptible to this neurotoxin .one interesting feature of the mptp mouse model of pd is the transient nature of the striatal damage in young mice . in contrast , administration of the drug to older mice would result in a permanent loss of nigrostriatal terminals , as well as cell bodies . this recovery potential in young mice allows for modeling of recovery .various techniques can be used to modulate the action of mptp to induce the pathological findings needed to reach the desired pd model . one of these techniques is to give probenecid with mptp to prolong its persistence in the tissues .in sporadic pd , the generation of neural precursor cells in the subependymal zone is impaired by da depletion , which is the hallmark of pd . mptp - induced degeneration of dopaminergic snpc - svz fibres impairs nsc proliferation in primates and in mice in a similar fashion to pd . he et al . raised the possibility that migrating neuroblasts in the svz may be more vulnerable to mptp than nigrostriatal dopaminergic neurons in the sn . furthermore , they suggested that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent.several mptp dosing regimens have been used . the acute regimen consists of multiple systemic administration of mptp ( usually four doses at 2-h intervals per day ) . the subacute regimen consists of a single systemic administration per day for several consecutive days ( usually 5 days ) and the chronic regimen through several weeks .the comparison of these different models indicated clearly that different schedules of administration of mptp mimic distinct stages of the disease and might induce different mechanisms of neuronal death .one of the limitations of mptp models is their inability to induce behavioral deficits apparent on standard motor mouse tests . however , new techniques for behavioral monitoring have shown promising results , for example , grid test and its modification , the vertical grid test and modified horizontal grid test , swim test , or the automated behavioural apparatus , laborastm ( laboratory animal behaviour observation , registration and analysis system ) .strain differences regarding the response to mptp has been questioned where c57/bl mice show maximum response compared to balb / c mice [ 85 , 93 , 94 ] . , who found prominent changes in balb / c response to mptp as balb / c mice still show a possible alternative , though less effective to c57/bl.an important property of the mptp - lesioned mouse and nonhuman primate is the potential for intrinsic recovery . this capacity would be of great help in identifying new therapeutic targets for the treatment of pd . many of the compounds in this class are , by design , neurotoxic . similarities between the insect and human nervous systems can lead to cross - toxicity of these compounds . organophosphatesalthough evidence supporting a role for ops in pd pathogenesis is scant , there are case reports of people with severe parkinsonism after op exposure ; however , the reversibility of the symptoms and lack of responsiveness to l - dopa are not consistent with pd . a recent family - based case - control study did implicate organophosphates and other insecticides in pd .however , it might be that the effects of op exposure probably involve disturbance of the balance between the dopamine and acetylcholine systems and not specific pathological changes in the dopamine system which makes them unsuitable in toxic modeling of pd . rotenonedue to its ability of inhibiting mitochondrial complex i ( nadh dehydrogenase ) , rotenone has become one of the toxic models used to study pd in animals [ 33 , 53].chronic systemic exposure to rotenone reproduced many features of pd , including nigrostriatal dopaminergic degeneration and the formation of cytoplasmic inclusions in these neurons . rats exposed to rotenone showed bradykinesia , postural instability , unsteady gait , and some evidence of tremors [ 54 , 55 ] . reported an improved rotenone model through giving 3 mg / kg daily with observation of behavioral changes including bradykinesia , postural instability / unsteady gait , and rigidity , which occurred maximally after 6 days of treatment where postmortem analysis ( immunohistochemistry of the th+ve neurons in substantia nigra ) confirmed the behavioral observations . others have shown the efficacy of rotenone as a model of pd in mice and suggested its use for assessing candidate anti - parkinson drugs .rotenone model can be used as an alternative to other classical pd models , for example , mptp and 6-ohda , especially when testing the neuroprotective effects of novel therapeutic modalities . a limiting factor of rotenone models would be the high mortality rate of the examined animals . another limitation would be the less - accurate results that can be obtained usually in cases of oral administered rotenone . organochlorinesthe first clue of a relation between organochlorines and pd was the presence of them in postmortem specimens of pd patients . kanthasamy et al . have postulated possible mechanisms for the influence of organochlorines on pd patients . effects on dopamine and dopamine transportersthe assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum).sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin - induced oxidative stressdieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64].chun et al . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin and mitochondrial dysfunctiondieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line .neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons .pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669].dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . 2.4.1.4c.v . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation.these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . pyrethroidsalthough pyrethroids are often considered environmentally labile , exposure of mice to the pyrethroid pesticides deltamethrin and permethrin has been demonstrated to increase dat - mediated dopamine uptake [ 7375 ] . acute toxicity of pyrethroids is primarily mediated through interaction with sodium channels , leading to prolonged depolarization and hyperexcitation of the nervous system .pyrethroids have also been shown to be potent releasers of neurotransmitters , including dopamine .although the mechanism by which pyrethroids are capable of increasing dat - mediated dopamine uptake is not clear , elwan et al . suggest that the effects of pyrethroids on dat are indirect and that longer - term exposures may be capable of damaging cells through an apoptotic mechanism.the use of pyrethroids in modeling of pd is still far from validation as frequent researches are needed to reach a final exposure protocol . the biometals , iron , copper , zinc , and manganese participate in many essential activities , and indeed deficiencies can be lethal . zinc and iron are increased and copper is decreased in the substantia nigra ( sn ) in parkinson 's disease ( pd ) . iron and pdof great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals .searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese : nontypical pd manifestationsmanganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . metals and -synucleinuversky et al . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation.the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice .however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . mptpthe potent parkinsonian neurotoxin , 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) causes pd in animals and is one of the most accepted models . this may be due to its capacity to show some of pd pathological findings ( sn neuron loss and striatal da depletion similar to pd ) .mptp - treated animals ( including humans ) do not exhibit lewy bodies ( lb ) , a hallmark neuronal inclusion of pd , but old treated primates have inclusions that are somewhat similar . humans and many other animal species including nonhuman primates , guinea pigs , mice , and cats are susceptible to this neurotoxin .one interesting feature of the mptp mouse model of pd is the transient nature of the striatal damage in young mice . in contrast , administration of the drug to older mice would result in a permanent loss of nigrostriatal terminals , as well as cell bodies . this recovery potential in young mice allows for modeling of recovery .various techniques can be used to modulate the action of mptp to induce the pathological findings needed to reach the desired pd model . one of these techniques is to give probenecid with mptp to prolong its persistence in the tissues .in sporadic pd , the generation of neural precursor cells in the subependymal zone is impaired by da depletion , which is the hallmark of pd . mptp - induced degeneration of dopaminergic snpc - svz fibres impairs nsc proliferation in primates and in mice in a similar fashion to pd . he et al . raised the possibility that migrating neuroblasts in the svz may be more vulnerable to mptp than nigrostriatal dopaminergic neurons in the sn . furthermore , they suggested that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent.several mptp dosing regimens have been used . the acute regimen consists of multiple systemic administration of mptp ( usually four doses at 2-h intervals per day ) . the subacute regimen consists of a single systemic administration per day for several consecutive days ( usually 5 days ) and the chronic regimen through several weeks .the comparison of these different models indicated clearly that different schedules of administration of mptp mimic distinct stages of the disease and might induce different mechanisms of neuronal death .one of the limitations of mptp models is their inability to induce behavioral deficits apparent on standard motor mouse tests . however , new techniques for behavioral monitoring have shown promising results , for example , grid test and its modification , the vertical grid test and modified horizontal grid test , swim test , or the automated behavioural apparatus , laborastm ( laboratory animal behaviour observation , registration and analysis system ) .strain differences regarding the response to mptp has been questioned where c57/bl mice show maximum response compared to balb / c mice [ 85 , 93 , 94 ] . , who found prominent changes in balb / c response to mptp as balb / c mice still show a possible alternative , though less effective to c57/bl.an important property of the mptp - lesioned mouse and nonhuman primate is the potential for intrinsic recovery . this capacity would be of great help in identifying new therapeutic targets for the treatment of pd . although evidence supporting a role for ops in pd pathogenesis is scant , there are case reports of people with severe parkinsonism after op exposure ; however , the reversibility of the symptoms and lack of responsiveness to l - dopa are not consistent with pd . a recent family - based case - control study did implicate organophosphates and other insecticides in pd . however , it might be that the effects of op exposure probably involve disturbance of the balance between the dopamine and acetylcholine systems and not specific pathological changes in the dopamine system which makes them unsuitable in toxic modeling of pd . due to its ability of inhibiting mitochondrial complex i ( nadh dehydrogenase ) , rotenone has become one of the toxic models used to study pd in animals [ 33 , 53 ] . chronic systemic exposure to rotenone reproduced many features of pd , including nigrostriatal dopaminergic degeneration and the formation of cytoplasmic inclusions in these neurons . rats exposed to rotenone showed bradykinesia , postural instability , unsteady gait , and some evidence of tremors [ 54 , 55 ] . reported an improved rotenone model through giving 3 mg / kg daily with observation of behavioral changes including bradykinesia , postural instability / unsteady gait , and rigidity , which occurred maximally after 6 days of treatment where postmortem analysis ( immunohistochemistry of the th+ve neurons in substantia nigra ) confirmed the behavioral observations . others have shown the efficacy of rotenone as a model of pd in mice and suggested its use for assessing candidate anti - parkinson drugs . rotenone model can be used as an alternative to other classical pd models , for example , mptp and 6-ohda , especially when testing the neuroprotective effects of novel therapeutic modalities . a limiting factor of rotenone models would be the high mortality rate of the examined animals . another limitation would be the less - accurate results that can be obtained usually in cases of oral administered rotenone . the first clue of a relation between organochlorines and pd was the presence of them in postmortem specimens of pd patients . kanthasamy et al . have postulated possible mechanisms for the influence of organochlorines on pd patients . effects on dopamine and dopamine transportersthe assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum).sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin - induced oxidative stressdieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64].chun et al . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin and mitochondrial dysfunctiondieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line .neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons .pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669].dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . 2.4.1.4c.v . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation.these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . the assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum ) . sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64 ] . . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line . neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons . pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669 ] . dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation . these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . although pyrethroids are often considered environmentally labile , exposure of mice to the pyrethroid pesticides deltamethrin and permethrin has been demonstrated to increase dat - mediated dopamine uptake [ 7375 ] . acute toxicity of pyrethroids is primarily mediated through interaction with sodium channels , leading to prolonged depolarization and hyperexcitation of the nervous system . pyrethroids have also been shown to be potent releasers of neurotransmitters , including dopamine . although the mechanism by which pyrethroids are capable of increasing dat - mediated dopamine uptake is not clear , elwan et al . suggest that the effects of pyrethroids on dat are indirect and that longer - term exposures may be capable of damaging cells through an apoptotic mechanism . the use of pyrethroids in modeling of pd is still far from validation as frequent researches are needed to reach a final exposure protocol . the biometals , iron , copper , zinc , and manganese participate in many essential activities , and indeed deficiencies can be lethal . zinc and iron are increased and copper is decreased in the substantia nigra ( sn ) in parkinson 's disease ( pd ) . iron and pdof great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals .searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese : nontypical pd manifestationsmanganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . metals and -synucleinuversky et al . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation.the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . of great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals . searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation . the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice .however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice . however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . the potent parkinsonian neurotoxin , 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) causes pd in animals and is one of the most accepted models . this may be due to its capacity to show some of pd pathological findings ( sn neuron loss and striatal da depletion similar to pd ) . mptp - treated animals ( including humans ) do not exhibit lewy bodies ( lb ) , a hallmark neuronal inclusion of pd , but old treated primates have inclusions that are somewhat similar . humans and many other animal species including nonhuman primates , guinea pigs , mice , and cats are susceptible to this neurotoxin . one interesting feature of the mptp mouse model of pd is the transient nature of the striatal damage in young mice . in contrast , administration of the drug to older mice would result in a permanent loss of nigrostriatal terminals , as well as cell bodies . various techniques can be used to modulate the action of mptp to induce the pathological findings needed to reach the desired pd model . one of these techniques is to give probenecid with mptp to prolong its persistence in the tissues . in sporadic pd , the generation of neural precursor cells in the subependymal zone is impaired by da depletion , which is the hallmark of pd . mptp - induced degeneration of dopaminergic snpc - svz fibres impairs nsc proliferation in primates and in mice in a similar fashion to pd . he et al . raised the possibility that migrating neuroblasts in the svz may be more vulnerable to mptp than nigrostriatal dopaminergic neurons in the sn . furthermore , they suggested that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent . the acute regimen consists of multiple systemic administration of mptp ( usually four doses at 2-h intervals per day ) . the subacute regimen consists of a single systemic administration per day for several consecutive days ( usually 5 days ) and the chronic regimen through several weeks . the comparison of these different models indicated clearly that different schedules of administration of mptp mimic distinct stages of the disease and might induce different mechanisms of neuronal death . one of the limitations of mptp models is their inability to induce behavioral deficits apparent on standard motor mouse tests . however , new techniques for behavioral monitoring have shown promising results , for example , grid test and its modification , the vertical grid test and modified horizontal grid test , swim test , or the automated behavioural apparatus , laborastm ( laboratory animal behaviour observation , registration and analysis system ) . strain differences regarding the response to mptp has been questioned where c57/bl mice show maximum response compared to balb / c mice [ 85 , 93 , 94 ] . , who found prominent changes in balb / c response to mptp as balb / c mice still show a possible alternative , though less effective to c57/bl . an important property of the mptp - lesioned mouse and nonhuman primate is the potential for intrinsic recovery . this capacity would be of great help in identifying new therapeutic targets for the treatment of pd . pd is a multifactorial disease , so despite role of environmental exposure , genetic predisposition in sporadic cases can be broadly grouped into four categories : genes involved in the metabolism of xenobiotics ( e.g. , cyp2d6 , nat2 , gsts ) , neurodegeneration ( e.g. , nos ) , the functioning of dopaminergic neurons ( e.g. , dopamine transporters and receptors ) , and linkage - derived genes ( e.g. , uchl1 , alpha - synuclein ) . there is increasing evidence that genes involved in inherited forms of the disease may act as predisposing factors in the sporadic forms of pd . so , new models of pd produced in vivo on transgenic animals and in vitro on transfected cell cultures are considered important because they may be helpful in discovering the pathophysiology of pd . meredith et al . have classified genetic models into three categories . first , mouse models based on the deletion of genes important for the development or maintenance of da neurons or their phenotypes . second , mouse or rat models based on expression or deletion of genes known to cause familial forms of pd . finally , a third class of genetic models is based on virally mediated expression of genes or mutations known to cause familial pd , usually in nigrostriatal da neurons . despite having some features of the human disease , genetic models do not provide an ideal choice for assessing regenerating capacity of new therapeutic approaches and so are of less use for pharmaceutical development . in our search for an ideal toxic model of pd , we could not find a perfect one . it seems that all toxins when used alone can precipitate only some characters of pd pathology but not the exact pd condition . these same findings may be the cause of the manning - bog and langston advice of using model fusion to overcome the single model limitation through combining genetic with toxic models . we would encourage the use of toxins combination in pd modeling as a new type of model fusion .

parkinson 's disease ( pd ) is one of the neurodegenerative diseases which we can by certainty identify its pathology , however , this confidence disappeares when talking about the cause . a long history of trials , suggestions , and theories tried linking pd to a specific causation . in this paper , a new suggestion is trying to find its way , could it be toxicology ? can we in the future look to pd as an occupational disease , in fact , many clues point to the possible toxic responsibility either total or partial in causing this disease . searching for possible toxic causes for pd would help in designing perfect toxic models in animals .

1. Introduction 2. Historical Background 3. Environment, Genetics, and PD 4. Conclusion

PMC4932075

however , insufficient body water due to continuous dehydration caused by prolonged exercise leads to a decrease in plasma volume and cardiac output and an increase in plasma osmolarity and core temperature1 , 2 . furthermore , inefficient heat dispersion caused by a decreased rate of blood flow to the skin can accelerate fatigue and deteriorate exercise performance by retaining body heat3 , 4 . therefore , to improve exercise performance in athletes and to enhance the effects of regular exercise for health maintenance and promotion , it is important to develop a method that can alleviate exercise - induced rise in body temperature . far infrared electromagnetic wavelengths of 5.61,000 m can activate cells and promote metabolism when absorbed by the human body . previous studies verified the efficacy of ge - coated clothing5 , 6 , and observed that ge - containing fibers emitted far infrared rays and anions and had an antibacterial effect . these results suggested that ge - containing fibers could promote blood circulation and had the potential to suppress fatigue and relieve thermal stress by wearing ge - containing and/or ge - coated clothing during exercise . however , a scientific approach to proving such an effect has been very limited . therefore , the present study aimed to investigate the effects of wearing ge - coated functional clothing during exercise on tympanic temperature ; thermal sensation ; heat shock protein 70 ( hsp70 ) , which is involved in homeostatic regulation in exercise - induced thermal stress ; and lactate , an indicator of fatigue . nine healthy and untrained male subjects ( age : 20.2 0.4 years ; height : 175.7 2.8 cm ; weight : 73.1 10.1 kg ; body mass index : 23.7 3.0 kg / m ; muscle mass : 34.6 2.9 kg ; fat mass : 12.4 6.2 kg ; percentage body fat : 16.3 6.3% ; resting heart rate : 70.9 3.6 beats / min ; systolic blood pressure : 118.6 7.3 mmhg ; diastolic blood pressure : 72.6 8.4 mmhg ) volunteered as subjects for the present study . the study was conducted in accordance with the standards set by the latest revision of the declaration of helsinki , and all the subjects provided written informed consent in accordance with the department of sport for all studies at soongsil university guidelines . anthropometric measurements included height , body composition , resting heart rate , and blood pressure . height and body composition were measured using a stadiometer ( seca213 ; seca , germany ) and a bioimpedance analysis device ( inbody720 ; biospace , korea ) , respectively . resting heart rate and blood pressure were measured in a seated position , using a heart rate monitor ( polar a5 ; polar , finland ) and a mercury sphygmomanometer ( trimline ; pymah , usa ) , respectively . the 9 subjects participated in control and experimental tests , both involving 60 min of running on a treadmill at an exercise intensity of 75% heart rate reserve in a laboratory with temperature and humidity of 18.5 0.5 c and 36.0 1.0% , respectively . the clothing worn by each subject consisted of a half - sleeve t - shirt and short pants . during the control test , the participants wore conventional clothing ( which had not received functionality treatment ) ; during the experimental test , they wore ge - coated functional clothing ( which had been prepared by coating the same clothing used for the control test with ge ) . according to the result of a comparison test using fourier transform infrared spectroscopy ( ft - ir ) and a black body at 37 c , which is almost the same as body temperature , the far infrared rays emitted by the ge - coated functional clothing characteristically showed an emission rate of 0.893 m and a wavelength of 3.44 10 w / mm . tympanic temperature was measured using infrared ear thermometry ( thermoscan irt-4520 ; braun , germany ) as a surrogate for core temperature . thermal sensation was measured using a disc score7 . using a 22-gauge needle , a serum separator tube ( becton dickinson , usa ) , and an ethylenediaminetetraacetic acid - coated tube ( becton dickinson , usa ) , 8 ml of blood was collected from the antecubital vein of each subject immediately before exercise ( ibe ) and immediately after exercise ( iae ) . the collected blood samples were centrifuged for 15 min at 3,000 rpm , and then stored at 80 c until analysis . the serum hsp70 levels were determined using a high - sensitivity , commercially available enzyme - linked immunosorbent assay ( elisa ) kit ( assay designs , usa ) . the absorbance was measured at 450 nm with a microplate reader ( emax ; molecular devices , usa ) . the plasma lactate levels were determined using a clinical chemistry analyzer ( ektachem dt 60 ; eastman kodak , usa ) . statistical analyses were performed using spss software , version 21.0 for windows ( spss inc . data are presented as mean standard deviation ( sd ) . for identifying differences in normally distributed results , a two - way repeated analysis of variance ( anova ) when significant time by trial interactions occurred , simple main effects were assessed using the independent and paired t - tests . the tympanic temperature , disc score , hsp70 , and lactate levels are presented in table 1table 1.germanium-coated functional clothing effects on tympanic temperature , disc score , hsp70 , and lactate levelsvariablecontrol ( n=9)experimental ( n=9)ibeiaeibeiaetym t 36.73 0.3037.16 0.66disc score1.33 0.872.44 1.13*1.22 0.971.11 1.17hsp70 ( ng / ml)0.184 0.0170.205 0.027 * 0.193 0.0170.210 0.019*lactate ( mmol / l)1.48 0.602.84 1.34 * 1.12 0.452.03 0.89*data are presented as mean sd . tym t : tympanic temperature ; ibe : immediately before exercise ; iae : immediately after exercise ; rating scale range , 3 to 4 ; * p<0.05 vs. ibe ; p<0.05 vs. control test . the tympanic temperature iae was significantly elevated compared to the temperature ibe in the control test ( p<0.05 ) , while no significant change was observed in the experimental test . in both tests , the hsp70 and lactate levels measured iae were significantly increased compared to the levels ibe ( p<0.05 ) . the disc score iae was significantly increased compared to the score evaluated ibe in both tests ( p<0.05 ) , and it was significantly higher iae in the control test compared to that in the experimental test ( p<0.05 ) . tym t : tympanic temperature ; ibe : immediately before exercise ; iae : immediately after exercise ; rating scale range , 3 to 4 ; * p<0.05 vs. ibe ; p<0.05 vs. control test organic ge has been reported to promote immune activation and play an important role in regulating antioxidative activities8 , 9 . for this reason , it has been widely utilized for manufacturing not only health functional foods but also bracelets , necklaces , and textile products such as underwear . to verify the effects of wearing ge - coated functional clothing during exercise on thermal stress , in the present study , the results in the control test ( wearing common clothing ) showed a significant increase in tympanic temperature measured iae ; however , the experimental test ( wearing ge - coated clothing ) did not show such an increase . in addition , the measurement of thermal sensation using the disc score showed significantly lower levels iae in the experimental test than that in the control test . the reasons for such results could be that ge - coated clothing alleviated exercise - induced thermal stress by promoting blood circulation during exercise . this interpretation is supported by the observations that increase in skin blood flow during exercise is an effective mechanism for dispersing heat accumulated in the body10 , and that ge - containing clothing had the effect of promoting blood circulation5 , 6 . in both tests in the present study , the serum hsp70 and plasma lactate levels showed a significant increase iae , but without a significant difference between the 2 tests . the result for hsp70 levels supports the results of previous studies that reported a significant increase after acute exercise11 , 12 , and suggests that not only high core temperature but also various other factors contribute to hsp70 expression . this interpretation is consistent with the reports of a positive correlation between hsp70 expression and core temperature13 ; however , hsp70 expression was also affected by various physiological and environmental stress factors such as heat , training status , oxidative stress , ph change in the body , and glucose depletion14 , 15 . conversely , the lactate level is considered independent of thermal stress , probably due to the same level of exercise intensity used for both tests ; this result suggests that wearing ge - coated clothing during exercise exerts no significant effect on exercise - induced fatigue . this interpretation is supported by ishii and nishida , who reported that circulating lactate concentrations were dependent on exercise intensity16 , and also by mohr et al . , who reported that exercise under different environmental conditions ( 21 c vs. 43 c ) showed no difference in plasma lactate concentration in spite of significant differences in core temperature17 . in conclusion , the present study suggests that wearing ge - coated clothing during endurance exercise may have the positive effect of alleviating thermal stress that accumulates during exercise .

[ purpose ] the present study investigated the effects of wearing germanium - coated functional clothing on tympanic temperature , thermal sensation , heat shock protein 70 ( hsp70 ) , and lactate during endurance exercise . [ subjects and methods ] nine healthy and untrained male subjects were enrolled . subjects ran for 60 min on a treadmill ( 75% heart rate reserve ) in the following 2 tests : 1 ) control test ( wearing conventional clothing ) and 2 ) experimental test ( wearing germanium - coated functional clothing ) . during each test , the tympanic temperature and thermal sensation were measured , and blood samples were collected immediately before exercise and immediately after exercise . thermal sensation was measured using a disc score . [ results ] the tympanic temperature immediately after exercise was significantly increased compared to the temperature immediately before exercise in the control test , while no significant change was observed in the experimental test . in both tests , the disc score and hsp70 and lactate levels immediately after exercise were significantly increased compared to those immediately before exercise . in addition , the disc score immediately after exercise was significantly higher in the control test than in the experimental test . [ conclusion ] wearing germanium - coated functional clothing during endurance exercise may have the positive effect of alleviating thermal stress that accumulates in the body during exercise .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3600345

being a primary care professional ( pcp ) means being at the forefront of managing chronic , complex , and difficult areas of health . working with patients who have cancer , those who are dying and those who experience or have experienced family and/or sexual violence can be difficult and emotionally confronting . in a day 's work , pcps see many complicated and complex patients , but those who experience family violence can be particularly demanding and challenging because of chronic health issues , psychological distress , relationship and authority issues , and coping strategies such as denial or somatization . if we then add to the potential emotional impact of survivor stories on pcps , the mental and emotional work of caring for these patients , the patient - professional interaction and boundary issues , and the long - term nature of the care required , we will have the perfect recipe for increased pcp stress . work - related stress is already experienced by family physicians and nurses , with burnout being the most investigated area [ 47 ] . prevalence rates of burnout for medical residents ( including family medicine residents ) have been reported between 18 and 82% and between 10 and 55% for family physicians [ 4 , 6 , 7 ] . a number of factors have been identified as important contributors to burnout ; these include the work environment , changing policy , changes in the practice of family medicine , workload , loss of autonomy , and feeling a lack of personal accomplishment [ 4 , 7 ] . personal characteristics of the pcps themselves , such as being high achievers and perfectionists , the relationship between work and self - esteem , gender , age , and family and support networks , may also contribute to their susceptibility to stress [ 4 , 7 , 8 ] . pcp 's recognition and support of patients who have experienced violence may improve the mental and physical health through early intervention and better service access [ 912 ] . working with family violence can , in itself , be difficult and confronting for pcps because of the patient 's experience within their family . pcps may also be both victims and perpetrators of family violence and this can influence their ability to work effectively with patients . an earlier study by sugg and inui reported that 15% of male doctors and 31% of female doctors had a personal history of child abuse or intimate partner violence . pcps may be concerned that family violence is a private no - go area that can not be discussed without offending the patient . they describe feeling overwhelmed and concerned about broaching the topic with patients , that they lack the time and the confidence to deal with the problem , that they might make the situation worse , and that they can not fix it personal safety issues often confront those who work with victimized children and adults . the traumatic nature of child abuse and adult violence with its negative effects on the emotional , mental , and physical health of victim / survivors is well documented . less discussed is the impact of working in this area on the well - being of the medical professionals and their staff . this paper outlines a theoretical framework for understanding the possible reactions of pcps who work in this area . we then discuss the emotional and physical safety issues while being at work and the social , emotional , and physical impacts of child abuse and violence at work . researching and working with victims of family violence can be emotionally challenging [ 1923 ] . working with children can make it even more so because of bearing witness to the child 's abuse experiences , conflicts with parents and carers , the inability to form an effective therapeutic relationship with the child or their family , a hostile working environment , lack of or failure of services to assist the child and the family , direct threats and concerns about personal safety , and the possibility of litigation [ 22 , 2426 ] . in addition , working with victimised children and adults can evoke strong emotional reactions [ 22 , 26 ] . the general practitioners ( gps ) below describe their encounters : a mother was breastfeeding when her son 's smile triggered her memories of incest . she told me the story of her pain caused long ago by the csa perpetrated by her father . her story was powerful , making me pay attention to her story amongst so many illness stories . as i reflect on that consultation now , i think it was the intensity of her fear and distress which alerted me to the importance of her experiences . she put her baby on one side of the consulting room , as far away as possible , and just cried . ( female gp , urban ) .there were two boys and a little girl and they all lived in the same street . i went and saw the parents of the two disabled boys [ who were abusing the little girl ] and they were n't prepared to do anything , they did not want to know about it , did not feel like supervising , perhaps they did not believe the story . i reported the case to child protection and the families all said we have to move out of town if you can not do anything and we do not want to leave . the kids do not want to leave the school and why should we have to go away to keep our children safe . [ ] that process was extremely painful , you need to go through with the families , families of the abused and the family of the abuser and see it brought out in the open , to the police and then watch them go through the pain , through the system . i started doing work [ with men who perpetrate violence ] , i can remember saying how lucky i was that i worked in an area where there was no abuse what a clever decision i 'd made , to pick such a good postcode , but obviously i just did not see it [ ] i think it 's more that you are aware that it [ abuse ] goes on and i 'm looking out for it more . in the past , if someone would say something , start hinting it , you 'd run a million miles , you 'd change the topic , you 'd start talking about something else and it 's easy to do that [ ] ( male gp , urban ) . she told me the story of her pain caused long ago by the csa perpetrated by her father . her story was powerful , making me pay attention to her story amongst so many illness stories . as i reflect on that consultation now , i think it was the intensity of her fear and distress which alerted me to the importance of her experiences . she put her baby on one side of the consulting room , as far away as possible , and just cried . there were two boys and a little girl and they all lived in the same street . i went and saw the parents of the two disabled boys [ who were abusing the little girl ] and they were n't prepared to do anything , they did not want to know about it , did not feel like supervising , perhaps they did not believe the story . i reported the case to child protection and the families all said we have to move out of town if you can not do anything and we do not want to leave . the kids do not want to leave the school and why should we have to go away to keep our children safe . [ ] that process was extremely painful , you need to go through with the families , families of the abused and the family of the abuser and see it brought out in the open , to the police and then watch them go through the pain , through the system . ( female gp small rural town ) . before i started doing work [ with men who perpetrate violence ] , i can remember saying how lucky i was that i worked in an area where there was no abuse what a clever decision i 'd made , to pick such a good postcode , but obviously i just did not see it [ ] i think it 's more that you are aware that it [ abuse ] goes on and i 'm looking out for it more . in the past , if someone would say something , start hinting it , you 'd run a million miles , you 'd change the topic , you 'd start talking about something else and it 's easy to do that [ ] ( male gp , urban ) . there is an increasing literature on the emotional work of caring and the toll this work takes on those who undertake it [ 18 , 24 , 3032 ] . the effects of being a direct or indirect witness to child abuse and adult violence can result in compassion fatigue , burnout , vicarious trauma and secondary traumatic stress . these terms are often used interchangeably but are , in fact , conceptually different . compassion fatigue is a general term applied to anyone who suffers as a result of acting in a helping capacity , a natural outcome of helping and knowing about trauma [ 33 , page 92 ] and [ 34 , page 9 ] . burnout occurs when a person 's health or outlook on life is negatively affected because of the impact of their work . figley described burnout as physical , emotional , and mental exhaustion caused by long - term involvement in emotionally demanding situations , as a gradual wearing down over time . workers with burnout report low job satisfaction , feelings of powerlessness , and being overwhelmed at work . vicarious trauma is defined as the transformation of the therapist 's or helper 's inner experience as a result of empathetic engagement with survivor clients and their trauma material [ 30 , page 31 ] and results in a profound shift in the worldview of the worker . the result of vicarious trauma can potentially include the disruption of the worker 's view of themselves , the self - changing nature of care giving [ 31 , page xxxviii ] , others and the world in general . child protection workers , physicians , nurses , therapists , social workers and child abuse researchers report such experiences . the traumatic experience can extend beyond those who work directly with the victim / survivor . traumatization has been reported in those who have indirect contact : in receptionists , supervisors , and managers , in those processing accounts or records , in family members of the treating professionals , and in those working with patient histories or reports . the impact is related to the trauma they are exposed to , their own characteristics and history , the research methods they use , their support systems , and context in which they do their research . it is described as a pervasive feature of working with the traumatised and a cumulative response to traumatic material . it can be triggered by either a one - off exposure to a significant issue or repeated exposure to a range of issues and incidents . it can have a profound impact on individuals and be as debilitating as the primary trauma . other contributing factors originally proposed by pearlman and saakvitne that apply to primary care across a range of settings internationally include the chronicity of the trauma work , combining service provision and research ( particularly in resource poor nations ) , increased violence to women and children in war and conflict areas , the individual professional 's capacity for empathy , and the individual 's personal history of trauma . the vicarious trauma has been criticised because it fails to explain why similar experiences cause vicarious trauma in some while others do not experience vicarious trauma . secondary traumatic stress is a similar concept to vicarious trauma , but there are differences as it is based on a set of clinical symptoms . secondary traumatic stress closely parallels posttraumatic stress disorder in its symptomatology and is a normal response to exposure to trauma through working with survivors of traumatic events . symptoms of secondary traumatic stress impact on psychological functioning , result from cognitive shifts and disturbances in relationships . they include intrusive symptoms such as pcps re - experiencing the survivors ' trauma through thoughts , feelings , and images ; avoidance and numbing symptoms such as avoiding working in areas that recall the trauma or forgetting parts of an interview ; and symptoms of hyperarousal such as palpitations and sweating and nightmares and sleep disturbances [ 38 , 39 ] . vicarious trauma and secondary stress have provided the conceptual framework for much of the literature that underpins the current understanding of the impact on those who work with survivors of trauma . more recent work has highlighted methodological issues and the need to measure all of these areas to test their independence . in medicine and nursing , burnout is defined as feelings of emotional exhaustion , depersonalization , and a perceived lack of personal accomplishment because of a long - term engagement with emotionally demanding situations [ 2 , 4 ] . burnout as a model has the advantage of combining the personal impacts and the impact on work , but the interrelationships between the three factors are unclear . preventing , understanding , and recognising vicarious trauma , secondary traumatic stress , and burnout in their work with family violence will assist pcps to respond more effectively to it and to reduce its potential to adversely affect the way in which they work and their view of the world in general . for those already working in the area , it will assist them to better understand their experience and for those in management positions , to put in place strategies to prevent the possibility of secondary traumatization for themselves and their staff . pcps must be trained to do this work at undergraduate and postgraduate levels and as part of their ongoing professional development because pcps still report being inadequately trained to do so . both training and professional experience have been associated with increased feelings of confidence in dealing with violence as well as increased comfort in discussions with patients making pcps feel more in control and reducing pcp stress [ 5 , 13 , 40 ] . the institutions and professional organisations responsible for the education pcps must ensure that students and professionals are trained well and develop the necessary knowledge skills and attitudes as well as the facilitating support for those working with family violence . organisations which were expected to champion the health needs of women and children but failed to do so , by marginalising the need to respond to violence , blaming the victim , not addressing the training needs of pcps , and discrediting the work of those responding to and supporting women were identified as particularly difficult for workers and researchers . hierarchical organisations have been identified as a more important predictor of trauma than the individual characteristics of the workers . the education of pcps should address the needs of patients first and foremost , but the pcps sense of vulnerability , fear , and inadequacy also need to be addressed for them to work effectively in this area . like other social problems and health risk behaviours , there is no easy cure to family violence and pcps need to reframe what they consider as a successful consultation rather than being frustrated by a lack of change . pcps report of positive changes after family violence is disclosed , including patient engagement with counselling and legal services .i asked a 40 year old new patient about child abuse which i was certain she had experienced . twelve months later she came in and said you know that question you asked me twelve months ago , i could n't talk then but i can today . in retrospect i asked a 40 year old new patient about child abuse which i was certain she had experienced . twelve months later she came in and said you know that question you asked me twelve months ago , i could n't talk then but i can today . in retrospect my first consultation was very successful , i just did not know it . traumatic reactions are related to the duration of exposure to traumatic material and the type of trauma experienced . while pcps can not control the content on acute consultations , they can manage and structure their work in terms of review appointments and should consider structuring their work so that they have regular breaks and opportunities to manage less demanding problems . support and supervision from more experienced or staff trained in responding to trauma may help in the management of distressing cases . pcps need to reflect on their ability to provide care for those who have experienced family violence . while all should have a basic competency in recognising and responding to violence in positive and helpful ways , not all may be willing or able to deliver care . in such cases , knowledge of appropriate providers of ongoing care and support services is essential . support systems and networks for pcps need to be considered because these are often not routinely available to pcps . setting up a recognised family violence team at work for all who work with family violence is useful , providing peer - peer support and the ability to discuss difficult or distressing cases . psychiatric and counselling services have a much more developed culture of providing support and supervision for those working with mental illness and traumatized clients . pcps may need to consider engaging with services outside of work for support and supervision if they are going to regularly work with family violence . networks can include coworkers within primary care and may extend to other agencies working with those who experience violence ; working as part of a team is often a much more positive experience than working alone . support systems that extend beyond the clinic should not be forgotten , with friends and family being reported as an important support [ 5 , 41 ] . a key factor in the ability to care for others is the ability to care for oneself a key factor in the ability to care for others is the ability to care for oneself although not all of us working with trauma will experience secondary trauma , we are all at risk . acknowledging how emotionally difficult working in this field can be , recognising that it will affect and may distress you , learning how to recognise symptoms of secondary trauma early and the importance of self - care are essential elements in managing and preventing secondary trauma . the literature highlights a number of responses that can be used at educational , organizational , and personal levels to prevent and respond to secondary trauma , and in turn , improve the quality of care provided by pcps to people experiencing family and sexual violence . at an educational level an understanding of burnout , secondary , or vicarious trauma and their implications should be integrated into training curricula of all helping professionals working with family and sexual violence [ 44 , 4648 ] . an understanding of burnout , secondary , or vicarious trauma and their implications should be integrated into training curricula of all helping professionals working with family and sexual violence [ 44 , 4648 ] . at an organisational level develop a workplace policy that recognises secondary trauma as a potential workplace health risk ; addresses related stigma ; normalises work place stress ; and outlines appropriate responses , including professional development ; workload and case management ; peer support and ongoing routine management meetings in which secondary trauma is discussed [ 4850].develop a culture of learning , sharing and support within the workplace [ 44 , 48].provide adequate support and supervision which includes discussion of secondary trauma and its implications .provide workplace development programmes that include secondary trauma and how to identify early warning signs between oneself and others [ 46 , 49].ensure managers who are appropriately trained to recognise and respond to secondary trauma in a supportive , nonpunitive manner .review workplace policies regularly . develop a workplace policy that recognises secondary trauma as a potential workplace health risk ; addresses related stigma ; normalises work place stress ; and outlines appropriate responses , including professional development ; workload and case management ; peer support and ongoing routine management meetings in which secondary trauma is discussed [ 4850 ] . develop a culture of learning , sharing and support within the workplace [ 44 , 48 ] . provide adequate support and supervision which includes discussion of secondary trauma and its implications . provide workplace development programmes that include secondary trauma and how to identify early warning signs between oneself and others [ 46 , 49 ] . ensure managers who are appropriately trained to recognise and respond to secondary trauma in a supportive , nonpunitive manner . develop an awareness of your own personal risk of burnout and secondary trauma and accept reactions as a normal response to specialised work [ 44 , 51 ] . promote personal well - being by being physically active , engaging socially , and volunteering in your community and adopt appropriate self - care strategies [ 3 , 48 ] ( box 1 ) . skill up in the field of trauma and family violence [ 47 , 52 ] . take responsibility for your own mental health and well - being .create opportunities to debrief with colleagues and decrease social isolation [ 48 , 52].develop and maintain clear client limits and boundaries [ 47 , 48 , 51].recognise potential personality traits that may increase risk of experiencing secondary trauma .find a sense of meaning in your work and develop a connection or spirituality to something that provides guidance for and develops a positive outlook on life . .tremendously satisfying because i believe that my work is a little drop of water which when added to others over time will become a roaring mighty ocean that will spur the wind of change that will improve the lives of women everywhere ( respondent number : 37 ) . develop an awareness of your own personal risk of burnout and secondary trauma and accept reactions as a normal response to specialised work [ 44 , 51 ] . promote personal well - being by being physically active , engaging socially , and volunteering in your community and adopt appropriate self - care strategies [ 3 , 48 ] ( box 1 ) . skill up in the field of trauma and family violence [ 47 , 52 ] . take responsibility for your own mental health and well - being . create opportunities to debrief with colleagues and decrease social isolation [ 48 , 52 ] . develop and maintain clear client limits and boundaries [ 47 , 48 , 51 ] . recognise potential personality traits that may increase risk of experiencing secondary trauma . find a sense of meaning in your work and develop a connection or spirituality to something that provides guidance for and develops a positive outlook on life . .tremendously satisfying because i believe that my work is a little drop of water which when added to others over time will become a roaring mighty ocean that will spur the wind of change that will improve the lives of women everywhere ( respondent number : 37 ) . recognising that we are under stress can be difficult , particularly when professional cultures discourage it . many pcps have trained in systems that encourage them to be in control , this can be problematic when pcps are stressed and hide it from their peers and colleagues and , conversely , can be an area that colleagues fail to see occurring . it has been suggested that partners , families , and friends are better at recognising when we are stressed than we are or our work colleagues are . while prevention is the best management , even with the best preparation some pcps who work with family violence will become stressed . related stress to the healthcare system , the occupational system , and the personal system . the authors ' previous work with researchers and staff who worked with sexual violence identified work - related management strategies and personal strategies as their main support framework . in terms of the health care system , policies and procedures that recognise the time and expertise required for pcps to support and provide long - term care for survivors are necessary . ensuring that adequate counselling and effective therapy services are available to adults and children within the community is essential to support the work of the pcps . in addition , ongoing pcp education must develop and reinforce basic knowledge skills and attitudes to ensure that they have the appropriate skills to recognise and support those who experience family violence and to enable these patients to access the appropriate trauma services . work - related management strategies in managing traumatic material include ongoing development of skills because studies suggest that becoming experienced and an expert reduced trauma . spreading the workload where possible is reported as helping . those with better relationships with their coworkers and higher numbers of support systems were less likely to develop secondary traumatic stress . self - care strategies assisted pcps ; these included physical fitness , psychological strategies , spirituality / advocacy and staying connected . various forms of physical activity and eating well have been described as well as using humour and laughter , avoiding retraumatizing material like films and newspapers , being pleased with small changes , being creative , travelling and exploring new places , gardening , talking with partners and family , eating and spending time with family and friends , going to church , praying , making a difference , and writing books , papers , and pamphlets to help [ 4 , 52 , 53 ] . elwood and colleagues while recognising the importance of responding to workplace health risks and secondary trauma suggest that we should proceed with some caution . the development and implementation of workplace policies on secondary and vicarious trauma must be informed by evidence and with cognisance of available resources . much of the research done to date is limited to high - income settings and based on unclear definitions and weak methodologies . recommend more methodologically sound ; analytical research on secondary trauma , its symptoms , and responses to ensure that we do not waste limited resources on implementing ineffective interventions that are based on a poorly defined construct . these need to be addressed at the undergraduate , prevocational , and practitioner levels on an ongoing basis to develop a skilled and competent workforce where the potential for stress is minimised . as pcps engage more with recognising and responding to family violence , it will be necessary to put in place the appropriate education and healthcare system , workplace , and personal support to enable the pcps to continue to work with patients who have experienced family violence and their ongoing chronic health needs . pcps need to be aware of their own support needs and the strategies they can put in place to engage well with those who have experienced family violence , providing safe long - term chronic care and access to the required services .

primary care professionals ( pcps ) are increasingly being expected to identify and respond to family and sexual violence as the chronic nature and severity of the long - term health impacts are increasingly recognized . this discussion paper reports the authors ' expert opinion from their experiences running international workshops to prevent trauma among those who work and research sexual violence . it describes the burnout and secondary traumatic stress literature which provides the evidence supporting their work . implications for practicing basic training in response to trauma and ongoing education are a key area for responding to family violence and preventing professional stress . a professional culture that supports and values caring well for those who have experienced family violence as well as caring for the carer is needed . working in teams and having more support systems in place are likely to protect pcps from secondary traumatic stress and burnout . undergraduate and postgraduate training of pcps to develop trauma knowledge and the skills to ask about and respond to family violence safely are essential . in addition , the healthcare system , workplace , and the individual practitioner support structures need to be in place to enable pcps to provide safe and effective long - term care and access to other appropriate services for those who have experienced family violence .

1. Introduction 2. Understanding the Potential for Trauma 3. How Can PCPs Prepare to Work with Patients Who Have Experienced Family Violence? 4. Preventing Burnout and Vicarious Trauma among PCPs 5. Recognising Stress and Responding to It 6. Conclusion

PMC3247183

the natural pyrethrin insecticides have the desirable environmental properties of being both non - toxic to mammals and non - persistent . chrysanthemic acid [ 2 , 2-dimethyl-3-(2-methyl-1-propenyl ) ] is one of the products from pyrethrin photodecomposition and is widely used as the acidic part of synthetic pyrethroidin insecticides [ 4 - 9 ] . the photochemical degradation of the acid components of pyrethrins has been examined in numerous studies . and ueda and matsui found that carbons 1 and 2 ( scheme 1 ) of the main cyclopropane group are cleaved , subsequently leading to the formation of a diradical according to elliot and janes . the photochemistry of simple cyclopropanes has been examined by several workers [ 13 - 18 ] and the fundamental photochemical transformations are generally observed . for instance , the photodegradation of pyrethrins in sunlight is rapid and results in the isomerization of the side - chains , photooxidation to a variety of carboxylic acids , and isomerization of the cyclopropane acids . the main objective of this project was to carry out a theoretical study to determine the reaction mechanism of the decomposition of chrysanthemic acid . this investigation may improve our knowledge about the potential energy surface ( pes ) of these important compounds . we explored the potential energy surfaces of the ground state and the lowest singlet and triplet excited states and the interplay between them . dft calculations have been used successfully in calculating transition structures and the reaction parameters of various reactions , such as pericyclic rearrangements , cycloadditions and bimolecular snreactions . in the present work , we use the density functional ( dft ) approach to undertake a theoretical investigation of the mechanism of photochemical decomposition of cha . more specifically , we calculate structures and relative energies of reactant , products , intermediate and transition states involved in all the three suggested pathways ( cis - trans isomerization , rearrangement , fragmentation ) . for a more complete study we also carried out the effect of solvent on the pes using the polarizable continuum model ( pcm ) . all calculations were performed with the gaussian03w program , running on a pentium iv personal computer . we used restricted and unrestricted b3lyp gradient corrected exchange - correlation functional in combination with the 6 - 311+g * * basis set [ 25 - 27 ] . in our computational investigation , we determined the location of the minima and transition structures of the singlet state surfaces . for equilibrium geometries and transition states , we carried out irc calculations to confirm that the transition stats connect to right minima . zero - point vibrational energy corrections ( zpve ) were estimated at the same theory level at which optimization was carried out and etotal was calculated as eopt ( optimization energy at equilibrium geometry ) + zpve . basis set superposition error ( bsse ) corrections were used to obtain more reasonable total energies in fragmentation reaction ( pathway 3 ) . subsequently , the polarizable continuum model ( pcm ) was applied considering water , ethanol , and cyclohexane as solvents ( = 78.3553 , 24.852 , and 2.0165 , respectively ) . all calculations were performed with the gaussian03w program , running on a pentium iv personal computer . we used restricted and unrestricted b3lyp gradient corrected exchange - correlation functional in combination with the 6 - 311+g * * basis set [ 25 - 27 ] . in our computational investigation , we determined the location of the minima and transition structures of the singlet state surfaces . for equilibrium geometries and transition states , we carried out irc calculations to confirm that the transition stats connect to right minima . zero - point vibrational energy corrections ( zpve ) were estimated at the same theory level at which optimization was carried out and etotal was calculated as eopt ( optimization energy at equilibrium geometry ) + zpve . basis set superposition error ( bsse ) corrections were used to obtain more reasonable total energies in fragmentation reaction ( pathway 3 ) . subsequently , the polarizable continuum model ( pcm ) was applied considering water , ethanol , and cyclohexane as solvents ( = 78.3553 , 24.852 , and 2.0165 , respectively ) . three pathways are possible for the decomposition reaction of chrysanthemic acid , as described in an introduction and as displayed in chart 1 : trans - cis isomerization ( p1-p3 ) , fragmentation , cleavage of the 1 , 3 and 1 , 2 bonds ( p1-p5 ) and rearrangement ( p2-p4 ) . this project was designed to explore the pes for the photodecomposition of chrysanthemum acid , which is a major product from the decomposition of pyrethrine pesticides . to reduce the time necessary for calculations , we replaced the methyl groups in cha with hydrogen atoms . atom labels for trans - chrysanthemic acid . to investigate the cis - trans isomerization mechanism of cha ( p1-p3 ) , the potential energy profile was generated by scanning the c12-c1-c3-c8 ( ) angle ( figure 1 ) from 0 to 180 in the s0 state . the different conformations located during the cis - trans isomerization of cha are displayed in figure 2 . the values of c12-c1-c3-c8 dihedral angles are 150.0 and 2.72 for p1 and p3 isomers , respectively . these results show that the two branched groups of cha are approximately in a plane in the cis form ( additional file 1 ) . by inspection figure 2 , one trans conformer associated with the dihedral angle c12-c1-c3-c8 = 180 is less stable than cis form , p1 by ~ 10 kcal / mol . however , the energy difference between the two minimum isomers ( p1 and p3 ) is only 0.577 kcal / mol . the potential curve calculated at the b3lyp/6 - 311+g * * level of theory possesses minima at = 2.72 and 150 in the cis and trans isomers , respectively . the structure with = 60 is predicted to be a transition state with an imaginary frequency of i1130.91 cm . no significant differences were found in all of the other geometrical parameters between the two isomers ( cis and trans ) , as shown in figure 3 . the optimized geometries of the reactants , transition state and products are shown in figure 3 . a more detailed and comparative analysis of the geometric parameters for the p1-p3 reaction revealed that a drastic geometric reorganization occurs from p1 to the transition state ( ts ) . the c1-c2 distance decreases from 1.539 in p1 to 1.492 in ts . the c3-c2 and c3-c1 distances increase from 1.512 and 1.499 in p1 to 1.531 and 1.518 in ts , respectively . the average total lengths of the three c - c bonds in the ring are exactly the same ( 1.515 ) , indicating that the ring does not open during rotation about the c12-c1-c3-c8 dihedral angle . these results indicate that the cis and trans forms of cha are found in similar proportions , in accordance with previous experimental observations . potential energy profiles along the rotation around the c12-c1-c3-c8 dihedral angle and some optimized structures ( angles and relative energies are given in angles and kcal / mol , respectively ) . geometries of the stationary points for the first suggested pathway ( p1-p3 ) ; distances are given in . the rearrangement of cha ( p1 ) to produce lactone ( p4 ) was investigated using the b3lyp/6 - 311+g * * level of theory . our computations predict that the pathway of the rearrangement of cha occurs in stepwise mechanism . as shown in figure 4 , the potential energy surface of rearrangement process contains two transition states and diradical intermediate . the electronic energies , the electronic energies plus the corresponding zero - point vibrational energy ( zpve ) corrections , and relative energies for all the species are reported in table 1 and take the corresponding reactant as a reference . all calculations are carried for all species in potential energy surface for rearrangement process using the restricted and unrestricted b3lyp/6 - 311+g * * level . the relative energies in figure 4 indicate that the rearrangement pathway for cha is endothermic reaction where the product is at 16.8 kcal / mol above reactant . the closed - shell first transition structure on this process , ts1 , is located 1.4 kcal / mol below the second transition state ts2 . according to the relative energies for the stationary points listed in figure 5 , the forward and back activation barriers are 47.5 and 32.1 kcal / mol , respectively . the previous studies on the rearangment of vinylcyclopropane have reported that only one transition state is located . the activation barrier in this studied had about 46.9 kcal / mol compared to 47.5 kcal / mol in our study . this result indicates that an influence of the carboxylic group on the activation energy of the rearrangement path is little . the energy difference between the final product and the intermediate species is 24.10 kcal / mol from dft calculations . when focused on the overall energetic of the reaction p1-p4 , it is evident that the reaction depends on the energetic balance due to the cleavage of a c1-c3 bond in the starting reactant p1and accompanied by the formation of a c3- o bond in the rearranged carbon centered diradical intermediate . this path is started with cleavage c1-c3 bond in trans conformation p1 with decreasing others c - c bonds except the terminal ch = ch2 . figure 5 displays the main geometrical parameters of the equilibrium and transition structures for the formation of lactone optimized at the b3lyp level with the basis set described above . the negative charge of oxygen atom in the carbonyl group increases from ts1 ( additional file 2 ) with -0.343 to reach -0.351 in ts2 . also , the positive charge on c1 in ts1 and ts2 is 0.286 and 0.315 , respectively . these values mean that the formation bond during this pathway occurs easily between oxygen of carbonyl group and c1 than other carbon atoms . in conclusion , the rearrangement in the cha followed the same route as that in the vinylcyclopropane with the slightly lowered potential barrier . the calculated relative energies in kcal / mol of the rearrangement of cha at the b3lyp/6 - 311+g * * level . calculated energies of the stationary points of rearrangement of cha ( p1-p4 ) . bond lengths and milliken charge distribution for optimized structures of the stationary points located for the rearrangement reactions ( p1-p4 ) . therefore , the most important channel for cha decomposition channels is the decomposition to carbene product via ts3 , the third pathway is the fragmentation , which gives a carbene compound ( p1-p5 ) via c1-c2 and c1-c3 bond cleavage , was also investigated with the b3lyp/6 - 311+g * * level of theory . the total energy of the conformations along the potential surface increases with cleavage of the c1-c2 bond , followed by cleavage of the c1-c3 bond to produce the products , as shown in figure 6 . inspection of the data in figure 6 reveals an increased dihedral angle c2-c1-c3-c12 and decreased c3-c1 bond length in ts2 , indicating that the reaction appears to be concerted . however , figures 7 and 8 show a noticeable shortening of the c2-c3 bond along the fragmentation pathway . the fragmentation process occurs after two steps of successive conformational changes via two transition states , ts2 and ts3 . calculations of the harmonic vibrational frequencies ( imaginary frequencies ) revealed that ts2 and ts3 are true transition states and identified the atoms undergoing the main displacement in the transition structure . because it is too endothermic , this fragmentation process ( p1-p5 ) is less favorable to proceed than the other two pathways . the high endothermicity of the third pathway prevents fragmentation because it is even higher than the energy barrier for the more feasible pathway through ts3 ( 47.5 kcal / mol ) , as shown in figure 7 . this required energy is significant when compared to the more feasible pathway , as shown in figure 4 ( 1.445 kcal / mol endothermic ) . the potential energy surface diagram of the fragmentation process via c1-c2 cleavage ( p1-p5 ) . a standard b3lyp hybrid functional was consistently employed for both geometry optimizations and td - dft calculations . we used the td - dft/6 - 311+g * * method to determine if the reactions occur in the ground or an excited state . if this energy is small , then the reaction probably occurs in the excited state . in further details about the electronic structure , we characterized the low - lying three singlet excited states within the current td - dft calculations as in table 2 . all excited states represent the p - p * and n - p * intramolecular charge transfer ( ct ) transitions . the first and second peaks originate from mixing orbitals , homo and homo-1 to lumo . the first intense peak , dominantly describable with homo-1 - lumo excitation , has large f of 0.195 ( additional file 3 ) . the homo is mainly delocalized on whole molecule whereas the homo-1 is located on carboxylic moiety , depicted in figure 9 . homo-1 - lumo excitation therefore leads to intramolecular ct from vinyl moiety to carboxylic moiety , as illustrated in figure 9 where the electron density increases mainly in the vinyl moiety and decreases in the carboxylic moiety . the third transition originates from homo-1 ( mainly distributed on carboxylic moiety ) to lumo with moderate f of 0.037 . by inspection of the fmo correlation diagram of p1 as depicted in figure 9 , the homo is stabilized than the lumo , which lead to the gap energy is high . the electron density is littler on c1-c3 bond in the ring this means that this bond is easy to break . our results showed that the promotion of an electron requires a large amount of energy relative to the activation energy and the energy of the reaction . the excitation energy of p1 is 136.46 kcal / mole ( table 2 ) , which is larger than the value of barrier of the three studied pathways . these results suggest that all of the studied reactions take place in their ground state rather than in an excited state . calculated low - energy singlet excitation energies , wavelengths , and oscillator strengths ( f ) for cha ( p1 ) using td - b3lyp/6 - 311 g * * level . frontier molecular orbital picture ( fmo ) ( homo , homo-1 , lumo ) of cha ( p1 ) we investigated the solvent effect using single - point scrf calculations on optimized structures . we used an scrf method with a polarizable continuum model ( pcm ) at the b3lyp/6 - 311+g * * level ( additional file 4 ) . the effect of different dielectric constants on the pes of the decomposition of cha was performed using three solvents ( water = 78.3553 , ethanol = 24.852 and cyclohexane = 2.0165 ) . the solvent effect on the pes for the reaction decomposition of cha at the dft level of theory the solvation energies in kcal / mol of the studied species in pes using pcm at the b3lyp/6 - 311+g * * level . the solvent does not affect the ordering stability of the potential energy surface species . however , the solvents increase the stability of all of the reaction species with increasing dielectric constants . to investigate the cis - trans isomerization mechanism of cha ( p1-p3 ) , the potential energy profile was generated by scanning the c12-c1-c3-c8 ( ) angle ( figure 1 ) from 0 to 180 in the s0 state . the different conformations located during the cis - trans isomerization of cha are displayed in figure 2 . the values of c12-c1-c3-c8 dihedral angles are 150.0 and 2.72 for p1 and p3 isomers , respectively . these results show that the two branched groups of cha are approximately in a plane in the cis form ( additional file 1 ) . by inspection figure 2 , one trans conformer associated with the dihedral angle c12-c1-c3-c8 = 180 is less stable than cis form , p1 by ~ 10 kcal / mol . however , the energy difference between the two minimum isomers ( p1 and p3 ) is only 0.577 kcal / mol . the potential curve calculated at the b3lyp/6 - 311+g * * level of theory possesses minima at = 2.72 and 150 in the cis and trans isomers , respectively . the structure with = 60 is predicted to be a transition state with an imaginary frequency of i1130.91 cm . no significant differences were found in all of the other geometrical parameters between the two isomers ( cis and trans ) , as shown in figure 3 . the optimized geometries of the reactants , transition state and products are shown in figure 3 . a more detailed and comparative analysis of the geometric parameters for the p1-p3 reaction revealed that a drastic geometric reorganization occurs from p1 to the transition state ( ts ) . the c1-c2 distance decreases from 1.539 in p1 to 1.492 in ts . the c3-c2 and c3-c1 distances increase from 1.512 and 1.499 in p1 to 1.531 and 1.518 in ts , respectively . the average total lengths of the three c - c bonds in the ring are exactly the same ( 1.515 ) , indicating that the ring does not open during rotation about the c12-c1-c3-c8 dihedral angle . these results indicate that the cis and trans forms of cha are found in similar proportions , in accordance with previous experimental observations . potential energy profiles along the rotation around the c12-c1-c3-c8 dihedral angle and some optimized structures ( angles and relative energies are given in angles and kcal / mol , respectively ) . geometries of the stationary points for the first suggested pathway ( p1-p3 ) ; distances are given in . the rearrangement of cha ( p1 ) to produce lactone ( p4 ) was investigated using the b3lyp/6 - 311+g * * level of theory . our computations predict that the pathway of the rearrangement of cha occurs in stepwise mechanism . as shown in figure 4 , the potential energy surface of rearrangement process contains two transition states and diradical intermediate . the electronic energies , the electronic energies plus the corresponding zero - point vibrational energy ( zpve ) corrections , and relative energies for all the species are reported in table 1 and take the corresponding reactant as a reference . all calculations are carried for all species in potential energy surface for rearrangement process using the restricted and unrestricted b3lyp/6 - 311+g * * level . the relative energies in figure 4 indicate that the rearrangement pathway for cha is endothermic reaction where the product is at 16.8 kcal / mol above reactant . the closed - shell first transition structure on this process , ts1 , is located 1.4 kcal / mol below the second transition state ts2 . according to the relative energies for the stationary points listed in figure 5 , the forward and back activation barriers are 47.5 and 32.1 kcal / mol , respectively . the previous studies on the rearangment of vinylcyclopropane have reported that only one transition state is located . the activation barrier in this studied had about 46.9 kcal / mol compared to 47.5 kcal / mol in our study . this result indicates that an influence of the carboxylic group on the activation energy of the rearrangement path is little . the energy difference between the final product and the intermediate species is 24.10 kcal / mol from dft calculations . when focused on the overall energetic of the reaction p1-p4 , it is evident that the reaction depends on the energetic balance due to the cleavage of a c1-c3 bond in the starting reactant p1and accompanied by the formation of a c3- o bond in the rearranged carbon centered diradical intermediate . this path is started with cleavage c1-c3 bond in trans conformation p1 with decreasing others c - c bonds except the terminal ch = ch2 . figure 5 displays the main geometrical parameters of the equilibrium and transition structures for the formation of lactone optimized at the b3lyp level with the basis set described above . the negative charge of oxygen atom in the carbonyl group increases from ts1 ( additional file 2 ) with -0.343 to reach -0.351 in ts2 . also , the positive charge on c1 in ts1 and ts2 is 0.286 and 0.315 , respectively . these values mean that the formation bond during this pathway occurs easily between oxygen of carbonyl group and c1 than other carbon atoms . in conclusion , the rearrangement in the cha followed the same route as that in the vinylcyclopropane with the slightly lowered potential barrier . the calculated relative energies in kcal / mol of the rearrangement of cha at the b3lyp/6 - 311+g * * level . calculated energies of the stationary points of rearrangement of cha ( p1-p4 ) . bond lengths and milliken charge distribution for optimized structures of the stationary points located for the rearrangement reactions ( p1-p4 ) . therefore , the most important channel for cha decomposition channels is the decomposition to carbene product via ts3 , the third pathway is the fragmentation , which gives a carbene compound ( p1-p5 ) via c1-c2 and c1-c3 bond cleavage , was also investigated with the b3lyp/6 - 311+g * * level of theory . the total energy of the conformations along the potential surface increases with cleavage of the c1-c2 bond , followed by cleavage of the c1-c3 bond to produce the products , as shown in figure 6 . inspection of the data in figure 6 reveals an increased dihedral angle c2-c1-c3-c12 and decreased c3-c1 bond length in ts2 , indicating that the reaction appears to be concerted . the c2-c3 bond is the same in the two transition states . however , figures 7 and 8 show a noticeable shortening of the c2-c3 bond along the fragmentation pathway . the fragmentation process occurs after two steps of successive conformational changes via two transition states , ts2 and ts3 . calculations of the harmonic vibrational frequencies ( imaginary frequencies ) revealed that ts2 and ts3 are true transition states and identified the atoms undergoing the main displacement in the transition structure . because it is too endothermic , this fragmentation process ( p1-p5 ) is less favorable to proceed than the other two pathways . the high endothermicity of the third pathway prevents fragmentation because it is even higher than the energy barrier for the more feasible pathway through ts3 ( 47.5 kcal / mol ) , as shown in figure 7 . this required energy is significant when compared to the more feasible pathway , as shown in figure 4 ( 1.445 kcal / mol endothermic ) . the potential energy surface diagram of the fragmentation process via c1-c2 cleavage ( p1-p5 ) . a standard b3lyp hybrid functional was consistently employed for both geometry optimizations and td - dft calculations . we used the td - dft/6 - 311+g * * method to determine if the reactions occur in the ground or an excited state . if this energy is small , then the reaction probably occurs in the excited state . in further details about the electronic structure , we characterized the low - lying three singlet excited states within the current td - dft calculations as in table 2 . all excited states represent the p - p * and n - p * intramolecular charge transfer ( ct ) transitions . the first and second peaks originate from mixing orbitals , homo and homo-1 to lumo . the first intense peak , dominantly describable with homo-1 - lumo excitation , has large f of 0.195 ( additional file 3 ) . the homo is mainly delocalized on whole molecule whereas the homo-1 is located on carboxylic moiety , depicted in figure 9 . homo-1 - lumo excitation therefore leads to intramolecular ct from vinyl moiety to carboxylic moiety , as illustrated in figure 9 where the electron density increases mainly in the vinyl moiety and decreases in the carboxylic moiety . the third transition originates from homo-1 ( mainly distributed on carboxylic moiety ) to lumo with moderate f of 0.037 . by inspection of the fmo correlation diagram of p1 as depicted in figure 9 , the homo is stabilized than the lumo , which lead to the gap energy is high . the electron density is littler on c1-c3 bond in the ring this means that this bond is easy to break . our results showed that the promotion of an electron requires a large amount of energy relative to the activation energy and the energy of the reaction . the excitation energy of p1 is 136.46 kcal / mole ( table 2 ) , which is larger than the value of barrier of the three studied pathways . these results suggest that all of the studied reactions take place in their ground state rather than in an excited state . calculated low - energy singlet excitation energies , wavelengths , and oscillator strengths ( f ) for cha ( p1 ) using td - b3lyp/6 - 311 g * * level . frontier molecular orbital picture ( fmo ) ( homo , homo-1 , lumo ) of cha ( p1 ) we used an scrf method with a polarizable continuum model ( pcm ) at the b3lyp/6 - 311+g * * level ( additional file 4 ) . the effect of different dielectric constants on the pes of the decomposition of cha was performed using three solvents ( water = 78.3553 , ethanol = 24.852 and cyclohexane = 2.0165 ) . the solvent effect on the pes for the reaction decomposition of cha at the dft level of theory the solvation energies in kcal / mol of the studied species in pes using pcm at the b3lyp/6 - 311+g * * level . . however , the solvents increase the stability of all of the reaction species with increasing dielectric constants . potential energy surface ( pes ) for three channels of decomposition of cha ( p1 ) were studied in the gas phase at the b3lyp/6 - 311+g * * level of theory . the solvent effect on pes , we conclude the following . 1 - the calculations rationalize and verify all experimental facts . the b3lyp/6 - 311+g**level of theory provides a reasonable way to investigate the decomposition channels of chrysanthemic acid . 2 - cis - trans isomerization pathway is energetically favorable than the fragmentation and rearrangement pathways . 3 - the solvent effect does not affect the shape of the potential energy surfaces . in other words , the solvent effect on the reaction is small and tends to stabilize all of the isomers . p1-opt . output optimized of p1 structure . p1-tddft . output uv absorption calculation using tddft method for p1 . this project was funded by the deanship of scientific research ( dsr ) king abdulaziz university , jeddah , under grant no .

backgroundchrysanthemic acid ( cha ) is a major product from the photodecomposition of pyrethrin which is an important class of pesticide compounds.in the following paper , hybrid density functional theory ( dft ) calculations of the potential energy surface ( pes ) for three possible channels decomposition of chrysanthemic acid ( cis - trans isomerization , rearrangement and fragmentation ) have been carried at the b3lyp/6 - 311+g * * level of theory . dft was employed to optimize the geometry parameters of the reactants , transition states , intermediates and products based on detailed potential energy surfaces ( pes).resultsour results suggest that all three pathways of cha are endothermic . dft calculations revealed that the activation barriers for cis - trans isomerization are low , leading to a thermodynamically favorable process than other two pathways . we also investigated the solvent effect on the pes using the polarizable continuum model ( pcm ) . in addition , time - dependent density functional theory ( tddft ) calculations showed that these reactions occur in the ground state rather than in an excited state.conclusionthe rearrangement process seems to be more favorable than the decomposition of cha to carbene formation . the solvent effect calculations indicated no changes in the shape of the pes with three continua ( water , ethanol and cyclohexane ) , although the solvents tend to stabilize all of the species .

Background Computational Details Results and discussion Cis-trans isomerization pathway Rearrangement Pathway Excitation energy Solvent Effect Conclusion Competing interests Authors' contributions Supplementary Material Acknowledgements

PMC4333581

good photochemical properties , chemical stability , and ease of synthesis make coumarins an important class of fluorescent probes for biological studies . in addition to being versatile fluorophores , coumarin chromophores can be used as light - removable protecting groups , so - called caging groups . caged molecules have been extensively applied in the optical control of cellular processes . in particular , the 6-bromo-7-hydroxycoumarinmethyl caging group undergoes fast two - photon photolysis at 740 nm and has been used to optically control neurotransmitters , secondary messengers , and oligonucleotides . two - photon irradiation enables optical activation of biological processes with enhanced tissue penetration of up to 1 mm . moreover , two - photon caging groups can be released with greater precision in three - dimensional space than simple one - photon caging groups . here we report the site - specific incorporation of three coumarin amino acids into proteins via genetic code expansion with unnatural amino acids ( uaas ) to integrate the optical properties of coumarin probes into cellular systems . genetic code expansion requires the addition of orthogonal translational machinery to achieve site - specific uaa incorporation into proteins . recent advances in engineering pyrrolysyl - trna synthetase / trna pairs for the incorporation of sterically demanding amino acids prompted us to synthesize coumarin lysines 13 ( figure 1a ) and to test their incorporation into proteins . the photochemical characteristics of these uaas complement and enhance the properties of caged and fluorescent amino acids that have been genetically encoded in bacterial and mammalian cells . lysines 13 were assembled in three steps from their corresponding coumarin alcohols ( supporting information , scheme s1 ) . briefly , the coumarin alcohols were activated with nitrophenyl chloroformate and coupled to commercially available boc - lysine . a global deprotection under acidic conditions furnished the corresponding coumarin derivatives 13 in good yields . all three coumarin lysines 13 contain identical benzopyrone cores as fluorescent probes . however , subtle substitutions result in a set of coumarin derivatives with unique photochemical properties . introduction of a bromine at the 6-position enables decaging not only with uv ( single photon ) light ( in case of 1 ) , but also near ir ( two - photon ) excitation ( in case of 2 ) . in contrast , extension of the coumarin carbamate linker by a single carbon atom results in coumarin lysine 3 , which does not undergo photolysis and thus represents a stable coumarin amino acid probe . thus , coumarin lysines 1 and 2 can be used as both fluorescent and light - activated probes for optochemical control of protein function using uv or near - ir light , while coumarin lysine 3 may serve as a stable fluorescent probe that does not decage under uv excitation . the methanosarcina barkeri pyrrolysyl trna synthetase / trnacua ( mbpylrs / trnacua ) is functional and orthogonal in a wide range of organisms , such as escherichia coli , yeast , mammalian cells , and animals such as caenorhabditis elegans and drosophila melanogaster . furthermore , wild - type pylrs recognizes several unnatural amino acids without accepting any of the 20 common amino acids as a substrate . the active site of the pylrs can be further engineered through directed evolution to enable the incorporation of additional unnatural amino acids with new functions , including post - translational modifications , bioconjugation handles , photo - cross - linkers , photocaging groups , and others . thus , we generated and screened a panel of mbpylrs mutants , guided by mutants that were previously reported , to direct the incorporation of 2 in response to a tag amber codon in mammalian cells using a mcherry - tag - egfp reporter . cells containing a mbpylrs mutant with only two amino acid mutations y271a and l274 m showed uaa - dependent expression of full - length mcherry - egfp - ha . the y271a mutation has previously been reported to direct the incorporation of n - carbamate - linked lysines , while the l274 m mutation was discovered to facilitate higher amber suppression activities with 2 in vivo , because it allows greater flexibility of the side chain and imposes less steric bulk at the back of the hydrophobic pocket . this synthetase , termed bhckrs , enabled the site - specific incorporation of not only 2 but also 1 and 3 in response to the amber codon tag within sfgfp - y151tag - his6 in e. coli ( figure 1 ) . this is not surprising , considering the very similar structures of 13 and previous observations of the high promiscuity of pylrs . to further rationalize the ability of bhckrs to incorporate 13 , molecular modeling was employed . the wild - type pylrs structure ( pdb : 2q7h ) was used as a starting template for which the y271a and l274 m mutations were introduced using modeller . the mutant structure was energy minimized in amber molecular dynamics before docking 13 into the active site pocket using autodock4 . as expected , 13 adopt very similar poses , reflecting their similarity in structure ( see supporting information , figure s1 ) . the mutated synthetase model reveals that the y271a and l274 m mutations greatly enlarge the binding pocket to accommodate the bulky bicyclic caging group , while also orienting it in a favorable -stacking interaction with w382 . this orientation also benefits from a favorable h - bond interaction between the coumarin hydroxyl group and d373 . similar to published crystal structures , the amino group s positioning is maintained by interactions with a structural water and y349 . it has been previously shown that interactions with n311 and r295 play an important role in amino acid recognition by the pylrs system . the docked structure maintains these key interactions with the carbamate carbonyl forming a h - bond with n311 , while the carboxylic acid forms a h - bond with r295 ( figure 1b , c ) . sds - page analysis reveals coumarin fluorescence of the expressed proteins containing the coumarin lysines 13 . no fluorescence is observed for wild - type sfgfp because its excitation wavelength ( 488 nm ) does not match that of 13 ( 365 nm ) and because of the denaturing conditions of the gel . the dependence of protein expression on the presence of 13 demonstrates that the engineered bhckrs synthetase has a high specificity for coumarin lysines and does not significantly incorporate any of the common 20 amino acids . similar results were obtained for the incorporation of 13 into ubiquitin and myoglobin in e. coli . electrospray ionization mass spectrometry ( esi - ms , supporting information , figures s2s4 ) showed that recombinantly expressed sfgfp-1 and -3 have a mass of 28446.22 and 28460.60 da , in agreement with the expected masses of 28446.03 and 28460.04 da , respectively . esi - ms analysis of sfgfp-2 showed a mass of 28445.97 da , indicating a partial loss of bromine during e. coli expression , possibly due to reductive dehalogenation . overall , these results demonstrate that 13 can be incorporated into proteins in e. coli in good yields ( 8.0 mg / l , 1.6 mg / l , and 2.5 mg / l , respectively , for sfgfp ) and with high specificity . ( a ) structures of the genetically encoded coumarin amino acids for fluorescence reporting and light activation of protein function . ( b ) crystal structure of pylrs ( 2q7h ) with the pyrrolysine substrate ( yellow ) in the active site . ( c ) ( d ) sds - page analysis of sfgfp - y151tag containing 13 through incorporation in e. coli . the gel was stained with coomassie blue ( top ) , and coumarin fluorescence was imaged via excitation at 365 nm ( bottom ) . ( e ) fluorescence micrographs of hek 293 t cells expressing the bhckrs / trnacua pair and mcherry - tag - egfp - ha in the presence or absence of 13 . ( f ) western blot analysis of cell lysates using an anti - ha antibody and a gapdh antibody as a loading control . full - length protein expression is only observed in the presence of 13 , and incorporation efficiency with all three amino acids is similar in mammalian cells . to demonstrate that the coumarin lysines 13 can also be genetically incorporated into proteins in mammalian cells , pbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt were cotransfected into human embryonic kidney ( hek ) 293 t cells . cells were incubated for 24 h in the absence of any unnatural amino acid and in the presence of 13 ( 0.25 mm ) . fluorescence imaging revealed egfp expression only in the presence of 13 , indicating specific incorporation of the coumarin lysines in response to the tag codon , without measurable incorporation of endogenous amino acids ( figure 1e ) . this was further confirmed by an anti - ha western blot on cell lysates from the same experiment ( figure 1f ) . furthermore , full - length mcherry - egfp protein was immunoprecipitated from hek 293 t cells using an immobilized antibody against the ha - tag and mass spectrometry sequencing confirmed that 13 are site - specifically incorporated into protein in mammalian cells ( supporting information , figure s5 ) . importantly , the presence of bromine was verified for protein containing 2 , confirming the genetic encoding of the bhc - caged lysine . because the coumarin groups on 1 and 2 are caging groups that can be removed via light exposure , loss of their intrinsic fluorescence can be used as an indicator of protein decaging through uv irradiation , as shown in figure 2a , b . this was demonstrated through a uv exposure time - course of purified sfgfp-1 , followed by sds - page analysis . the coumarin fluorescence intensity of sfgfp-1 gradually decreases with extended uv exposure as more of the coumarin caging group is removed from the protein , while the continued presence of the coomassie - stained protein band indicates stability of the protein . in a cellular context , this may enable experiments that allow for the determination of protein expression , protein localization , and protein decaging using a single optochemical probe in a single experiment . in contrast , insertion of an extra methylene unit between the lysine and the fluorophore fully abrogates photocleavage and thus establishes 3 as a stable amino acid for the site - specific fluorescent labeling of proteins . no change in coumarin fluorescence is observed after uv exposure of sfgfp-3 for 20 min ( figure 2c , d ) . due to the identical fluorophores in 1 and 3 and the stability of 3 to the uv irradiation conditions , the loss of protein fluorescence in sfgfp-1 is due to decaging and not due to photobleaching . this is further supported by mass spectroscopic analysis of the proteins before and after uv exposure ( see supporting information , figures s2 and s4 ) . to demonstrate the ability of the genetically encoded coumarin lysines to act as reporters for protein localization in live cells , we investigated their utility as a protein nuclear localization marker . a plasmid was constructed to express egfp - ha with an n - terminal nls ( nuclear localization signal , pnls - linker - egfp - ha ) , which reliably localizes egfp to the nucleus ( supporting information , figure s6 ) . a tag amber codon was introduced in the linker between the nls and egfp , allowing for site - specific unnatural amino acid incorporation without affecting egfp formation or nuclear translocation . cells cotransfected with the pnls - ktag - egfp and bhckrs / pyltrnacua plasmid pair in the presence of 1 ( 0.25 mm ) were analyzed for coumarin fluorescence ( 405 nm excitation , 450480 nm emission ) and egfp fluorescence ( 488 nm excitation , 490520 nm emission ) by confocal microscopy . the observation of complete colocalization of both fluorophores in the nucleus ( merged micrographs ) demonstrates the ability to use 1 as a reporter of protein localization ( figure 2e and supporting information , movie s1 and figure s7 ) . sds - page fluorescence analysis shows photodecaging of sfgfp-1 while sfgfp-3 is stable to uv exposure . ( a ) loss of coumarin fluorescence after extended sfgfp-1 in - gel decaging for 050 min ( 365 nm , transilluminator ) . ( b ) coomassie staining reveals identical sfgfp-1 protein amounts in all lanes . ( c ) no loss of coumarin fluorescence is observed , since sfgfp-3 does not decage . ( e ) nuclear colocalization of coumarin and egfp fluorescence in cho k1 cells cotransfected with pnls - tag - egfp - ha and the bhckrs / pylt pair ( pbhckrs-4pylt ) in the presence of 1 ( 0.25 mm ) . a dic image and a merged image of all three channels to apply the coumarin lysines 13 in the optical control of protein function in live cells , firefly luciferase ( fluc ) was selected as an initial target because bioluminescence measurements afford low background , high sensitivity , and easy quantification . on the basis of the fluc crystal structure , a critical lysine residue , k206 , was identified , which is positioned at the edge of the substrate - binding pocket ( figure 3b ) . it has been proposed that this residue stabilizes and orients atp in the active site . the -amino group on k206 provides a hydrogen - bond interaction with the -phosphate of atp and promotes the adenylation reaction with luciferin , thus being essential for catalytic activity as shown by the dramatic decrease in enzymatic activity displayed by the k206r mutant . therefore , we hypothesized that a sterically demanding coumarin caging group placed on k206 would prevent the interaction with atp and limit the overall access of the substrates to the active site ( figure 3a ) . photolysis of the coumarin lysine would remove the caging group and produce a native lysine residue , restoring the catalytic activity of the enzyme ( figure 3b ) . a genetically encoded photocaged lysine at k206 would enable the enhanced regulation of the catalytic activity of firefly luciferase via light activation . site - directed mutagenesis of the corresponding k206 residue to the amber codon ( tag ) enabled incorporation of 13 into firefly luciferase in mammalian cells . hek 293 t cells were cotransfected with the mutated firefly luciferase plasmid ( pgl3-k206tag ) and the mbbhckrs / pyltrnacua pair ( pbhckrs-4pylt ) in the presence of 13 ( 0.25 mm ) . after 24 h incubation , the cells were either irradiated for 4 min ( 365 nm , 25 w ) or kept in the dark . the incorporation of 13 into fluc caused complete inhibition of luciferase activity before uv irradiation , as determined by a bright - glo luciferase assay , comparable to the negative control ( no unnatural amino acid ) . after uv irradiation , 1 and 2 were decaged to produce native lysine , resulting in the activation of firefly luciferase by 34-fold and 31-fold , respectively ( figure 3c ) . as expected , 3 did not show any activation of luciferase enzymatic activity upon illumination , as it does not undergo decaging . therefore , the activity of firefly luciferase can be tightly optochemically regulated by incorporation of a coumarin lysine residue into the active site of the luciferase protein . interestingly , attempts to apply 1 and 2 at position k529 , another site that can be used for optical control of luciferase function , led to greatly diminished luciferase activity , while introduction of our previously reported o - nitrobenzyl - caged lysine worked at both positions k206 and k529 . western blots confirmed that both fluc - k206 1 and fluc - k529 1 were expressed at similar levels in mammalian cells ( supporting information , figures s8 and s9 ) . engineering of an optochemically controlled photinus pyralis firefly luciferase through unnatural amino acid mutagenesis . ( a ) caging groups at position k206 are blocking access to the binding pocket by luciferin and atp and are disrupting a required hydrogen bonding network . ( b ) after decaging , wild - type fluc is generated and the substrates can now enter the active site . ( c ) bright - glo luciferase assay of cells that were either kept in the dark or irradiated ( 365 nm , 4 min ) . no enzymatic activity was observed for the caged proteins , and significant increases in luminescence were observed after photolysis of luciferase containing 1 or 2 , while the k206 3 mutant was permanently deactivated , as expected . error bars represent standard deviations from three independent experiments . to observe the optical triggering of protein function via decaging of 1 and 2 in real time , enhanced green fluorescent protein ( egfp ) was selected as a second target protein for caging . egfp consists of an 11-stranded -barrel and a central -helix with the thr65-tyr66-gly67 chromophore . correctly folded egfp is a prerequisite for mature chromophore formation , with a number of lysine residues being essential to its successful folding . most notable is that only 1 lysine ( k85 ) out of 20 is buried within the protein . k85 forms a salt bridge with d82 and h - bonding interactions with the backbone of c70 and s72 , all of which are in close proximity to the chromophore ( figure 4a ) . it has been shown that c70 , s72 , and d82 are key residues for control of chromophore formation and oxidation . we hypothesized that introduction of coumarin - caged lysines 1 and 2 at k85 would affect d82 , c70 , and s72 , interrupting the -helix bending and thus indirectly inhibiting chromophore maturation . to this end , we envisioned that uv activation would yield native egfp that rapidly undergoes maturation . an egfp mutant with an amber codon at position k85 ( pegfp - k85tag ) was generated as a fusion construct with mcherry , to provide a second reporter for successful plasmid transfection and incorporation of 1 and 2 . hek 293 t cells were cotransfected with pegfp - k85tag - mcherry and the bhckrs / pyltrna pair in the presence of 1 and 2 ( 0.25 mm ) . after 24 h , the cells were washed and incubated in fresh media for 1 h. cells expressing mcherry were observed by fluorescence imaging to confirm that egfp-1/2-mcherry is generated in the presence of 1 or 2 . cells were irradiated for 30 s at 365 nm , and fluorescence was imaged by time - lapse microscopy . after photolysis of egfp-1 , green fluorescence started to appear around 10 min , and over time the fluorescence intensity gradually increased , reaching a plateau at 120 min ( figure 4d and supporting information , movie s2 ) . a half - life of 49 min was observed , matching reports of egfp chromophore maturation as the rate - limiting step . previous measurements of egfp folding and maturation have been exclusively performed in test tubes . no cellular studies have been conducted , as a precise starting point for kinetic analysis could not be provided . ( a ) location of k85 ( yellow ) and interactions with d82 , c70 , and s72 in egfp . ( b ) schematic of the pegfp - k85tag - mcherry construct and its application in light activation studies . ( c ) fluorescence imaging of hek 293 t cells expressing egfp - k85tag - mcherry , 90 min after irradiation at 365 nm ( 30 s , dapi filter , 358365 nm ) in the presence of 1 ( nikon a1r confocal microscope , 20 objective , 2-fold zoom ) . ( d ) normalized egfp fluorescence as a function of time after 365 nm light activation ( error bars represent standard deviations from the measurement of three independent cells , t1/2 = 49 min ) . given that the coumarin lysines have relatively broad absorption bands in the 300420 nm range that enable decaging at longer wavelengths , we speculated that irradiation at 405 nm may efficiently activate 1 and 2 . thus , activation through blue light irradiation using a standard laser - scanning confocal microscope was tested . as expected , exposure at 405 nm induced fluorophore formation of egfp-1 and -2 ( figure 5a , b ) . because attempts to decage a previously incorporated nitrobenzyloxycarbonyl lysine and nitrobenzyl tyrosine at 405 nm were not successful on comparable time scales and at comparable illumination power ( data not shown ) , the caged lysines 1 and 2 may enable multiwavelength activation of proteins caged with the two different optical probes . taking advantage of the two - photon decaging feature of 2 , photocontrol of egfp folding by two - photon activation of egfp-2 was performed . hek 293 t cells were cotransfected with pegpf - k85tag - mcherry and pbhckrs-4pylt in the absence or presence of 1 and 2 ( 0.25 mm ) . after a 24 h incubation , the cells were washed and incubated in fresh media for 1 h and irradiated with a multiphoton laser ( 760 nm , 130 mw , 2 m / s dwell time , 30 cycles , olympus fluoview fv1000 mpe multiphoton laser scanning microscope fv10-asw , maitai dsbb - ol ir pulsed laser ) . images were acquired before and after two - photon irradiation using both egfp ( 488 nm ) and mcherry ( 561 nm ) excitation . gratifyingly , an egfp fluorescent signal was observed after photolysis of 2 at 760 nm ( figure 5c ) . the cells expressing egfp containing 1 , as a control , were also exposed to two - photon excitation ( 760 nm ) and imaged in the same fashion ( figure 5d ) ; no egfp activation was observed . in addition to the increased three - dimensional resolution that is provided through two - photon excitation , effectively shifting the activation wavelength to the near - ir will enable multiwavelength activation in conjunction with other optically triggered biological processes , while also preventing any overlap with established fluorescent reporter proteins . fluorescence confocal imaging of cos-7 cells expressing egfp - ktag - mcherry , before and after irradiation at 405 nm ( 30 mw diode laser , 20% laser power , 12.6 s dwell time , 8 cycles ) in the presence of 2 ( a ) or 1 ( b ) ( zeiss confocal lsm710 microscope , 40 water objective ) . similar light - activation experiments before and after irradiation of hek 293 t cells at 760 nm ( 130 mw , 2 m / s dwell time , 30 cycles , olympus fluoview fv1000 mpe , maitai dsbb - ol ir pulsed laser ) , in the presence of 2 ( c ) or 1 ( d ) , imaged with a olympus fluoview1000 , 40 oil objective . the site - specific genetic incorporation of three new coumarin lysine analogues 13 into proteins was achieved in bacterial and mammalian cells using an engineered bhckrs synthetase system . the genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization , and the small size of these coumarin lysines is expected to minimally perturb protein structure and function , unless they are placed at critical sites . in addition to their small size , the spectral properties of 13 do not interfere with common fluorescent proteins ( e.g. , egfp ) . while the amino acid 3 showed stability under irradiation conditions , the coumarins 1 and 2 were readily decaged , generating wild - type lysine residues . as a proof - of - principle , photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue , and excellent off to on light - switching ratios were observed for 1 and 2 . as expected furthermore , two - photon and single - photon optochemical control of egfp maturation was demonstrated , enabling the use of different , potentially orthogonal , excitation wavelengths ( 365 , 405 , and 760 nm ) for the sequential activation of protein function in live cells . while the caged lysine 2 could be activated using two - photon irradiation at 760 nm , the lysine 1 was stable under these conditions . however , decaging of 1 was readily achieved with blue light of 405 nm , while a previously encoded o - nitrobenzyl - caged lysine requires uv activation . these results demonstrate that coumarin lysines are a new and valuable class of optical probes that can potentially be used for the investigation and regulation of protein structure , dynamics , function , and localization in live cells . the small size of coumarin , the application as both a light - activated caging group and a fluorescent probe , and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a unique and multifunctional tool for cellular biology . the ability to incorporate all three coumarin lysines with the same pylrs / trnacua pair further facilitates their application . ( 1 ) construction of pnls - tag - egfp - ha : the ptag - egfp - ha fragment was amplified from pmcherry - tag - egfp - ha using the pcr primers g1/g2 , digested with hindiii and bglii , and ligated into pegfp - n1 ( clontech ) , generating the ptag - egfp - ha plamid . the pnls pcr fragment was obtained by using primers n1/n2 and then ligated into the hindiii and xbai sites of ptag - egfp - ha to generate the pnls - tag - egfp - ha plasmid . ( 2 ) construction of pnls - wt - egfp - ha : plasmids were obtained by converting the tag codon of pnls - tag - egfp - ha into an aag ( lys ) codon using primers qc1/qc2 and a quikchange site - directed mutagenesis kit ( agilent ) . ( 3 ) construction of pbhckrs-4pylt : the plasmid was obtained by ligating the p4cmve - u6-pylt fragment from pmbpylt between the restriction sites nhei and mfei sites of pmbbhckrs . the plasmid , pbad - sfgfp - y151tag - pylt was cotransformed with pbk - bhckrs into e. coli top10 cells . a single colony was grown in lb media overnight , and 500 l of the overnight culture was added to 25 ml of lb media , supplemented with 1 mm of the designated unnatural amino acid and 25 g / ml of tetracycline and 50 g / ml of kanamycin . cells were grown at 37 c , 250 rpm , and protein expression was induced with 0.1% arabinose when the od600 reached 0.6 . after overnight expression at 37 c , cells were harvested and washed by pbs . the cell pellets were resuspended in 6 ml of phosphate lysis buffer ( 50 mm , ph 8.0 ) and triton x-100 ( 60 l , 10% ) , gently mixed , and incubated for 1 h at 4 c . the cell mixtures were sonicated , and the cell lysates were centrifuged at 4 c , 13 000 g , for 10 min . the supernatant was transferred to a 15 ml conical tube , and 100 l of ni - nta resin ( qiagen ) was added . the resin was then collected by centrifugation ( 1000 g , 10 min ) , washed twice with 400 l of lysis buffer , and followed by two washes with 400 l of wash buffer containing 20 mm imidazole . the protein was eluted with 400 l of elution buffer containing 250 mm imidazole . the purified proteins were analyzed by 10% sds - page and stained with coomassie blue . two different instruments were used : ( a ) protein samples were analyzed using capillary lc esi - tof ms . the protein samples were loaded onto a prlp - s column ( thermo fisher 5 m , 1000 a , 300 m i.d . 100 mm ) on an lc system ( ultimate 3000 , dionex , sunnyvale , ca ) . the lc system was directly coupled to an electrospray ionization time - of - flight mass spectrometer ( microtof , brukerdaltonics , billerica , ma ) . chromatographic separation was performed at a constant flow rate of 3.5 l / min using a binary solvent system ( solvent a : 2.5% acetonitrile and 0.1% formic acid ; solvent b : 80% acetonitrile and 0.1% formic acid ) and a linear gradient program ( 05 min , 5% b ; 510 min , 530% b ; 1030 min , 3075% b ; 3035 min , 75100% b ; 3545 min , 1005% b ; 4560 min , 5% b ) . mass spectra were acquired in positive ion mode over the mass range m / z 50 to 3000 . esi spectra were deconvoluted with the maxent algorithm ( data analysis 3.3 , bruker daltonics , billerica , ma ) , obtaining molecular ion masses with a mass accuracy of 12 da . ( b ) high - resolution exact mass measurement were conducted on an agilent technologies ( santa clara , ca ) 6210 lc - tof mass spectrometer . samples were analyzed via a 1 l flow injection at 300 l / min in a water : methanol mixture ( 25:75 v / v ) with 0.1% formic acid . the mass spectrometer was operated in positive ion mode with a capillary voltage of 4 kv , nebulizer pressure of 35 psi , and a drying gas flow rate of 12 l / min at 350 c . reference ions of purine at m / z 121.0509 and hp-0921 at m / z 922.0098 were simultaneously introduced via a second orthogonal sprayer and used for internal calibration . human embryonic kidney ( hek ) 293 t cells were grown in dmem ( dulbecco s modified eagle medium , gibco ) supplemented with 10% fbs ( gibco ) , 1% pen - strep ( corning cellgro ) , and 2 mm l - glutamine ( alfa aesar ) in 96-well plates ( costar ) in a humidified atmosphere with 5% co2 at 37 c . hek 293 t cells were transiently transfected with the pmbbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt at 75% confluency in the presence or absence of 1 , 2 , and 3 ( 0.25 mm ) in 96-well plates . after an overnight incubation at 37 c , the cells were washed by pbs and imaged with a zeiss axio observer.z1microscope ( 10 objective ) . to confirm the expression of the fusion protein and also differentiate between expression levels , hek 293 t cells were cotransfected with pmbbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt in the presence or absence of 1 , 2 , and 3 ( 0.25 mm ) in six - well plates . after 24 h of incubation , the cells were washed by chilled pbs , lysed in mammalian protein extraction buffer ( ge healthcare ) with complete protease inhibitor cocktail ( sigma ) on ice , and the cell lysates were cleared at 13 200 rpm centrifugation ( 4 c , 20 min ) . the protein lysate was boiled with loading buffer and then analyzed by 10% sds - page . after gel electrophoresis and transfer to a pvdf membrane ( ge healthcare ) , the membrane was blocked in tbs with 0.1% tween 20 ( fisher scientific ) and 5% milk for 1 h. the blots were probed and incubated with the primary antibody , -ha - probe ( y-11 ) rabbit polyclonal lgg ( sc-805 , santa cruz biotech ) , overnight at 4 c , followed by a fluorescent secondary antibody , goat--rabbit lgg cy3 ( ge healthcare ) , for 1 h at room temperature . hek 293 t cells were transfected with pbhckrs - mcherry - tag - egfp - ha and p4cmve - u6-pylt in a 10 cm petri dish and incubated with dmem containing 1 , 2 , or 3 ( 0.25 mm ) for 24 h. cells were lysed with extraction buffer ( ge healthcare ) and the mcherry-1/2/3-egfp - ha protein was immunoprecipitated using the pierce ha tag ip / co - ip kit ( pierce ) according to manufacturer s protocol . the proteins were separated on sds - page gels and stained with silver stain . regions corresponding to the expected molecular weight of mcherry - egfp - ha were excised , washed with hplc water , and destained with 50% acetonitrile/25 mm ammonium bicarbonate until no visible staining . gel pieces were dehydrated with 100% acetonitrile and reduced with 10 mm dithiothreitol at 56 c for 1 h , followed by alkylation with 55 mm iodoacetamide at room temperature for 45 min in the dark . gel pieces were then again dehydrated with 100% acetonitrile to remove excess alkylating and reducing agents and rehydrated with 20 ng/l trypsin/25 mm ammonium bicarbonate and digested overnight at 37 c . the resultant tryptic peptides were extracted with 70% acetonitrile/5% formic acid , speed - vac dried , and reconstituted in 18 l of 0.1% formic acid . tryptic digests were analyzed by reverse - phased lc - ms / ms using a nanoflow lc ( waters nanoacquity uplc system , waters corp . , milford , ma ) coupled online to an ltq / orbitrap velos hybrid mass spectrometer ( thermo - fisher , san jose , ca ) . separations were performed using a c18 column ( picochip column packed with 10.5 cm reprosil c18 3 m 120 chromatography media with a 75 m i d column and a 15 m tip , new objective , inc . , mobile phase a was 0.1% formic acid in water , and mobile phase b was 0.1% formic acid in acetonitrile . samples were injected onto a trap column ( nanoacquity uplc trap column , waters corp . , milford , ma ) and washed with 1% mobile phase b at a flow rate of 5 l / min for 3 min . peptides were eluted from the column using a 90 min gradient running at 300 nl / min ( 5% b for 3 min , 536% b in 62 min , 3695% b in 2 min , 95% b for 8 min , 95%5% b in 1 min , 5% b for 16 min ) . the ltq / orbitrap instrument was operated in a data - dependent ms / ms mode in which each high resolution broad - band full ms spectra ( r = 60 000 at mass to charge ( m / z ) 400 , precursor ion selection range of m / z 300 to 2000 ) was followed by 13 ms / ms scans in the linear ion trap where the 13 most abundant peptide molecular ions dynamically determined from the ms scan were selected for tandem ms using a relative collision - induced dissociation ( cid ) energy of 35% . dynamic exclusion was enabled to minimize redundant selection of peptides previously selected for cid . ms / ms spectra were searched with the mascot search engine ( version 2.4.0 , matrix science ltd . ) against a uniprot jellyfish proteome database ( june 2014 release ) from the european bioinformatics institute ( http://www.ebi.ac.uk/integr8 ) combined with endogenous mcherry - egfp fasta sequences . the following modifications were used : static modification of cysteine ( carboxyamidomethylation , + 57.0214 da ) and variable modification of methionine ( oxidation , + 15.9949 da ) for all searches , variable modifications of lysine for mcherry - egfp - ha ( 1 , + 218.17 da ; 2 , + 295.93 da ; 3 , + 231.03 da ) . the mass tolerance was set at 20 ppm for the precursor ions and 0.8 da for the fragment ions . peptide identifications were filtered using peptideprophet and proteinprophet algorithms with a protein threshold cutoff of 99% and peptide threshold cutoff of 95% implemented in scaffold ( proteome software , portland , or ) . hek 293 t cells were cultured in dmem ( dulbecco s modified eagle medium , gibco ) supplemented with 10% fbs ( gibco ) , 1% pen - strep ( gibco ) , and 2 mm l - glutamine ( alfa aesar ) in 96-well plates ( bd falcon ) in a humidified atmosphere with 5% co2 at 37 c . at 8090% confluency , cells seeded on plates were transfected and the medium was changed to fresh dmem supplemented without or with 1 , 2 , or 3 ( 0.25 mm ) . the plasmid pmbbhckrs-4pylt was constructed containing both cmv - mbbhckrs and 4cmve - u6-pylt . a tag amber stop codon was introduced at the k206 site using primers gl1/gl2 and a quikchange mutagenesis kit ( agilent technologies ) . a pgl3-control plasmid containing the gene encoding p. pyralis firefly luciferase with the tag amber mutation at residue k206 ( pgl3-k206tag ) was cotransfected into cells with the plasmid pbhckrs-4pylt using linear pei according to the manufacturer s protocol ( millipore ) . after double transfection and 24 h incubation , the medium was changed to dmem without phenol red , and the cells were irradiated with uv light ( 365 nm ) for 4 min using a 365 nm uv lamp ( high performance uv transilluminator , uvp , 25 w ) or kept in the dark . cells were lysed by addition of 100 l of substrate solution ( promega ) in a 96-well plate ( bd falcon ) , and luminescence was measured on a synergy 4 multimode microplate reader with an integration time of 2 s and a sensitivity of 150 or on a tecan m1000 microplate reader with an integration time of 1 s. cho k1 cells were plated into a polylysine - coated four - well chamber slide ( lab - tek ) and , after incubation to 75% confluency , were transfected with 1 g of pnls - ktag - egfp and pbhckrs-4pylt each . after 16 h incubation at 37 c/5% co2 in dmem with 10% fbs in the presence of 1 ( 0.25 mm ) , cells were washed with dmem without phenol red and then incubated for 2 h. the cells were washed with pbs , fixed with 4% formaldehyde , and stained with rhodamine phalloidin ( life technologies ) . the chamber slide was dried in the dark overnight and cells were imaged on a zeiss 710 confocal microscope ( 40 water objective ) . hek 293 t cells were plated into a poly - d - lysine - coated eight - well chamber slide ( lab - tek ) . after incubation to 70% confluency , cells were transfected with pegfp - k85tag - mcherry and pbhckrs-4pylt ( 200 ng each ) . after a 20 h incubation at 37 c/5% co2 in dmem with 10% fbs in the presence of 1 ( 0.25 mm ) , cells were washed with dmem without phenol red and then incubated for 1 h. before light activation , mcherry - expressing cells were identified using the txred channel , and imaged with a nikon a1rsi confocal microscope ( 20 objective , 2-fold zoom , egfp ( ex . subsequently , cells were illuminated for 15 s at 365 nm light ( dapi filter , 358365 nm ) , and egfp and mcherry fluorescence was acquired by time - lapse imaging ( every 1 min for the first 15 min , every 5 min for the following 150 min , scan resolution 512 512 , scan zoom 2 , dwell time 1.9 ms ) . hek 293 t cells were plated into a polylysine - coated -dish ( ibidi ) , and after incubation to 50% confluency , the cells were transfected with 1 g each of pegfp - ktag - mcherry and pbhckrs-4pylt . after a 20 h incubation at 37 c/5% co2 in dmem with 10% fbs in the presence of 1 or 2 ( 0.25 mm , 0.5% dmso ) , cells were washed with dmem without phenol red and then incubated for 1 h. cells were imaged with an olympus fluoview confocal microscope before two - photon irradiation ( 40 oil objective , egfp ( ex . 560 nm ) channels ) , imaging positions for mcherry - expressing cells were recorded , and the cell -dish was transferred to an olympus multiphoton microscope for irradiation ( olympus fluoview fv1000 mpe ) . cells were localized at the previously recorded positions , focused using the mcherry channel , and then irradiated using a 760 nm laser ( 130 mw , 5% of laser power , 30 cycles of scanning , 2 m / s dwell time , maitai dsbb - ol ir pulsed laser ) . after irradiation , the cell -dish was transferred back to the original microscope for imaging . the initial template structure of pdb 2q7h was chosen as a starting point for all modeling . the missing loops were remodeled using modeler and the two point mutations ( y271a and l274 m ) were constructed using the mutate_model.py script provided by modeler . superposition of pdb 2q7 g on top of 2q7h provided the coordinates for the incorporation of atp and magnesium ions into the newly mutated structure . the atp and magnesium ions were parametrized in antechamber using previously developed parameters . the mutated structure was imported into amber12 software using the amber ff99sbildn force field . the protein was placed into a cubic box with a 12.0- border , solvated with 17 316 water molecules , and charge neutralized with the addition of six sodium ions . this system was energy minimized first with 5000 steps steepest descent method , followed by 15 000 steps conjugate gradient method with 5 kcal / mol restraints on all atoms . this was followed by another 5000 steps steepest descent method , followed by 15 000 steps conjugate gradient method with 2 kcal / mol restraints on all atoms except y271a and l274 m . the resulting energy - minimized structure was used as the starting structure for all our docking experiments . all amber12 computational experiments were completed on the center for simulation and modeling ( sam ) frank supercomputer at the university of pittsburgh . the energy - minimized mutant structure was prepared for docking with autodock4 by removing all sodium ions , and all water molecules except for a single water molecule which exists in the active site pocket of the protein . this structural water molecule is present in all available crystal structures and plays an important role in amino acid recognition . the side chains for residue l274 m were treated as flexible , while all other side chains were kept rigid . the unnatural amino acid ligands were constructed using chembiodraw3d , and the molecular geometry was optimized using the mmff94 force field . the ligand input files were prepared for docking using autodock tools as well . lamarkian genetic algorithm was used for docking with the following parameters : number of runs : 75 , ga_pop_size 150 , ga_num_evals 250 000 000 , ga_num_generations 27 000 were set , all other parameters were kept default . each coumarin lysine yielded a low energy cluster with binding scores of 9.63 kj / mol , 6.18 kj / mol , and 6.14 kj / mol for 1 , 2 , and 3 , respectively .

the site - specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl - trna synthetase system . the genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function . as a proof - of - principle , photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue , and excellent off to on light - switching ratios were observed . furthermore , two - photon and single - photon optochemical control of egfp maturation was demonstrated , enabling the use of different , potentially orthogonal excitation wavelengths ( 365 , 405 , and 760 nm ) for the sequential activation of protein function in live cells . these results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure , dynamics , function , and localization in live cells . the small size of coumarin , the site - specific incorporation , the application as both a light - activated caging group and as a fluorescent probe , and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology .

Introduction Results and Discussion Summary Experimental Section

PMC3390908

one major goal of proteomic profiling is an accurate quantitation of proteins in samples with high complexity and high dynamic range of protein concentrations , such as body fluids ( serum , plasma , csf , etc ) . because high confidence peptide / protein identification and at the same time high confidence quantitation is a highly challenging task , multiple analytical approaches have been developed based on separation of proteins in - gel ( 2de dige ) and/or gel - free platform utilizing various methods of metabolic or chemical labeling . each quantitative platform , including isobaric tags for relative and absolute quantitation ( itraq ) , has strengths and limitations that have been experimentally compared . itraq was developed in the early 2000s to be applied for the multidimensional protein identification technology ( mudpit ) approach in which proteins are fragmented with trypsin or other proteolytic enzyme and subsequently chemically labeled with isobaric tags . this platform became a central technology in modern proteomics research ; it is being widely used in all areas of research with great utility . as much as this approach seems to be straightforward , many aspects of this proteomic platform add sources of variability , and these limit the confidence in the output of protein identification and quantitation . the variability is introduced in multiple steps of sample preparation , efficiency of chemical tagging , performance of instrumentation , and method of acquisition used , as well as software ( algorithms ) and thresholds defined for database searches . importantly , 4-plex and 8-plex tags provide overlapping mass of reporter ions ; however , their balance groups are different , which has been postulated to have an impact on yield of fragmentation in collision induced dissociation ( cid ) leading to bias in quantitation . nevertheless , itraq - based quantitation is an attractive method in global proteomic quantitation . first , it can be used after processing of any sample , e.g. , cell lysates and proteins obtained from organelles , and as such is not limited to only those systems that can accommodate incorporation of stable isotopes during cell culture . third , software for protein identification and quantitation is fairly well developed and tested in numerous experimental settings . recently pichler and co - workers found that peptide labeling with 4-plex tags yields higher identification rates compared to 8-plex tags . this conclusion is of concern since experimental designs using 8-plex allow a much greater level and ease of comparison . for example , a study of one control and seven experimental conditions can be performed in one 8-plex experiment but would require at least three 4-plex experiments ( running the control and up to three experimental samples in each ) . this increases the amount of control sample needed , labor and supplies , and chromatography and mass spectrometry time and likely introduces a source of variability . the goal of this study was to compare experimental ratios of highly complex samples tagged with 4-plex versus 8-plex reagents in controlled ratios . we used proteinpilot 4.0 software for data analysis , which is associated with the absciex 4800 maldi - tof / tof mass spectrometer . ammonium phosphate , -cyano-4-hydroxycinammic acid ( chca ) , and trifluoroacetic acid ( tfa ) were from sigma aldrich ( st . louis , mo , usa ) . hplc grade water and acetonitrile ( mecn ; acn ) were from fisher scientific ( pittsburgh , pa , usa ) . human plasma samples were shipped on dry ice from university of california san diego ( ucsd ) to university of nebraska medical center ( unmc ) and on arrival remained frozen . hiv was inactivated in all samples by addition of 10 l of freshly prepared 10% triton x-100 and 50 l of a cocktail of protease inhibitors ( sigma - aldrich st . after 30 min samples were aliquoted , and those unused were stored at 80 c . a 250 l portion from each sample was filtered using a 0.2 m spin filter and immunodepleted using an igy14 column ( sigma - aldrich ) to remove the following proteins : albumin , 1-antitrypsin , igm , haptoglobin , fibrinogen , 1-acid glycoprotein , apolipoprotein a - i and a - iii , apolipoprotein b , igg , iga , transferrin , 2-macroglobulin , and complement c3 . flow - through fractions containing unbound proteins were concentrated using a vivaspin 15r ( sartorius , aubagne , france ) . protein concentration was determined using a nanodrop spectrophotometer ( thermo scientific , san jose , ca ) . a total of 400 g of proteins was pooled , and then aliquots of 50 g of proteins were used in order to perform the itraq labeling . a 50 g sample of proteins was precipitated with ethanol , by adding 10 vol of cold ethanol ( 200 proof ) to each sample , incubating for 3 h at 20 c , and centrifuging at 13,000 g for 15 min at 4 c . proteins pellets were washed with 1 ml of 70% ethanol and dried in a speedvac ( thermo scientific ) . subsequent solutions were provided by itraq reagent kits ( applied biosystem , carlsbad , ca ) . dried proteins were solubilized with dissolution solution , and proteins were denaturated with 1 l of denaturant reagent . protein reduction with reducing reagent was performed for 1 h at 60 c . according to the manufacturer protocol , samples used for itraq 4-plex were alkylated with 84 mm iodoacetamide for 30 min at room temperature , whereas for itraq 8-plex we used the cysteine blocking solution from the itraq kit for 10 min at room temperature . trypsin from abi was reconstituted at 1 g/l with milli - q water , and 10 g of trypsin was added to each sample . after digestion , peptides were labeled with itraq label reagent ( abi ) ; 4-plex labeling was performed for 1 h at room temperature , and after the incubation the reaction was quenched with 100 l of mq water for 30 min at room temperature . the 8-plex labeling was performed for 2 h at room temperature . labeled peptides were combined in one tube ; we mixed a known quantity of peptides from each tag ( see experimental design , figure 1 ) . samples used in all three experiments ( 400 , 600 and 650 g ) were taken from the same larger pool of immunodepleted plasma samples ( see materials and methods for details of immunodepletion ) . in all experiments regardless how much tagged peptides were used for analyses , 50 g of peptide digest was always used for itraq tagging to eliminate any effect of the tag to peptide ratio between experiments . samples were cleaned up using mixed cation exchange ( mcx ) column ( water corp . labeled peptides were solubilized with 1 ml of 0.1% formic acid , passed through the column , and then the column was washed with 5% methanol , 0.1% formic acid solution , and then with hplc grade methanol . then , 360 l of reconstituted sample was supplemented with 1.44 ml of offgel solution . next , samples were fractionated on the basis of their isoelectric point ( pi ) using 3100 offgel fractionator ( agilent , inc . offgel strips were rehydrated for 15 min at room temperature with 40 l of offgel solution . peptide samples were loaded onto gel strips , splitting them equally between all 12 wells . collected fractions were cleaned with c-18 spin columns , according to the manufacturer s protocol . briefly , fractions were adjusted to 5% acetonitrile ( acn ) and 0.5% trifluoracetic acid ( tfa ) and passed through activated columns . columns were washed twice with a 5% acn , 0.5% tfa solution , and peptides were eluted with a 70% acn , 0.1% tfa solution . subsequent fractionation of offgel fractions was performed off - line using tempo lc system with automatic high density spotting onto maldi target plates . peptides were solubilized in 12 l of 0.1% tfa , and 10 l of samples were loaded onto a proteocol c18 trap cartridge ( michrom biosources , auburn , ca ) and washed for 20 min at 9 l / min . gradient of separation was obtained using a ratio between two buffers : water / acn / tfa ( 98:2:0.1 ) ( buffer a ) and water / acn / tfa ( 2:98:0.1 ) ( buffer b ) . to perform the separation , the subsequent gradient was applied by altering buffer b percentage : time 05 min , 5% to 15% ; 552 min , 15% to 35% ; 5254 min , 35% to 80% ; 5464 min , 80% ; 6465 min , 80% to 5% ; and 6572 , min 5% . peptide elution was monitored with a uv cell at 214 nm absorbance . after the uv cell , eluted peptides were mixed with a matrix solution ( 1.2 mg / ml in 75% acn and 0.1% tfa solution ) at a flow rate 1 l / min using a harvard apparatus syringe pump . fractions were spotted every 30 s , and the voltage applied to the plate during spotting was 2.8 kv . spotted fractions were submitted for data acquisition on a 4800 maldi - tof / tof mass spectrometer ( abi ) . ms spectra were acquired from 800 to 3000 m / z , for a total of 1000 laser shots by an nd : yag laser operating at 355 nm and 200 hz . ms / ms analyses were performed using 2 kv collision energy with air as cid gas . metastable ions were suppressed , for a total of 1000 laser shots . protein identification and quantification the search parameters were as follows : itraq 4-plex ( peptide labeled ) , carbamidomethylation of cysteine , ncbi database ( created on december 2011 ) restricted to homo sapiens , itraq 8-plex ( peptide labeled ) , methylthioalkylation of cysteine , ncbi database ( created on december 2011 ) restricted to homo sapiens , for itraq 4-plex and 8-plex , respectively . our experimental design ( figure 1 ) used one large pool of human plasma immunodepleted of the 14 most abundant proteins . regardless of how much of the resulting peptides was used to create final ratios , we always used 50 g during the reaction for itraq labeling . this approach eliminated potential variability that might be associated with efficiency of chemical labeling when ratios to tag and peptides are not uniform . we used 114 , 115 , 116 , and 117 tags from 4-plex and from 8-plex kits and combined the following amount of labeled peptides ( separately for the 4-plex and 8-plex ) to achieve a 1:2:3:4 ratio from each kit : 10 g ( 114 ) , 20 g ( 115 ) , 30 g ( 116 ) , and 40 g ( 117 ) . in experiment 2 we repeated these conditions and added a third sample in which the 113 , 118 , 119 , and 121 tags from the 8-plex kit were used and peptides again mixed in a 1:2:3:4 ratio . however , in experiment 3 we compared labeling of 114 , 115 , 116 , and 117 tags from the 8-plex kit to labeling with all eight tags from the same 8-plex kit . relative to the first two experiments , we scrambled tag assignment to the amount of peptides used , which allowed us to limit another potential bias ( tag effect ) . in experiment 3 we also added 50 g of non - labeled peptides to the sample labeled with four tags to make up for the difference between amounts of peptides between those labeled with all eight tags . in all three experiments we used the same conditions for fractionation based on isoelectric point and subsequently rp - hplc in tempolc plate spotter . all data were processed by the same version of proteinpilot with the same version of database . in figure 2 ideally we should observe ratios of 0.25 ( 114:117 ) , 0.5 ( 115:117 ) , and 0.75 ( 116:117 ) . first , as confidence of protein identification decreases ( plotted on the x - axis ) , the ratios for individual proteins ( plotted on the y - axis ) become more dispersed and the groups start to overlap . second , when we used tags from the 4-plex kit , ratios of 114:117 showed lower than expected values , whereas when we used tags from the 8-plex kit the ratio of 114:117 was as expected ( 0.25 ) . the two other ratios were very similar for both kits , and all were slightly higher than expected . proteins were plotted by decreasing value of confidence of identification ( x axis ) and ratios that were calculated as relative to 117 reporter ion ( y axis ) . ( a ) plot for 114 , 115 , and 116 m / z reporter ions from itraq 4-plex kit . ( b ) ratios for the same m / z set of reporter ions from itraq 8-plex kit . in figure 3 we present a comprehensive comparison of ratios derived from all three experiments as a box - plot analysis . as shown in panel a , comparison of the ratios from experiment 2 shows a greater dispersion of data when tags from the 4-plex kit were used . comparison of the box ( containing the values from 25% to 75% of the ratios ) reveals that the tags from the 8-plex that have reporter masses similar to those of the 4-plex have a tighter distribution than those from the 4-plex . in panel b we present analysis of the spread of ratios for tags 115 , 116 , and 117 from the 8-plex kit relative to the 114 tag when labeled samples were mixed with an equal amount of non - labeled peptides . in this experiment measured ratios indicated that the presence of non - labeled peptides skewed results toward lower than expected values , which would have been 0.75 ( 115/114 ) , 0.50 ( 116/114 ) , and 0.25 ( 117/114 ) , respectively . dispersion of ratios was highest for 115/114 and lowest for 117/114 . in panel c we show comparison of ratios from the second part of experiment 3 in which we used all tags from the 8-plex kit ; however , samples were mixed in 1:1 , 1:2 , 1:3 , and 1:4 ratios and all were calculated as relative to the 113 tag . in this data set experimental ratios matched expected values for the following tags : 115/113 was 0.75 , 116/113 was 0.5 . ratios for tags 117/113 and 121/113 were comparable to each other ; however , both were below the 0.2 mark while we expected them to be at the 0.25 mark . ratios of 118/113 , 119/113 , and 114/113 were skewed to lower values quite substantially in some instances . besides the fact that higher ratios had a larger spread of values in the top and bottom quartiles , there was no obvious pattern of systematic skew of data in either the top or bottom quartiles across all comparisons . box - plot of the ratios comparing effect of tags from itraq 4-plex and 8 plex kits . data presented are from experiment 2 in panel a and from experiment 3 in panels b and c. ( a ) . ratios were calculated as relative to the highest amount of peptides tagged with 117 for 4-plex and 8-plex and 121 for 8-plex only . therefore , the first three box - plots represent a 1:4 ratio ( expected value 0.25 ) , the second three box - plots represent a 1:2 ratio ( expected value 0.5 ) , and the third set of box - plots represents a 3:4 ratio ( expected value 0.75 ) . box - plot analysis of ratios from tagging peptides with 114 , 115 , 116 , and 117 tags from 8-plex kit and mixed with an equal amount ( 50 g ) of non - labeled peptides . ( c ) . box - plot analysis of ratios of peptides tagged with all 8 tags from 8-plex kit . the amount of peptide digest was the same ( 100 g ) as in panel b. while manually analyzing ratios for individual peptides labeled with the different tags , we have found three predominant patterns of ratios regardless of whether the 4-plex or 8-plex assay was used ( figure 4b ) . among them , linear dependence ( pattern 1 , figure 4b ) is the most desirable and expected and is representative of more than 80% of all patterns found ( figure 4a ) . more than 90% of peptides ( figure 4a ) are included when these three patterns are combined . although more than 80% of peptides showed linear ratios , we were interested in what impact the non - linear patterns have on the overall quantitative ratio of a protein and whether these non - linear patterns may skew quantitation . to investigate such possibility we selected two proteins : serpin peptidase inhibitor , clade g , member 1 precursor and hemopexin precursor . the peptides that contributed to the overall ratios of these two proteins contained a mixture of the three predominant peptide ratios as presented in figure 4 . results of this analysis are presented in figure 5 and indicate that despite having a mixture of all three patterns of peptide ratios , protein quantitation still shows linearity . observed patterns of peptides ratios . ideally , all peptides labeled with itraq tags should show quantitative and linear ratios representing controlled mixing of protein samples . in reality , we have found seven non - linear or no change patterns representing less than 20% of the total number of peptides ( a ) . expected linear pattern is shown in panel b , and the two most predominant non - linear patterns are shown in panels c and d , respectively . when combined , these three patterns represent more than 90% of peptides . two proteins , serpin peptidase inhibitor , clade g , member 1 precursor and hemopexin precursor , were selected for this comparison . selection was based on the fact that in both cases overall protein ratios were calculated based on peptides representing a mixture of patterns 1 , 2 , and 3 shown in figure 4 . in both instances , serpin peptidase inhibitor , clade g , member 1 precursor ( a ) and hemopexin precursor ( b ) proteins showed overall linearity of ratios despite a mixture of linear and non - linear peptide ratios . one point of interest that pichler and co - workers found was that for samples labeled with itraq 8-plex , the number of peptide - spectrum matches and unique peptides was more than 70% lower and the number of protein groups more than 60% lower , as compared to itraq 4-plex . we identified 72 proteins with 99% and 98 with 66% confidence , respectively , using the 4-plex itraq kit , and 64 and 90 proteins for the respective thresholds when we used the 8-plex itraq kit . when we used a 50% confidence cutoff level , we found that samples labeled with 8-plex showed a decrease in identifications of only 13% at the peptide level and 19% at the protein level , and when compared to 4-plex these differences were not statistically significant . performed their quantitation and identification using a ltq orbitrap and cid - hcd hybrid method and searches were performed using mascot and proteome discoverer . we used maldi 4800 with proteinpilot 4.0 with paragon algorithm , which has an impact on the number of proteins identified . itraq , as with any other analytical tool , is under continuous scrutiny by scientists looking for ways of most accurate measurements in quantitative proteomics . because global profiling with quantitation is a multistep experiment , the final output may depend on wide range of factors ultimately contributing to skewed or even false positive results . one solution to prevent contribution of errors originating from itraq data analyses is tightening thresholds ; however , this approach must be used with caution because it may easily lead to loss of important information . therefore , itraq is being constantly evaluated and each study emphasizes different aspects of this approach . gan and co - workers assessed the reliability of itraq from perspective of different types of replicate analyses and took into account technical , experimental , and biological variations . mahoney and co - workers reported that measured variability was a function of mean abundance , fold changes were biased toward the null , and variance of a fold change was a function of protein mass and abundance . ow and co - workers evaluated the quantitative dynamic range of itraq quantitation in high- and low - complexity samples . although their study has similarities in experimental design , there are also important differences , including the use of non - mammalian samples to create a high complexity background , spiking in strategy to measure ratios , strong cation exchange ( scx ) separation in first dimension , and a qtof mass spectrometry platform . in a subsequent paper the authors used a similar strategy of spiking in known proteins to evaluate accuracy and precision of itraq based quantitation and proposed spiking as a method to address accuracy and variance - stabilizing normalization to address the issue of precision . in another study evaluating accuracy of quantitation using itraq ratios thingholm and co - workers used whole hela cell lysate as a model sample and focused on phosphopeptides after enrichment on a tio2 column . the authors used esi as ionization mode and a ltq xl orbitrap as mass spectrometry platform . they reported correlation between reductions in identification efficiency with the size of the isobaric tag . taking all these studies together , we have gained knowledge into understanding the itraq platform ; however , our study presented here offers insight from a different perspective . here we perform a calculated experiment using immunodepleted human plasma , a body fluid that is highly complex and has a high dynamic range of protein concentration . another way our study differs significantly is that we use a maldi - tof / tof platform , followed by proteinpilot 4.0 data analysis , both of which are offered from and supported by absciex , the manufacturers of itraq . other groups focused on software and mathematical models for itraq data analyses and comparing algorithms across many platforms . despite the collective effort , many outstanding questions related to accuracy and sources of variability in itraq technique remain to be addressed , and more systematic studies with direct comparisons across mass spectrometry platforms , complexity of samples , and sample preparation methods are needed to fully understand bias resulting from itraq quantitation . we were intrigued by the report of pichler and co - workers showing that peptide labeling with 4-plex tags yields higher identification rates compared to 8-plex tags . the authors used a ltq orbitrap mass spectrometer , cid - hcd hybrid method , and proteome discoverer software ( thermo scientific ) . the authors attributed the differences in yields to differences in chemical structures of balance groups and concluded that balance groups used in 8-plex tags are less susceptible to fragmentation . however on the basis of our previous experience we found not only that were the differences in peptide and protein identifications low but that the 8-plex tags resulted in increased confidence of quantitation with limited impact on protein identification . taking this together we decided to test this effect using a systematic experimental approach to examine this as well as the accuracy of ratios obtained from intensities of the reporter ions with simplified experimental design to remove as much bias as possible . we intentionally chose human plasma because of our past work on biomarker discovery in body fluids ( csf , serum , and plasma analyses ) and because it constitutes a highly complex mixture of proteins with high dynamic range of relative concentrations . plasma / serum and csf have been used in many biomarker discovery studies , including using itraq platform ; however , in many instances validation using larger population of clinical samples was disappointing , leaving questions about the sources of such disconnect unanswered . we used one large sample of immunodepleted plasma securing identical material for all experiments . biological variability , although very important , was not an objective of our study , and pooling multiple samples averaged levels of proteins in the mix . importantly , we used a maldi - tof / tof 4800 mass spectrometer and proteinpilot software with paragon algorithm , which are different than those used by pichler and co - workers . we have found that 8-plex tags performed with higher quantitation accuracy than the same ( by m / z of reporter ions ) tags from 4-plex reagent , providing experimental ratios closer to theoretical ratios without dramatically affecting peptide or protein identification . also , when confidence of protein identification decreases , the spread of ratios increases in both instances , however , to a lesser extent when 8-plex tags are used ( figure 3a ) . therefore we conclude that more consistent ratios would be due to more complete cid fragmentation of tags using maldi mass spectrometry . box - plot analysis of ratios from subsequent experiments showed that spread of ratios is much tighter in two middle quartiles when 8-plex tags are used . additionally , labeling peptides with 8-plex tags yielded more peptides with linear dependence of calculated itraq ratios , thus better reflecting the ratio of controlled mixing . skewing of the measured ratios in the 1:1 mixture of tagged and nontagged peptides was an unexpected effect considering that the same amount of peptides tagged with 8-plex yielded ratios close to their theoretical values . in the mixture of tagged and nontagged peptides , for each peptide there were two different precursor ions that yielded identical or very similar fragmentation spectra . all spectra could contribute to confidence of protein identification ; however , only half of the spectra contributed to quantitation . if peptide fragmentation used for protein identification and fragmentation of tags used for quantitation are processed by algorithm as separate events and results are merged at the final step , such effect should not be observed . on the other hand , if quantitation and identification is considered by algorithm as one event , 50% of spectra with null quantitation may induce a systematic skew in the calculation of ratios . therefore , completeness of tagging may have a quite profound effect on quantitative output even if such incomplete tagging is proportional to all of the peptides in the sample . also results from experiment 1 may also suggest that ratio can be affected by either low level of precursor ion and thus more fragment ions were under background level and/or poor fragmentation during cid . we observed in other itraq experiments examples in which the intensity of reporter ions was clearly above background providing good quantitation , but cid fragmentation of the tagged peptide was so poor that identification was calculated with confidence below 1% ( data not shown ) . summarizing , we provide here experimental evidence that under our experimental conditions , 8-plex tagging is advantageous over 4-plex tagging in two aspects . first , 8-plex tagging provides more consistent ratios without compromising on protein identification . second , the 8-plex system of tagging allows investigation of eight experimental conditions in one analytical experiment . a question that remains to be addressed is whether , during itraq data acquisition , the peptide and reporter ion fragmentation that leads to identification and quantitation , respectively , should be considered as two separate events or dependent on each other . this would need to be addressed formally in subsequent experiments .

methods for isobaric tagging of peptides , itraq or tmt , are commonly used platforms in mass spectrometry based quantitative proteomics . these two methods are very often used to quantitate proteins in complex samples , e.g. , serum / plasma or csf supporting biomarker discovery studies . the success of these studies depends on multiple factors , including the accuracy of ratios of reporter ions reflecting quantitative changes of proteins . because reporter ions are generated during peptide fragmentation , the differences of chemical structure of itraq balance groups may have an effect on how efficiently these groups are fragmented and thus how differences in protein expression will be measured . because 4-plex and 8-plex itraq reagents do have different structures of balanced groups , it has been postulated that indeed differences in protein identification and quantitation exist between these two reagents . in this study we controlled the ratios of tagged samples and compared quantitation of proteins using 4-plex versus 8-plex reagents in the context of a highly complex sample of human plasma using absciex 4800 maldi - tof / tof mass spectrometer and proteinpilot 4.0 software . we observed that 8-plex tagging provides more consistent ratios than 4-plex without compromising protein identification , thus allowing investigation of eight experimental conditions in one analytical experiment .

Introduction Materials and Methods Results Discussion

PMC4120211

ureteropelvic junction ( upj ) obstruction has been classically treated through the standard open approach with outstanding results . since anderson - hynes ( ah ) reported the first dismembered pyeloplasty , a great number of authors have published excellent results , with overall success rates of 90 - 100% . their technique respected the basic principles of the open classical approach while providing less morbidity and faster recovery . since then , transperitoneal and retroperitoneal approaches have been described and advocated by several authors , with excellent results . in order to ease laparoscopic repair and decrease surgical time , many alternative time saving maneuvers and even robot assistance have been developed . these alternative techniques helped in bringing the operative time close to that of open surgery and made laparoscopic pyeloplasty a more desirable alternative . we evaluated the transmesocolic technique as a way to reduce operative time and facilitate repair by avoiding colon displacement and compare it with conventional retrocolic laparoscopic pyeloplasty ( rlp ) in cases of left sided ureteropelvic junction obstruction ( upjo ) . between september 2006 and may 2012 , a total 130 laparoscopic pyeloplasties were performed . only left sided pelvi - ureteric junction obstruction were enrolled for study . a total of 38 transmesocolic laparoscopic pyeloplasty ( tmp ) and 41 left sided rlp were performed . the data were recorded in a prospective manner that included age , pelvic volume , presence of stone [ figure 1a and b ] , aberrant vessel , [ figure 1b ] operative time , analgesics requirement ( paracetamol 15 mg / kg body weight for children more than 10 kg of weight and 7.5 mg / kg body weight for children less than 10 kg of weight ) , time to accept oral feeds , drain removal . a dismembered ah pyeloplasty was performed in all patients . double j stent ( djs ) was placed in 73.68% of patients in tmp group while 82.92% patients in rlp group . based on pelvic volume , patients were divided into three groups : group 1 : less than 50 ml ( 16 patients in rlp and 24 patients in tmp)group ii : 50 - 100 ml ( 14 patients in rlp and 10 patients in tmp)group iii : greater than 100 ml ( 11 patients in rlp and 4 patients in tmp ) . group 1 : less than 50 ml ( 16 patients in rlp and 24 patients in tmp ) group ii : 50 - 100 ml ( 14 patients in rlp and 10 patients in tmp ) group iii : greater than 100 ml ( 11 patients in rlp and 4 patients in tmp ) . all patients with significant obstruction ( obstructed pattern and t1/2 more than 20 min ) as per the well - tempered diuretic renogram were taken for surgery . all cases were done by a single surgeon who has experience of more than two hundred laparoscopic pyeloplasty including fifty cases of transmesocolic pyeloplasty . written consent of all patients was taken . the patient was placed in the lateral decubitus position . using hasson 's canula by open technique , pneumoperitoneum up to 8 - 10 mm hg two 3 mm or 5 mm ports were placed subcostally in the midclavicular line and mid - way between spinoumbilical lines respectively . a mesocolic window was opened by dissecting the mesocolon with the harmonic scalpel . this space was limited medially by the inferior mesenteric vein and laterally by the medial margin of the descending colon . a 5 cm incision was then made longitudinally , lateral to the inferior mesenteric and gonadal veins , in between the medial and left colonic artery . this area was free of vessels and the incision frequently enabled direct visualization of the lumbar ureter . sometimes small branches of the left colonic artery were present and were clipped and sectioned , with no impairment of the colonic vascularization . in this way , we created a peritoneal window that allowed a direct access to the renal pelvis . whenever a wider operative field was necessary , the superior margin of the incision was suspended to the lateral abdominal wall with a suture . needle aspiration of renal pelvis was done if pelvic capacity was more than 50 ml as to minimize the size of mesocolic window . further , in larger pelvises , a stay suture passed percutaneously helps in better stabilization of the former . the ureter and renal pelvis were carefully dissected to avoid unnecessary damage to their vasculature , whereas for the same reason , the kidney and ureter were not mobilized . the upj was divided proximal to the renal pelvis and the obstructed part was left attached to the ureter . if necessary , the ureter was transposed over any aberrant vessels [ figure 1b ] . the ureteral stent was placed by introducing the guide wire through a 16-gauge intravenous catheter sheath placed percutaneously under direct laparoscopic control . then the ureter was spatulated , and a running suture on a small and half- circle [ sh ] needle was placed . we used djs according to age ( length of jj = age of the child in years + 10 cm ) . the anterior plane of the anastomosis was completed using the same suturing technique as for the posterior layer . in some cases , we used a 2 mm percutaneous retractor on the anterior flap of the sectioned renal pelvis in order to stabilize the loose pelvis and facilitate the anastomosis . the mesocolic window was usually closed with two or three hem - o - lok clips or by using a 3 - 0 polyglactin interrupted suture after placement of the suction tube . the procedure began by incising at toldt 's line , dissecting the left colon from gerota 's fascia and reflecting it medially to visualize the renal hilum and upj . median follow - up period for transmesocolic group was 12.5 months ( 9.5 - 62 months ) and 14 months ( 8 - 66 months ) for retro colic group . follow - up included renal dynamic scan , first at 6 weeks after stent removal , then at 3 months and further 2 scans at 6 monthly intervals , resolution of symptom and or resolution of hydronephrosis on ultrasound . between september 2006 and may 2012 , a total 130 laparoscopic pyeloplasties were performed . only left sided pelvi - ureteric junction obstruction were enrolled for study . a total of 38 transmesocolic laparoscopic pyeloplasty ( tmp ) and 41 left sided rlp were performed . the data were recorded in a prospective manner that included age , pelvic volume , presence of stone [ figure 1a and b ] , aberrant vessel , [ figure 1b ] operative time , analgesics requirement ( paracetamol 15 mg / kg body weight for children more than 10 kg of weight and 7.5 mg / kg body weight for children less than 10 kg of weight ) , time to accept oral feeds , drain removal . a dismembered ah pyeloplasty was performed in all patients . double j stent ( djs ) was placed in 73.68% of patients in tmp group while 82.92% patients in rlp group . based on pelvic volume , patients were divided into three groups : group 1 : less than 50 ml ( 16 patients in rlp and 24 patients in tmp)group ii : 50 - 100 ml ( 14 patients in rlp and 10 patients in tmp)group iii : greater than 100 ml ( 11 patients in rlp and 4 patients in tmp ) . group 1 : less than 50 ml ( 16 patients in rlp and 24 patients in tmp ) group ii : 50 - 100 ml ( 14 patients in rlp and 10 patients in tmp ) group iii : greater than 100 ml ( 11 patients in rlp and 4 patients in tmp ) . all patients with significant obstruction ( obstructed pattern and t1/2 more than 20 min ) as per the well - tempered diuretic renogram were taken for surgery . all cases were done by a single surgeon who has experience of more than two hundred laparoscopic pyeloplasty including fifty cases of transmesocolic pyeloplasty . written consent of all patients was taken . the patient was placed in the lateral decubitus position . using hasson 's canula by open technique , pneumoperitoneum up to 8 - 10 mm hg two 3 mm or 5 mm ports were placed subcostally in the midclavicular line and mid - way between spinoumbilical lines respectively . a mesocolic window was opened by dissecting the mesocolon with the harmonic scalpel . this space was limited medially by the inferior mesenteric vein and laterally by the medial margin of the descending colon . a 5 cm incision was then made longitudinally , lateral to the inferior mesenteric and gonadal veins , in between the medial and left colonic artery . this area was free of vessels and the incision frequently enabled direct visualization of the lumbar ureter . sometimes small branches of the left colonic artery were present and were clipped and sectioned , with no impairment of the colonic vascularization . in this way , we created a peritoneal window that allowed a direct access to the renal pelvis . whenever a wider operative field was necessary , the superior margin of the incision was suspended to the lateral abdominal wall with a suture . needle aspiration of renal pelvis was done if pelvic capacity was more than 50 ml as to minimize the size of mesocolic window . further , in larger pelvises , a stay suture passed percutaneously helps in better stabilization of the former . the ureter and renal pelvis were carefully dissected to avoid unnecessary damage to their vasculature , whereas for the same reason , the kidney and ureter were not mobilized . the upj was divided proximal to the renal pelvis and the obstructed part was left attached to the ureter . if necessary , the ureter was transposed over any aberrant vessels [ figure 1b ] . the ureteral stent was placed by introducing the guide wire through a 16-gauge intravenous catheter sheath placed percutaneously under direct laparoscopic control . then the ureter was spatulated , and a running suture on a small and half- circle [ sh ] needle was placed . we used djs according to age ( length of jj = age of the child in years + 10 cm ) . the anterior plane of the anastomosis was completed using the same suturing technique as for the posterior layer . in some cases , we used a 2 mm percutaneous retractor on the anterior flap of the sectioned renal pelvis in order to stabilize the loose pelvis and facilitate the anastomosis . the mesocolic window was usually closed with two or three hem - o - lok clips or by using a 3 - 0 polyglactin interrupted suture after placement of the suction tube . the procedure began by incising at toldt 's line , dissecting the left colon from gerota 's fascia and reflecting it medially to visualize the renal hilum and upj . median follow - up period for transmesocolic group was 12.5 months ( 9.5 - 62 months ) and 14 months ( 8 - 66 months ) for retro colic group . follow - up included renal dynamic scan , first at 6 weeks after stent removal , then at 3 months and further 2 scans at 6 monthly intervals , resolution of symptom and or resolution of hydronephrosis on ultrasound . between september 2006 and may 2012 , a total 130 laparoscopic pyeloplasties were performed . only left sided pelvi - ureteric junction obstruction were enrolled for study . a total of 38 transmesocolic laparoscopic pyeloplasty ( tmp ) and 41 left sided rlp were performed . the data were recorded in a prospective manner that included age , pelvic volume , presence of stone [ figure 1a and b ] , aberrant vessel , [ figure 1b ] operative time , analgesics requirement ( paracetamol 15 mg / kg body weight for children more than 10 kg of weight and 7.5 mg / kg body weight for children less than 10 kg of weight ) , time to accept oral feeds , drain removal . a dismembered ah pyeloplasty was performed in all patients . double j stent ( djs ) was placed in 73.68% of patients in tmp group while 82.92% patients in rlp group . based on pelvic volume , patients were divided into three groups : group 1 : less than 50 ml ( 16 patients in rlp and 24 patients in tmp)group ii : 50 - 100 ml ( 14 patients in rlp and 10 patients in tmp)group iii : greater than 100 ml ( 11 patients in rlp and 4 patients in tmp ) . group 1 : less than 50 ml ( 16 patients in rlp and 24 patients in tmp ) group ii : 50 - 100 ml ( 14 patients in rlp and 10 patients in tmp ) group iii : greater than 100 ml ( 11 patients in rlp and 4 patients in tmp ) . all patients with significant obstruction ( obstructed pattern and t1/2 more than 20 min ) as per the well - tempered diuretic renogram were taken for surgery . all cases were done by a single surgeon who has experience of more than two hundred laparoscopic pyeloplasty including fifty cases of transmesocolic pyeloplasty . written consent of all patients was taken . under general anesthesia , the patient was placed in the lateral decubitus position . using hasson 's canula by open technique , pneumoperitoneum up to 8 - 10 mm hg two 3 mm or 5 mm ports were placed subcostally in the midclavicular line and mid - way between spinoumbilical lines respectively . a mesocolic window was opened by dissecting the mesocolon with the harmonic scalpel . this space was limited medially by the inferior mesenteric vein and laterally by the medial margin of the descending colon . a 5 cm incision was then made longitudinally , lateral to the inferior mesenteric and gonadal veins , in between the medial and left colonic artery . this area was free of vessels and the incision frequently enabled direct visualization of the lumbar ureter . sometimes small branches of the left colonic artery were present and were clipped and sectioned , with no impairment of the colonic vascularization . in this way , we created a peritoneal window that allowed a direct access to the renal pelvis . whenever a wider operative field was necessary , the superior margin of the incision was suspended to the lateral abdominal wall with a suture . needle aspiration of renal pelvis was done if pelvic capacity was more than 50 ml as to minimize the size of mesocolic window . further , in larger pelvises , a stay suture passed percutaneously helps in better stabilization of the former . the ureter and renal pelvis were carefully dissected to avoid unnecessary damage to their vasculature , whereas for the same reason , the kidney and ureter were not mobilized . the upj was divided proximal to the renal pelvis and the obstructed part was left attached to the ureter . if necessary , the ureter was transposed over any aberrant vessels [ figure 1b ] . the ureteral stent was placed by introducing the guide wire through a 16-gauge intravenous catheter sheath placed percutaneously under direct laparoscopic control . then the ureter was spatulated , and a running suture on a small and half- circle [ sh ] needle was placed . we used djs according to age ( length of jj = age of the child in years + 10 cm ) . the anterior plane of the anastomosis was completed using the same suturing technique as for the posterior layer . in some cases , we used a 2 mm percutaneous retractor on the anterior flap of the sectioned renal pelvis in order to stabilize the loose pelvis and facilitate the anastomosis . the mesocolic window was usually closed with two or three hem - o - lok clips or by using a 3 - 0 polyglactin interrupted suture after placement of the suction tube . the procedure began by incising at toldt 's line , dissecting the left colon from gerota 's fascia and reflecting it medially to visualize the renal hilum and upj . median follow - up period for transmesocolic group was 12.5 months ( 9.5 - 62 months ) and 14 months ( 8 - 66 months ) for retro colic group . follow - up included renal dynamic scan , first at 6 weeks after stent removal , then at 3 months and further 2 scans at 6 monthly intervals , resolution of symptom and or resolution of hydronephrosis on ultrasound . in tmp group , 32 patients were boys and 6 were girls while 36 patients were boys and 5 were girls in rlp group . djs was put in 73.68% patients in tmp group while 82.92% patients in rlp group . in rlp group , the mean operative time was 75.84 min ( time from port insertion to pyeloplasty ) and 135.4 min ( total operative time ) while it was 44.82 min and 104.82 min respectively in tmp group . on average , tmp was 30 min shorter than classic rlp ( 104.8 min vs. 135.4 min ; p 0.05 ) . there were no intra - operative complications or open conversions . in post - operative period three patients had increased drain output , 1 patient had fever and hematuria , 1 patient had wound infection in rlp group while 1 patient in tmp group had increased drain output . even patients with pelvic volume greater than 100 ml could be safely managed with prior aspiration [ figure 2a ] . patients characteristic a case of left ureteropelvic junction obstruction with ( a ) secondary stones managed with transmesocolic approach , ( b ) crossing vessel ( arrow ) managed with transmesocolic approach ( a ) needle ( aspiration ) aspiration of dilated pelvis ( > 50 ml ) managed with transmesocolic approach . ( b ) antegrade jj stenting after completion of posterior wall anastomosis the mean hospital stay , mean drain removal time , mean time for oral intake and mean analgesic dose requirement was significantly lower in tmp group as compare to rlp . all patients in rlp group had unobstructed drainage with static split renal function while in tmp group , 1 patient who had base line renal function of 10% , had further deterioration in function and needed nephrectomy . in recent years , several minimally invasive procedures for pyeloplasty have emerged in order to decrease the morbidity associated with the classic open approach . at present , laparoscopic pyeloplasty has a success rate of 88 - 100% , which is similar to open surgery . these authors described their technique , as a way of easing renal pedicle access and decrease operative time . when a transperitoneal approach is chosen for a right - side lp , the access to the upj is simple as the surgeon has only to lift up the liver to identify the renal pelvis . thus , the pyeloplasty can be performed through a small incision in the posterior peritoneum . conversely , on the left side , mobilization of the descending colon is mandatory to identify the underlying renal fascia and to access the upj . however , the descending colon can reduce the width of the operative field , especially in obese patients or when the colon is distended . in these cases , the introduction of a fourth trocar is often required to improve the exposure . to avoid the mobilization of the descending colon and all the inherent problems associated with bowel manipulation , a direct transperitoneal access to the left upj castillo et al . , who reported series of 18 and 11 patients , respectively . crossing vessels were present in 15.78% patients of tmp group while 18.42% patients in rlp group . . stated , tmp is effective even in the presence of a large renal pelvis or abnormal polar vessels . in our study , on an average , tmp was 30 min shorter than classic rlp with a 22% reduction of operative time . reported a reduction of 23% and romero et al . reported a reduction of 22.5% , which is similar to our study the classic rlp approach needs mobilization of the descending colon and its mesentery , which is time consuming . reported 3.6 days and 2.9 days in rlp and tmp group respectively . a study carried out by braga et al . in 2007 coded the mean hospital stay as 2.5 days . the reduction of 19.2% was reported by romero et al . while in our study it was 29.7% . the mean hospital stay in our study was 5.89 days ( range 3 - 17 days ) in rlp group while 4.14 days ( range 3 - 6 days ) in tmp group . higher values are due the fact that poor sanitary condition at home and poor wound care would increase infection and morbidity , owing to which discharging of the patient is delayed . mean analgesic dose requirement was significantly less in tmp group than rlp group ( p < 0.001 ) . manipulation of the colon and adjacent abdominal wall can cause visceral pain . but generally tmp causes less pain when compared to rlp . however , castillo et al . reported no difference in post - operative pain in either group . mean time for oral intake and drain output was significantly less in tmp group than rlp ( p < 0.05 ) . this data supports the facts that tmp is a feasible and reproducible technique that allows shorter operative times without increasing morbidity . different comparative studies for tmp and rlp the limitations of this study were high mean age in both groups and no pain score was recorded during the study . however , randomized prospective nature of the study and a substantial number of the cases compared in both arms contributed to the strength of this study . moreover , inclusion of left sided of upjo avoided any sidewise bias for the operator . transmesocolic approach for laparoscopic pyeloplasty enables a significant shorter operative time without increasing morbidity with comparable result to that of conventional retrocolic approach even in the presence of large renal pelvis , aberrant vessel and associated renal stones .

objective : this prospective randomized study was designed to evaluate the feasibility and outcome of transmesocolic laparoscopic pyeloplasty ( tmp ) and compare it with retrocolic laparoscopic pyeloplasty ( rlp ) in pediatric and adolescent patients.materials and methods : between september 2006 to may 2012 , data of pediatric and adolescent patients undergoing laparoscopic pyeloplasty were recorded in a prospective manner . data included age , pelvic volume , presence of stones , aberrant vessels , operative time , analgesics requirement and time to accept oral feeds and drain removal . patients with left side pelviureteric junction obstruction with any size of pelvic volume , with or without renal stones and aberrant vessels were included in the study . patients were assigned into two groups by simple randomization technique . a total of 38 tmp and 41 left sided rlp were performed . median follow - up period for transmesocolic group was 12.5 months ( 9.5 - 62 months ) and 14 months ( 8 - 66 months ) for retro colic group . outcome for this study was adequate drainage on renal scan , improvement in symptom and or resolution of hydronephrosis on ultrasound . statistical analysis was performed using the mann - whitney test.results:the mean patient age was 8.73 years in rlp and 7.73 years in tmp . in rlp group the mean operative time was 75.84 min ( time from port insertion to pyeloplasty ) and 135.4 min ( total operative time ) while it was 44.82 min and 104.82 min respectively in tmp group . compared with classic rlp , tmp cases showed a significant reduction in operative time.conclusions:the transmesocolic approach for left sided pyeloplasty enables a shorter operative time even in the presence of large pelvis , aberrant vessel and stones without increasing morbidity in comparison to rlp approach .

INTRODUCTION MATERIALS AND METHODS None Patients and data collection Surgical technique RESULTS DISCUSSION CONCLUSIONS

PMC4183358

hamiltonian mapping demonstrates how the standard wham formalism can be modified and used to extrapolate information about a biased hamiltonian from the unmodified reference hamiltonian , h0(x ) , where x represents the molecular coordinates . wham is typically used along with umbrella sampling , in which a set of l additional restraining potentials , { v } = v1(x ) , ... , vl(x ) , are added to h0(x ) in order to enhance conformational sampling in rarely visited regions of phase space . in this case , the modified hamiltonian takes the form1where { } is the set of coupling parameters used for scaling individual biasing potentials , 0 = 1 , and h0(x ) = v0(x ) . then , for r independent simulations , each performed at a temperature t = 1/kb ( where kb is the boltzmann constant ) and with differing { } , the bias is removed by solving the following pair of wham equations self - consistently2and3 t may be constant or vary across the r simulations . ) is a particular value along a progress variable of interest , nm is the total number of configurations in the mth simulation , and nk({v}, ) is the histogram count of configurations with { v } and . finally , fm is related to the helmholtz free energy for the mth simulation . in contrast to standard wham , in the hamiltonian mapping framework , we proceed in the opposite direction ; we perform one or more simulations of the system under the original hamiltonian h0(x ) and then add a bias to the statistical averages computed from h0(x ) to examine thermodynamic behavior under a modified hamiltonian . for this scenario , eq 2 is recast as4essentially , the histogram counts nk now come from the sampling of h0(x ) , and the sum of the scaled restraining potentials in the denominator reduces to h0(x ) . briefly , in this model , each residue is represented by a single bead centered at the c position and with the mass of the corresponding amino acid . an additive potential describes the bonded and non - bonded interactions between the beads . virtual bonds and angles are defined by harmonic potentials with reference values determined by the c coordinates in the experimental structure . a potential for virtual dihedral angles is based upon backbone dihedral angle probability distributions from the protein data bank ( pdb ) for the 400 possible amino acid pairs and thus is independent of the specific topology of the system . residue pairs separated by three or more bonds interact through the following potential5where rij is the distance between residues i and j , ij corresponds to the distance between the two residues at which vij is minimum , and ij is the interaction strength at ij . residues located in close proximity in the experimental structure ( i.e. , forming a native contact ) interact favorably with ij values based on the statistical contact energies of miyazawa and jernigan , while residue pairs not in close proximity experience a slight repulsive interaction that takes the form of a typical 126 lennard - jones potential . further description of the go-like model and details of the simulation setup can be found in refs ( 25 ) and ( 8) , respectively . we further consider the non - bonded interaction potential ( eq 5 ) for native contacts between the idp and the receptor in tuning the model to match the experimental kd . for this set of intermolecular contacts , we scale the interaction strength , ij , by a factor so as to control the proportion of bound and unbound states throughout simulation . from the fraction of unbound states , pu , the kd is computed as6where [ protein ] is the concentration of protein in moles per liter ( 10 mol / l in the current study ) . a configuration is considered unbound if zero intermolecular native contacts are formed and if the distance between the centers of mass of the idp and of the target protein is greater than the cutoff for calculating the non - bonded interactions ( 25 ) . we also note that the strength of intramolecular contacts within the idp are tuned in a similar manner to reproduce the experimental estimate of residual helical structure in the unbound state .

molecular recognition by intrinsically disordered proteins ( idps ) plays a central role in many critical cellular processes . toward achieving detailed mechanistic understanding of idp target interactions , here we employ the hamiltonian mapping methodology , which is rooted in the weighted histogram analysis method ( wham ) , for the fast and efficient calibration of structure - based models in studies of idps . by performing reference simulations on a given hamiltonian , we illustrate for two model idps how this method can extrapolate thermodynamic behavior under a range of modified hamiltonians , in this case representing changes in the binding affinity ( kd ) of the system . given sufficient conformational sampling in a single trajectory , hamiltonian mapping accurately reproduces kd values from direct simulation . this method may be generally applied to systems beyond idps in force field optimization and in describing changes in thermodynamic behavior as a function of external conditions for connection with experiment .

Computational Methods

PMC3214428

general physical examination at age 15 years showed : height 113 cm ( <3 percentile ) , weight 10 kg ( <3 percentile ) , and head circumference 48 cm . he was apparently well up to the age of 18 months , later he developed failure to thrive , loss of hair , prominent eyes and joints , and senile appearance . systemic examination revealed craniofacial disproportion , mandibular and maxillary hypoplasia , prominent scalp veins , plucked bird appearance , thin lips , irregular and carious teeth , dental crowding , beaked nose , protruding ears with absent ear lobes , pyriform thorax , thin limbs , prominent and stiff joints , wide - based gait and shuffling , thin and high - pitch voice , incomplete sexual maturation , generalized alopecia , absence of subcutaneous fat , thin and wrinkled x - ray survey of the skeletal system showed radiolucent terminal phalanges ; coxa vulga , enlarged metaphysis of long bones , short dystrophic clavicle and osteoporosis [ fig . 4a and b ] . his best corrected visual acuity was 20/20 , n6 in both eyes ( be ) with -1.25 cylinders at 161 in right eye ( re ) and -0.50 cylinders at 41 in left eye ( le ) . external photograph of a 15-year - old hutchinson - guilford progeria male patient ( a ) showing disproportionately small face in comparison to the head , micrognathia , prominent eyes , both upper eyelids retraction , beaked nose , thin lips . ( b ) prominent scalp veins , alopecia with grey and sparse hairs , and protruding ears with absent earlobe ( a ) upper jaw dental crowding and dental caries ( b ) lower jaw dental crowding and dental caries prominent joints of hand ( red arrow : metacarpophalangeal joints , blue arrow : interphalangeal joints ) with loss of subcutaneous fat , nail dystrophy and sclerodematous skin ( a ) x - ray hand showing radiolucent terminal phalanges ( white arrow ) and osteoporotic changes ( black arrow ) . ( b ) x - ray shoulder joint showing dystrophic and short clavicle ( white arrow ) external ocular examination showed excessive wrinkling of forehead , superior sulcus deformity , inner canthal distance 20 mm , outer canthal distance 58 mm , inter - pupillary distance 40 mm , vertical palpebral fissure 12 mm , horizontal palpebral fissure 20 mm , lagophthalmos 2 mm , upper eyelid retraction 2.5 mm , upper lid lag in down gaze 5 mm , in be , nearly total loss of eyebrows and eyelashes , a few present eyelashes and eyebrows showed poliosis [ fig . thus , in view of the characteristic clinical presentation , imaging studies , and urine analysis , the child was diagnosed to have hgp syndrome . ( a ) both upper lids lag in down gaze , superior sulcus deformity , loss of eyelashes and eyebrows . the hgp syndrome is an extremely rare , fatal , genetic disorder of childhood with striking features resembling premature aging . the hgp syndrome has a slight male predilection ; the male - to - female ratio is 1.5:1 . the incidence of the hgp syndrome has been estimated at 1 in 4 - 8 million live births . the clinical diagnosis of the hgp syndrome is based on recognition of characteristic features and exclusion of other progeroid syndromes . children with progeria usually have a normal physical appearance in early infancy . at approximately one to two years of age , affected children develop severe growth retardation , resulting in short stature , low weight and delayed anterior fontanel closure . these children develop a distinctive facial appearance characterized by a disproportionately small face in comparison to the head , micrognathia , delayed dentitions , dental malformation and crowding , beaked nose , prominent eyes , nasolabial circumoral cyanosis and loss of scalp hair , eyebrows , and eyelashes . other characteristic features include generalized atherosclerosis , cardiovascular diseases and stroke , hip dislocations , prominent scalp veins , lipodystrophy , dystrophy of nails , joint stiffness , skeletal defects , and/or other abnormalities . the most common reported ocular features are prominent eyes , loss of eyebrows and eyelashes , and lagophthalmos . other rare ocular manifestations of the hgp syndrome are bands of skin running from the upper lid to the cornea , senile ectropion , ptosis with marcus - jaw - winking phenomenon , dry - eye syndrome , corneal dryness , keratopathy , iridocorneal adhesions , corneal opacities , corneal clouding , cataract , strabismus , irregular nystagmoid movements , myopia , hyperopia , retinal arteriolar narrowing and tortuosity , and retinal angiosclerosis.[468 ] many of these findings are based on individual case reports and are absent in the present case . the ophthalmic anthropometric measurements such as horizontal palpebral fissure length , interpupillary distance , inner canthal distance , outer canthal distance in our patient are less than aged - matched normal indian values . eyelid retractions , lagophthalmos , lid lag in down gaze , are due to inelastic and taut skin . poor pupillary dilatation may be due to ischemic changes in the iris muscles secondary to microvascular insufficiency induced by atherosclerosis , however , visible atrophic changes were absent on slit - lamp examination in our patient . in the hgp syndrome eyes looks prominent ( pseudoproptosis ) probably due to lid retraction , although there is no true proptosis . interestingly , patients with hgps do not develop other ocular features associated with aging , such as presbiopia , arcus senilis or age - related macular degeneration . the hgp syndrome should be differentiated from other progeroid syndromes such as werner 's syndrome ( adult progeria ) , wiedemann - rautenstrauch syndrome ( neonatal progeria ) , hallermann - streiff syndrome , mandibuloacral dysplasia , cockayne 's syndrome , rothmund - thomson syndrome , metageria and acrogeria . the average life expectancy is 13 years , with an age range of 7 - 27 years . the most common cause of death is myocardial infarction or congestive cardiac failure , secondary to widespread atherosclerosis . at present ophthalmic manifestations / signs / features / findings , eye , premature ageing syndrome and various combinations thereof . all cross - references in citing literature were included in our search and referenced . to the best of our knowledge , lid retraction , lid lag in down gaze , superior sulcus deformity and poor pupillary dilatation

the hutchinson - gilford progeria ( hgp ) syndrome is an extremely rare genetic condition characterized by an appearance of accelerated aging in children . the word progeria is derived from the greek word progeros meaning prematurely old. it is caused by de novo dominant mutation in the lmna gene ( gene map locus 1q21.2 ) and characterized by growth retardation and accelerated degenerative changes of the skin , musculoskeletal and cardiovascular systems . the most common ocular manifestations are prominent eyes , loss of eyebrows and eyelashes , and lagophthalmos . in the present case some additional ocular features such as horizontal narrowing of palpebral fissure , superior sulcus deformity , upper lid retraction , upper lid lag in down gaze , poor pupillary dilatation , were noted . in this case report , a 15-year - old indian boy with some additional ocular manifestations of the hgp syndrome is described .

Case Report Discussion

PMC5076654

consumers interest in the relationship between diet and health has increased the demand for information about the specific health characteristics of food . it is well known that food can contain ingredients that may have physiological benefits and/or reduce the risk of chronic disease , or even represent significant risk factors influencing ill health . in cheese , compounds like amino acids are freed during proteolysis ; they are substrates for secondary catabolic reactions by means of bacteria with aminoacyl decarboxylase activity that may yield bioactive compounds . some of them present useful nutraceutical properties , such as -aminobutyric acid ( gaba ) , while others , such as biogenic amines ( ba ) , may have negative effects on human health . over the last decade attention has been paid to the presence of gaba in cheese and dairy product , as a result of its health - related benefits ( mills et al . , 2009 ; wang et al . , 2010 ; nejati et al . , 2013 gaba is a non - protein amino acid acting as one of the main inhibitory neurotransmitters in the sympathetic mammalian nervous system and exerts positive effects in the treatment of sleeplessness , depression , chronic alcohol - related symptoms ( oh et al . , 2003 ) and parkinson s disease ( de jong et al . , 1984 ) . in addition , it has shown antitumorigenic activity ( thaker et al . , 2005 ) and , since it is a strong secregatogue of insulin from the pancreas , it can prevent diabetes ( hagiwara et al . , 2004 ) . gaba is produced by the decarboxylation of glutamate catalysed by the enzyme l - glutamic acid decarboxylase ( gad ) . this enzyme is present in different species of microorganism that could be used for the development of probiotic cheese ( wang et al . , 2010 ) . the most interesting are the lactic acid bacteria ( lab ) , in particular several strains of lactobacillus brevis ( mills et al . , 2009 ) , lactobacillus plantarum , lactobacillus delbrueckii subsp . lactis ( siragusa et al . , 2007 ; mills et al . , 2009 ; wang et al . , 2010 ) . the enterococcus spp . and streptococcus spp . also include strains that have shown gad activity ( hayakawa et al . , 1997 ) . other research has shown that these species of bacteria are also involved in the production of ba ( galgano et al . , 2001 ; linares et al . , 2012 ; lorencov et al . , 2012 ) , considered as a serious health problem when present at significant levels ( stratton et al . , 1991 ) . tyramine , -phenylethylamine , histamine , tryptamine , cadaverine , putrescine , spermine and spermidine are considered as the most important amines occurring in cheese . in cheese , ba are considered as an indicator of poor hygienic conditions of raw material and/or manufacturing practices since their production and accumulation is often associated with the activity of contaminant bacteria ( loizzo et al . , 2013 ) . in general , artisanal sheep cheeses are rich in gaba ( siragusa et al . , 2007 ) but also in ba ( loizzo et al . , 2013 ; in fact , microbial decarboxylase activity requires the availability of free amino acid precursors , produced as an outcome of proteolysis , and favourable ph and temperature conditions that can be achieved during ripening ( siragusa et al . some research has reported that gaba is also present in pecorino sardo ( siragusa et al . , 2007 ) , as are ba ( manca et al . , 2000 ) , even if these studies were limited to a very few samples . for these reasons this study has concerned the simultaneous determination of gaba and ba in pecorino sardo produced in cheese factories , pecorino produced in farmhouses , and casu marzu , made from insect larvae ( piophila casei ) , in order to assess the variability of these nitrogenous compounds important to define cheese quality , in products obtained with different technological traits . the study was carried out on traditional cheeses made in sardinia ( italy ) from whole ewe s milk . the types of cheese considered were pecorino sardo protected designation of origin ( pdo ) , produced at cheese factories ( 13 samples with ripening time ranging from 30 - 360 days ) , farmhouse pecorino ( 12 samples with ripening time ranging from 60 - 360 days ) , and farmhouse casu marzu ( 9 samples with ripening time ranging from 60 - 90 days ) . pecorino sardo pdo produced in cheese factories is a semi - cooked cheese made from thermised milk inoculated with a starter culture and coagulated with calf rennet . ripening takes place in ripening rooms , the temperature ( between 6 - 12c ) and relative humidity ( between 80 - 95% ) of which are measured and controlled automatically . pecorino cheese obtained from farmhouses is a semi - cooked cheese made from raw milk without a starter culture and coagulated with calf rennet . ripening takes place in rooms with no control over humidity or temperature , which may even reach room values . casu marzu ( also called casu modde , casu cunddu , or casu frzigu in sardinian language , which translates rotten cheese ) is a sardinian cheese produced with the use of larvae of the cheese fly piophila casei . it is prepared in summer , when higher temperatures favour the life - cycle of the fly , and derived from pecorino cheese that producers place in the warmer rooms of the plant , uncovered , so that flies have better access to lay their eggs in it . after 60 - 90 days of ripening the cheese is ready to eat . to extract the nitrogenous compounds considered , an amount of 1 g of ground cheese was weighed directly in a centrifuge tube and 20 ml of hcl 0.1 m added . the mixture was then homogenised in an ultra turrax homogeniser ( zipperer , staufen , germany ) for 5 min . the two acid extracts were combined and diluted to 50 ml with hcl 0.1 m. one ml of the extracts was diluted to 10 ml with hcl 0.1 m and then an aliquot of 400 l was derivatised with dns - cl . to prepare dansyl derivatives of free amino acids ( faa ) and ba , the method described by vinci and antonelli ( 2002 ) , was followed , with some modifications . 40 l of saturated sodium carbonate solution at 20c , 200 l of dansyl chloride solution ( 1.5% w / v in acetone ) and 300 l of acetonitrile were added to 400 l of standard solutions or sample extracts . the vial containing the reaction mixture was capped , vortexed and then incubated at 60c for 30 min under stirring in a sh 2000-dx thermo mixer ( finepcr , seoul , korea ) . in order to eliminate the excess of dansyl chloride the mixture was treated with 50 l of l - asparagine solution ( 2.2% w / v in water ) , then 10 l of glacial acetic acid was added to remove the excess of carbonate . the solution was filtered through a 0.22 m pvdf syringe filter ( millipore , bedford , ma , usa ) then 10 l were injected in high - performance liquid chromatography . simultaneous separation of dansyl derivatives of gaba , protein faa and ba was performed in a varian ( walnut creek , ca , usa ) chromatography system equipped with a prostar 230 solvent delivery system , a prostar 410 autosampler , and an lc 305 fluorescent detector ( linear instruments , reno , nv , usa ) . the column used was an alltima c18 column , 150 mm x 4.6 mm , 3 m [ alltech italia , sedriano ( mi ) , italy ] , fitted with an alltima c18 guard column , 7.5 mm x 4.6 mm x 5 m , thermostated at 40c . detection was carried out with the fluorescent detector operating at 340 and 520 nm as excitation and emission wavelengths respectively . free amino acids and ba were determined by the liquid chromatographic method described by minocha and long ( 2004 ) . the solvents used for separation were eluent a : 100% acetonitrile ( acn ) and eluent b : 20 mm ammonium acetate buffer ( ph 5.9 ) containing 3% v / v 2-propanol . a gradient programme was implemented as follows : time=0 , a : b ( 10:90 ) , flow=0.5 ml min ; time=1 min , a : b ( 10:90 ) , flow=0.5 ml min ; time=50 min , a : b ( 65:35 ) , flow=0.5 ml min ; time=65 min , a : b ( 100:0 ) , flow=0.5 ml min ; time=66 min , a : b ( 100:0 ) , flow=2 ml min ; time=70 min , a : b ( 10:90 ) , flow=0.5 ml min . the identification of the nitrogenous compounds was performed by comparison of the retention times of peaks in the samples to those of standard solutions and by addition of the suspected compound to the samples . a calibration curve was obtained by analysing standard solutions at 9 different concentrations and calibration graphs were constructed by plotting the peak area versus each analyte concentrations . statistical analyses of the data were performed using the software package spss 14 ( spss inc . , statistical analyses of the data were performed using the software package spss 14 ( spss inc . , chicago , il , usa ) . the results were expressed as means of two replications . on the basis of the original method designed for the analysis of faa and polyamines ( minocha and long , 2004 ) , we developed a method that allowed extending the analysis to other seven ba . to do this , it was necessary to extend the chromatographic running time from 56 to 70 min . in figure 1 the chromatograms of a cheese sample and of a standard solution of amino acids and amines dansyl amino acids and dansyl ba were eluited in the following order : aspartic acid , glutamic acid , serine , threonine , glycine , alanine , arginine , proline , -amino butyric acid , valine , methionine , isoleucine , leucine , phenylalanine , ornithine , cysteine+cystine , lysine , histidine , tyrosine , agmatine , tryptamine , phenylethylamine , putrescine , cadaverine , hystamine , serotonin , tiramine , spermidine and spermine . the content of the compounds analysed in the three types of ewe s milk cheese considered are shown in table 1 . the highest level of gaba was found in casu marzu ( ranging from 34.4 to 1001.3 mg 100 g ) and in pecorino ( ranging from 0.0 to 378.1 mg 100 g ) , while the samples of pecorino sardo pdo showed a lower content ( ranging from 2.3 to 52.0 mg 100 g ) . the results showed great variability in gaba content not only between the three types of cheese but also within the same type . by comparing these sardinian products with other ewe s milk cheeses it was noted that in most of the samples of both casu marzu and pecorino the gaba content was from 2 to 10 times higher than the maximum level ( 39.1 mg 100 g ) measured in other types of italian cheese ( siragusa et al . , 2007 ) . in particular , three samples of casu marzu had a content that was as much as 20 times greater than that reported in the above - mentioned work . the differences in gaba content appear to be dependent on the different degree of proteolysis observed between the three types of cheese . in fact , considering the content of total faa as an index of proteolysis level , calculated as the sum of the individual faa measured , a significant correlation with gaba was found ( pearson coefficient 0.555 at p=0.01 ) . as for gaba , the higher levels of total faa were found in the samples of casu marzu . this is most probably due to the high proteolytic activity induced by the piophila casei larvae , and to the high temperature used for cheese storage ( mazzette et al . , 2010 ) . the farmhouse samples of pecorino tended to have a total faa content greater than those of pecorino sardo pdo from cheese factories . as reported in the literature , such differences can be attributed to changes in manufacturing processes ( pintado et al . , 2008 ; schirone et al . , 2011 ) . by comparison , in cheese samples with the same age , great variability in total faa content was also found within the same type of cheese , highlighting that production processes can vary from one cheesemaker to another . in pecorino sardo , as observed in a previous work ( manca et al . , 1999 ) , variability in the faa content was due to a production protocol that allowed the use of different technological features . considering the amino acid profile ( percentage of the total faa for each amino acid ) shown in table 2 , it is possible to highlight that in three samples of pecorino and in three of casu marzu , gaba is the amino acid present in the highest percentage with respect to the total faa ( from 12.3 to 18.5 and from 14.2 to 22.9% respectively ) . in almost all the other samples , including the factory products , glutamic acid , the precursor of gaba , is the amino acid mostly represented , followed by leucine , valine and lysine . this faa profile is similar to that found in other ewe s milk cheese ( izco et al . , 2000 ; 1995 ) , while to our knowledge , gaba has never been found as the major faa in sheep s cheese . as for gaba and total faa , great variability in the total content of ba was found between the different types of cheese , but also within the same type . casu marzu showed the highest content of total ba ; in particular , two samples presented values of 1035.7 and 923.0 mg 100 g respectively . the other casu marzu samples had values ranging from 62.9 to 282.1 mg 100 g for this parameter , more similar to that found in other artisanal italian cheeses , such as formaggio di fossa ( mascaro et al . , 2010 ) and pecorino di farindola ( schirone et al . , 100 g have been proposed for total ba in food ( spanjer and van roode , 1991 ; ten brink et al . , 1990 ) , casu marzu can be considered unsafe , especially for patients treated with monoamine oxidase inhibitor drugs ( maois ) . also four samples of pecorino showed a total ba content ( between 79.0 and 287.8 mg 100 g ) that exceeded the threshold proposed , while the other samples presented values ranging from 9.4 and 73.4 mg 100 g that did not represent a risk for consumers health . however , the samples of pecorino had , in general , a higher level of total ba than those of pecorino sardo pdo , whose values varied from 3.1 to 33.5 mg 100 g. seeing that ba are considered an index of poor hygienic conditions , the high content of these compounds measured in casu marzu and pecorino could be related to less than optimal environmental conditions during the production process ( mazzette et al . , 2010 ) . the use of insects , as in casu marzu , and of raw milk , combined with a high storage temperature , seems to favour the production of ba , as already shown in other artisanal sheep s cheeses ( loizzo et al . 2010 ) . on the contrary , in pecorino sardo pdo , where the milk used is thermised and the temperature and humidity conditions under control , the products showed a low content of total ba . the variability observed in ba content , as observed by novella - rodriguez et al . ( 2008 ) in other types of cheeses , could be attributable to differences in the manufacturing process . a positive correlation was found between total ba and total faa ( pearson coefficient=0.513 at p=0.01 ) , confirming the findings of giraffa et al . ( 1995 ) who observed an increase in ba corresponding to an increase in proteolysis . tyramine , cadaverine and putrescine were the main amines in all the types of cheese considered , even if their content varied to a great extent : in this respect the statements made previously are valid for the total content of ba . in casu marzu the highest levels found for tyramine , cadaverine and putrescine were 231.4 , 470.7 and 165.8 mg 100 g respectively . , 2013 ; schirone et al . , 2013 ) , some samples of casu marzu seem to present the highest contents found in cheeses up to now . in blue cheese one of the richest in ba ( loizzo et al . , 2013 ; novella - rodriguez et al . , 2003 ) the highest levels of tyramine , cadaverine and putrescine were 158.5 , 210.4 and 25.7 mg 100 g respectively . regardless of the type of cheese , histamine , triptamine and pphenylethylamine were present in only about 50% of the samples and also for these amines the highest concentrations were found in samples of casu marzu and pecorino . the polyamines spermidine and spermine were present only in pecorino sardo pdo , with a level ranging from 0.0 to 11.1 and from 0.0 to 5.4 mg 100 g respectively , similar to that found in blue cheese ( novella - rodriguez et al . , this is in accordance with previous works in which these amines are usually present at low levels or are not detected in cheese ( loizzo et al . , 2013 ; the pearson test showed a significant correlation ( p=0.01 ) between gaba and the amines putrescine , cadaverine and triptamine ( pearson correlation coefficients 0.779 , 0.795 and 0.763 respectively ) ( table 3 ) . therefore , it may be assumed that factors responsible for ba production , such as temperature , ph , availability of faa and , primarily , microorganisms with amino acid decarboxylase activity ( pinho et al . , 2001 ) , may also affect gaba synthesis ( siragusa et al . the literature highlights that in cheese and other dairy products some microbial species responsible for the synthesis of one or more ba are also involved in gaba production . brevis , and lactococcus lactis , active in gaba production ( siragusa et al . , 2007 ) , 2012 ; galgano et al . , 2001 ) ; likewise a streptococcus thermophilus strain can form gaba , tyramine and histamine . cadaverine , putrescine and histamine ( maifreni et al . , 2013 ) , but also gaba could be produced by species belonging to the genera enterococcus ( dhakal et al . , 2012 ) . paracasei , lactococcus lactis , streptococcus thermophilus and enterococus spp , were found as components of the microflora of traditional pecorino sardo cheese ( mannu et al . , 2002 ; 2006 ) . therefore , the types of sheep s cheese considered seem to present the ideal conditions for favouring the production and accumulation of both gaba and ba . the pearson test showed a significant correlation ( p=0.01 ) between gaba and the amines putrescine , cadaverine and triptamine ( pearson correlation coefficients 0.779 , 0.795 and 0.763 respectively ) ( table 3 ) . therefore , it may be assumed that factors responsible for ba production , such as temperature , ph , availability of faa and , primarily , microorganisms with amino acid decarboxylase activity ( pinho et al . , 2001 ) , may also affect gaba synthesis ( siragusa et al . the literature highlights that in cheese and other dairy products some microbial species responsible for the synthesis of one or more ba are also involved in gaba production . brevis , and lactococcus lactis , active in gaba production ( siragusa et al . , 2007 ) , 2012 ; galgano et al . , 2001 ) ; likewise a streptococcus thermophilus strain can form gaba , tyramine and histamine . cadaverine , putrescine and histamine ( maifreni et al . , 2013 ) , but also gaba could be produced by species belonging to the genera enterococcus ( dhakal et al . , 2012 ) . paracasei , lactococcus lactis , streptococcus thermophilus and enterococus spp , were found as components of the microflora of traditional pecorino sardo cheese ( mannu et al . , 2002 ; madrau et al . , therefore , the types of sheep s cheese considered seem to present the ideal conditions for favouring the production and accumulation of both gaba and ba . great differences were found for gaba , total faa and ba content in the three types of cheese considered . farmhouse casu marzu and pecorino are the types of cheese with a high content of gaba , a compound with beneficial functions , but also of ba , in particular tyramine , cadaverine and putrescine that in high concentrations represent a health hazard for consumers . the pearson correlation test revealed that variability in the content of gaba and ba was correlated with the total faa , indicating that their production depends on the intensity of the proteolytic phenomena and thus on environmental and manufacturing factors that affect this biochemical event . given the significant correlation highlighted by the pearson test ( p=0.01 ) between gaba and the most frequently represented ba , it was supposed that the production of all these compounds is affected by the same factors . furthermore , it was shown that the cheeses considered , in particular those of farmhouse casu marzu and pecorino , presented ideal conditions for developing microorganisms and for their carboxylase activity . considering the interest of the food industry in functional food , casu marzu and pecorino microflora can potentially be useful to enrich cheese or milk - fermented products with gaba , but further studies are needed to assess whether microbial strains are present that are capable of producing a high content of gaba but not of ba and to individuate which technological factors may favour the grows of these bacteria .

the bioactive compounds -aminobutyric acid ( gaba ) and biogenic amines ( ba ) , together with protein - free amino acids , were measured by high - performance liquid chromatography in ewe s milk cheeses produced in sardinia with different technological traits . the study included three types of cheese : pecorino sardo pdo , pecorino and casu marzu . farmhouse casu marzu and pecorino showed gaba content ( maximum levels : 1001.3 and 378.1 mg 100 g1 respectively ) that had never been found so high in cheese before , suggesting that these types of cheese present ideal conditions to produce gaba . these two types of cheese also showed high levels of ba ( their total maximum levels were 1035.7 and 288.0 mg 100 g1 respectively ) . pearson correlation analysis detected significant correlation between gaba and the main ba present in the cheeses ( tyramine , cadaverine and putrescine ) , suggesting that the factors affecting the production of gaba are the same as those influencing ba formation .

Introduction Materials and Methods Statistical analysis Results and Discussion Relationship between -aminobutyric acid and biogenic amines Conclusions

PMC1566277

when using the mass balance equation to model indoor air quality , the primary assumption is that of uniform mixing . different points in a single compartment are assumed to have the same instantaneous pollutant concentrations as all other points . although such an assumption may be unrealistic , under certain conditions predictions ( or measurements ) of exposures at single points in a room are still within acceptable limits of error ( e.g. , 10% ) . in this article , three studies of the mixing of environmental tobacco smoke ( ets ) pollutants are reviewed , and data from several other ets field studies are presented . under typical conditions for both short sources ( e.g. , 10 min ) and the continuous sources of ets in smoking lounges , i find that average exposure concentrations for a single point in a room represent the average exposure across all points in the room within 10% for averaging times ranging from 12 to 80 min . i present a method for determining theoretical estimates of acceptable averaging times for a continuous point source.imagesfigure 1figure 2figure 3figure 6

Images

PMC4809460

patients fulfilling criteria for ftd were consecutively recruited from the centre for aging brain and neurodegenerative disorders , university of brescia , italy , from december 2001 to july 2014 . all patients underwent somatic and neurologic evaluation , routine laboratory examination , and a complete mental status evaluation . each patient was screened for monogenic inherited disease , such as grn , mapt , or c9orf72 mutations . stringent exclusion criteria were applied as follows : ( 1 ) cerebrovascular disorders , previous stroke , hydrocephalus , and intracranial mass documented by mri ; ( 2 ) history of traumatic brain injury or another neurologic disease ; ( 3 ) relevant medical problems ( e.g. , poorly controlled diabetes or hypertension ; cancer within the past 5 years ; clinically important hepatic , renal , cardiac , or pulmonary disorders ) ; ( 4 ) history of major depressive disorder , bipolar disorder , schizophrenia , substance abuse disorder , or mental retardation according to dsm - iv criteria ; ( 5 ) csf -amyloid / tau amyloid profile of alzheimer disease type ( available in almost 60% of patients ) to avoid confounding effects of different neuropathologic substrates ; and ( 6 ) logopenic variant of ppa ( lvppa ) . a comprehensive neuropsychological and behavioral assessment , including basic activities of daily living and instrumental activities of daily living , was carried out . the neuropsychological testing was performed by a standardized neuropsychological battery including mini - mental state examination , frontotemporal dementia - modified clinical dementia rating scale ( ftd - modified cdr ) , raven colored progressive matrices , controlled oral word association test and category fluency , clock drawing test , rey complex figure copy and recall , story recall test , digit span , trail making test a and b , token test , and de renzi imitation test . in addition , the aachener aphasie test was further administered to patients with ppa ( data not shown ) . the lvppa diagnosis was supported by the pattern of atrophy in posterior / presylvian or parietal regions at the mri examination ; -amyloid 42 and total tau csf determinations were also considered . the work conformed to the helsinki declaration and was approved by the local ethics committee of brescia hospital , italy . three single nucleotide polymorphisms located within kiaa0319/ttrap / them2 locus ( rs17243157 g / a ) , dcdc2 ( rs793842 a / g ) , and cntnap2 ( rs17236239 a / g ) genes were evaluated . the amplification protocols were designed as follows : 5 minutes at 95c for the first cycle , denaturation at 95c for 30 seconds , annealing ranging from 59c to 66c for 30 seconds ( depending on the analyzed polymorphism ) , extension at 72c for 30 seconds for the subsequent 35 cycles , and a final extension at 72c for 5 minutes . the pcr products were analyzed on 2% agarose with 0.005% of ethidium bromide to reveal the reaction and to verify their size . to evaluate rs17243157 and rs793842 polymorphisms , denaturing high performance liquid chromatography ( dhplc ) analysis on the wave nucleic acid fragment analysis system was performed ( transgenomic , santa clara , ca ) . samples with an altered dhplc profile were purified with microcon centrifugal filter devices ( amicon bioseparations ; millipore corp , billerica , ma ) and sequenced . nucleotide direct sequencing was performed on genomic dna for both strands by the abi 3500xl dna analyzer ( applied biosystems , foster city , ca ) and analyzed using chromas ( technelysium pty ltd , south brisbane , australia ) . to assess rs17236239 , direct sequencing was performed for both strands from purified pcr on the abi 3500 dna genetic analyzer ( applied biosystems ) , according to the manufacturer 's instructions , and analyzed using chromas ( technelysium pty ltd ) . brain images were collected using 2 different mr scanners : ( 1 ) 1.5 t mr scanner ( siemens symphony , erlangen , germany ) , equipped with a circularly polarized transmit receive coil to acquire 3d magnetization - prepared rapid gradient echo ( mprage ) t1-weighted scan ( repetition time [ tr ] = 2,010 milliseconds , echo time [ te ] = 3.93 milliseconds , matrix = 1 1 1 , in - plane field of view [ fov ] = 250 250 mm , slice thickness = 1 mm , flip angle = 15 ) ; and ( 2 ) 1.5 t mr scanner ( siemens avanto ) to acquire 3d mprage t1-weighted scan ( tr = 2,050 milliseconds , te = 2.56 milliseconds , matrix = 1 1 1 , in - plane fov = 256 256 mm , slice thickness = 1 mm , flip angle = 15 ) . preprocessing and statistical analyses were performed using the statistical parametric mapping ( spm8 ) software package ( wellcome department of imaging neuroscience , london , england , http://www.fil.ion.ucl.ac.uk/spm ) running on matlab 7.1 ( mathworks , natick , ma ) . for voxel - based morphometry ( vbm ) analysis , images were spatially normalized to a reference stereotactic template ( montreal neurological institute [ mni ] ) and smoothed by a gaussian kernel of 10 10 10 mm full width at half maximum . gray matter ( gm ) and white matter ( wm ) differences according to genotype status for each polymorphism were evaluated by vbm analyses in all ftd groups ( behavioral variant of ftd [ bvftd ] and ppas together ) and in ppas , respectively . an analysis of covariance model design was applied by entering 4 groups , i.e. , ( 1 ) bvftd carrying at - risk genotype , ( 2 ) bvftd not carrying at - risk genotype , ( 3 ) ppa carrying at - risk genotype , and ( 4 ) ppa not carrying at - risk genotype , and the covariates of no interest , i.e. , age , sex , disease severity ( as measured by ftd - modified cdr ) , presence of monogenic disease , and scanner type . total intracranial volume , i.e. , gm + wm + csf , was introduced in all the statistical analyses as a global nuisance variable to avoid the confounding effect on cortical atrophy . correlation analysis between cognitive performances and regional gm and wm atrophy clusters was carried out . for this , the clusters of gm and wm atrophy obtained in the previous analysis ( comparison between the at - risk group and not - at - risk group ) were selected as regions of interest ( rois ) , and the mean density was calculated for each patient . this approach relies on the assumption that functionally correlated brain regions show a greater concordance in gm and wm volumes as a result of mutually trophic influences or common experience - related plasticity . for this , the clusters of gm and wm atrophy obtained in the previous analysis ( comparison between the at - risk group and not - at - risk group ) were used as seed regions , exploring the pattern of covariance between the gm volume of each seed and the gm volume throughout the whole brain . for all the aforementioned analyses , the threshold was established at p < 0.001 ( uncorrected for multiple comparisons ) and a cluster size threshold was set at 200 voxels . patients were analyzed according to clinical phenotype ( bvftd or ppa ) or genotype status for each evaluated polymorphism . patients fulfilling criteria for ftd were consecutively recruited from the centre for aging brain and neurodegenerative disorders , university of brescia , italy , from december 2001 to july 2014 . all patients underwent somatic and neurologic evaluation , routine laboratory examination , and a complete mental status evaluation . each patient was screened for monogenic inherited disease , such as grn , mapt , or c9orf72 mutations . stringent exclusion criteria were applied as follows : ( 1 ) cerebrovascular disorders , previous stroke , hydrocephalus , and intracranial mass documented by mri ; ( 2 ) history of traumatic brain injury or another neurologic disease ; ( 3 ) relevant medical problems ( e.g. , poorly controlled diabetes or hypertension ; cancer within the past 5 years ; clinically important hepatic , renal , cardiac , or pulmonary disorders ) ; ( 4 ) history of major depressive disorder , bipolar disorder , schizophrenia , substance abuse disorder , or mental retardation according to dsm - iv criteria ; ( 5 ) csf -amyloid / tau amyloid profile of alzheimer disease type ( available in almost 60% of patients ) to avoid confounding effects of different neuropathologic substrates ; and ( 6 ) logopenic variant of ppa ( lvppa ) . a comprehensive neuropsychological and behavioral assessment , including basic activities of daily living and instrumental activities of daily living , was carried out . the neuropsychological testing was performed by a standardized neuropsychological battery including mini - mental state examination , frontotemporal dementia - modified clinical dementia rating scale ( ftd - modified cdr ) , raven colored progressive matrices , controlled oral word association test and category fluency , clock drawing test , rey complex figure copy and recall , story recall test , digit span , trail making test a and b , token test , and de renzi imitation test . in addition , the aachener aphasie test was further administered to patients with ppa ( data not shown ) . the lvppa diagnosis was supported by the pattern of atrophy in posterior / presylvian or parietal regions at the mri examination ; -amyloid 42 and total tau csf determinations were also considered . the work conformed to the helsinki declaration and was approved by the local ethics committee of brescia hospital , italy . three single nucleotide polymorphisms located within kiaa0319/ttrap / them2 locus ( rs17243157 g / a ) , dcdc2 ( rs793842 a / g ) , and cntnap2 ( rs17236239 a / g ) genes were evaluated . the amplification protocols were designed as follows : 5 minutes at 95c for the first cycle , denaturation at 95c for 30 seconds , annealing ranging from 59c to 66c for 30 seconds ( depending on the analyzed polymorphism ) , extension at 72c for 30 seconds for the subsequent 35 cycles , and a final extension at 72c for 5 minutes . the pcr products were analyzed on 2% agarose with 0.005% of ethidium bromide to reveal the reaction and to verify their size . to evaluate rs17243157 and rs793842 polymorphisms , denaturing high performance liquid chromatography ( dhplc ) analysis on the wave nucleic acid fragment analysis system was performed ( transgenomic , santa clara , ca ) . samples with an altered dhplc profile were purified with microcon centrifugal filter devices ( amicon bioseparations ; millipore corp , billerica , ma ) and sequenced . nucleotide direct sequencing was performed on genomic dna for both strands by the abi 3500xl dna analyzer ( applied biosystems , foster city , ca ) and analyzed using chromas ( technelysium pty ltd , south brisbane , australia ) . to assess rs17236239 , direct sequencing was performed for both strands from purified pcr on the abi 3500 dna genetic analyzer ( applied biosystems ) , according to the manufacturer 's instructions , and analyzed using chromas ( technelysium pty ltd ) . in the present study , brain images were collected using 2 different mr scanners : ( 1 ) 1.5 t mr scanner ( siemens symphony , erlangen , germany ) , equipped with a circularly polarized transmit receive coil to acquire 3d magnetization - prepared rapid gradient echo ( mprage ) t1-weighted scan ( repetition time [ tr ] = 2,010 milliseconds , echo time [ te ] = 3.93 milliseconds , matrix = 1 1 1 , in - plane field of view [ fov ] = 250 250 mm , slice thickness = 1 mm , flip angle = 15 ) ; and ( 2 ) 1.5 t mr scanner ( siemens avanto ) to acquire 3d mprage t1-weighted scan ( tr = 2,050 milliseconds , te = 2.56 milliseconds , matrix = 1 1 1 , in - plane fov = 256 256 mm , slice thickness = 1 mm , flip angle = 15 ) . preprocessing and statistical analyses were performed using the statistical parametric mapping ( spm8 ) software package ( wellcome department of imaging neuroscience , london , england , http://www.fil.ion.ucl.ac.uk/spm ) running on matlab 7.1 ( mathworks , natick , ma ) . for voxel - based morphometry ( vbm ) images were spatially normalized to a reference stereotactic template ( montreal neurological institute [ mni ] ) and smoothed by a gaussian kernel of 10 10 10 mm full width at half maximum . gray matter ( gm ) and white matter ( wm ) differences according to genotype status for each polymorphism were evaluated by vbm analyses in all ftd groups ( behavioral variant of ftd [ bvftd ] and ppas together ) and in ppas , respectively . an analysis of covariance model design was applied by entering 4 groups , i.e. , ( 1 ) bvftd carrying at - risk genotype , ( 2 ) bvftd not carrying at - risk genotype , ( 3 ) ppa carrying at - risk genotype , and ( 4 ) ppa not carrying at - risk genotype , and the covariates of no interest , i.e. , age , sex , disease severity ( as measured by ftd - modified cdr ) , presence of monogenic disease , and scanner type . total intracranial volume , i.e. , gm + wm + csf , was introduced in all the statistical analyses as a global nuisance variable to avoid the confounding effect on cortical atrophy . correlation analysis between cognitive performances and regional gm and wm atrophy clusters was carried out . for this , the clusters of gm and wm atrophy obtained in the previous analysis ( comparison between the at - risk group and not - at - risk group ) were selected as regions of interest ( rois ) , and the mean density was calculated for each patient . this approach relies on the assumption that functionally correlated brain regions show a greater concordance in gm and wm volumes as a result of mutually trophic influences or common experience - related plasticity . for this , the clusters of gm and wm atrophy obtained in the previous analysis ( comparison between the at - risk group and not - at - risk group ) were used as seed regions , exploring the pattern of covariance between the gm volume of each seed and the gm volume throughout the whole brain . for all the aforementioned analyses , the threshold was established at p < 0.001 ( uncorrected for multiple comparisons ) and a cluster size threshold was set at 200 voxels . patients were analyzed according to clinical phenotype ( bvftd or ppa ) or genotype status for each evaluated polymorphism . a total of 118 patients fulfilled inclusion / exclusion criteria and entered the present study , namely 84 patients with bvftd and 34 with ppa ( 24 agrammatic variant ppa and 10 semantic variant ppa ) . no differences between bvftd and ppa were found , with the exception of sex and frontal behavioral inventory scores . demographic and clinical characteristics of patients with ftd no difference of genotype distribution and allele frequency of kiaa0319 ( rs17243157 g / a ) , dcdc2 ( rs793842 a / g ) , and cntnap2 ( rs17236239 a / g ) polymorphisms between bvftd and ppa was observed ( table e-2 ) . we evaluated the effect of studied functional polymorphisms on gm damage , i.e. , kiaa0319 ( gg vs ga / aa ) , dcdc2 ( gg vs ga / aa ) , and cntnap2 ( gg vs ga / aa ) , by vbm . as reported in figure 1a and table e-3 , patients carrying kiaa0319 a * ( ga or aa ) showed greater gm atrophy in the left inferior and middle temporal gyri as compared with kiaa0319 gg . ( a ) gray matter ( gm ) atrophy in patients with ftd carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( b ) mean scores obtained by plotting middle temporal gyrus values in the 4 considered groups . ( c ) correlation analysis between the gm volume of the middle temporal gyrus and semantic fluency . bvftd = behavioral variant of frontotemporal dementia ; ftd = frontotemporal dementia ; ppa = primary progressive aphasia . as shown in figure 1b , the main effect in the left temporal region was driven more by ppa than by bvftd . patients with ppa carrying kiaa0319 a * had the greatest damage in the left middle temporal gyrus compared with patients who had bvftd carrying kiaa0319 a * and those carrying the gg genotype . the same results were obtained when the left inferior temporal gyrus was considered ( data not shown ) . gm damage of the aforementioned atrophic regions was related to the decreasing semantic fluency scores ( r = 0.37 , p < 0.001 , figure 1c ) . the analysis of wm damage in patients with ftd grouped according to kiaa0319 genotype led to comparable results . kiaa0319 a * carriers showed greater wm involvement of the left middle temporal gyrus , as compared with kiaa0319 gg ( figure 2a and table e-3 ) . as for gm damage , this effect was mainly due to ppa kiaa0319 a * ( figure 2b ) . wm atrophy of the left middle temporal gyrus was related to the decreasing semantic fluency ( r = 0.34 , p < 0.001 ) and short story ( r = 0.38 , p < 0.001 ) scores ( figure 2c ) . ( a ) white matter atrophy in patients with ftd carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( b ) mean scores obtained by plotting middle temporal gyrus values in the 4 considered groups . ( c ) correlation analysis between the gray matter volume of the middle temporal gyrus and short story / semantic fluency . the results are superimposed on a 2d - standardized t1 brain template , p < 0.001 uncorrected , clusters > 200 voxels . bvftd = behavioral variant of frontotemporal dementia ; ftd = frontotemporal dementia ; ppa = primary progressive aphasia . the inverse comparisons ( greater gm / wm volume in gg < a * ) did not show any clusters above the pre - established threshold . no differences of gm and wm atrophy in patients with ftd grouped according to dcdc2 ( gg vs ga / aa ) and cntnap2 ( gg vs ga / aa ) genotypes were found at the pre - established threshold . as the effect of kiaa0319 polymorphism was mainly found in ppas , further analyses were carried out in this group . patients with ppa carrying kiaa0319 a * had greater involvement of both gm and wm in the left middle temporal gyrus as compared with kiaa0319 gg ( figure 3 and table e-3 ) . ( a ) gray matter ( gm ) atrophy in patients with primary progressive aphasia ( ppa ) carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( b ) correlation analysis between the gm volume of the middle temporal gyrus and semantic fluency / short story . ( c ) white matter ( wm ) atrophy in patients with ppa carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( d ) correlation analysis between the wm volume of the middle temporal gyrus and semantic fluency . structural correlation analysis to assess interhemispheric and intrahemispheric connectivity according to kiaa0319 genotype was carried out . in patients with ppa carrying at - risk kiaa0319 a * genotype , decreased structural gm correlation between the left middle temporal gyrus ( peak mni coordinates x , y , z : 56 , 2 , 20 ) and other both interhemispheric and intrahemispheric brain regions was highlighted , as compared with patients with ppa carrying kiaa0319 gg . the same pattern was seen for wm density ( figure 4 and table e-4 ) . pattern of gray matter ( a ) and white matter ( c ) structural correlation of the middle temporal gyrus with other regions of the brain in primary progressive aphasia ( ppa ) kiaa0319 a * vs ppa kiaa0319 gg . linear correlation of decreased gray matter ( b ) and white matter ( d ) structural association between brain regions in ppa kiaa0319 a * ( dashed lines ) vs ppa kiaa0319 gg ( continuous lines ) . a total of 118 patients fulfilled inclusion / exclusion criteria and entered the present study , namely 84 patients with bvftd and 34 with ppa ( 24 agrammatic variant ppa and 10 semantic variant ppa ) . no differences between bvftd and ppa were found , with the exception of sex and frontal behavioral inventory scores . demographic and clinical characteristics of patients with ftd no difference of genotype distribution and allele frequency of kiaa0319 ( rs17243157 g / a ) , dcdc2 ( rs793842 a / g ) , and cntnap2 ( rs17236239 a / g ) polymorphisms between bvftd and ppa was observed ( table e-2 ) . we evaluated the effect of studied functional polymorphisms on gm damage , i.e. , kiaa0319 ( gg vs ga / aa ) , dcdc2 ( gg vs ga / aa ) , and cntnap2 ( gg vs ga / aa ) , by vbm . as reported in figure 1a and table e-3 , patients carrying kiaa0319 a * ( ga or aa ) showed greater gm atrophy in the left inferior and middle temporal gyri as compared with kiaa0319 gg . ( a ) gray matter ( gm ) atrophy in patients with ftd carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( b ) mean scores obtained by plotting middle temporal gyrus values in the 4 considered groups . ( c ) correlation analysis between the gm volume of the middle temporal gyrus and semantic fluency . bvftd = behavioral variant of frontotemporal dementia ; ftd = frontotemporal dementia ; ppa = primary progressive aphasia . as shown in figure 1b , the main effect in the left temporal region patients with ppa carrying kiaa0319 a * had the greatest damage in the left middle temporal gyrus compared with patients who had bvftd carrying kiaa0319 a * and those carrying the gg genotype . the same results were obtained when the left inferior temporal gyrus was considered ( data not shown ) . gm damage of the aforementioned atrophic regions was related to the decreasing semantic fluency scores ( r = 0.37 , p < 0.001 , figure 1c ) . the analysis of wm damage in patients with ftd grouped according to kiaa0319 genotype led to comparable results . kiaa0319 a * carriers showed greater wm involvement of the left middle temporal gyrus , as compared with kiaa0319 gg ( figure 2a and table e-3 ) . as for gm damage , this effect was mainly due to ppa kiaa0319 a * ( figure 2b ) . wm atrophy of the left middle temporal gyrus was related to the decreasing semantic fluency ( r = 0.34 , p < 0.001 ) and short story ( r = 0.38 , p < 0.001 ) scores ( figure 2c ) . ( a ) white matter atrophy in patients with ftd carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( b ) mean scores obtained by plotting middle temporal gyrus values in the 4 considered groups . ( c ) correlation analysis between the gray matter volume of the middle temporal gyrus and short story / semantic fluency . the results are superimposed on a 2d - standardized t1 brain template , p < 0.001 uncorrected , clusters > 200 voxels . bvftd = behavioral variant of frontotemporal dementia ; ftd = frontotemporal dementia ; ppa = primary progressive aphasia . the inverse comparisons ( greater gm / wm volume in gg < a * ) did not show any clusters above the pre - established threshold . no differences of gm and wm atrophy in patients with ftd grouped according to dcdc2 ( gg vs ga / aa ) and cntnap2 ( gg vs ga / aa ) genotypes were found at the pre - established threshold . as the effect of kiaa0319 polymorphism was mainly found in ppas , further analyses were carried out in this group . patients with ppa carrying kiaa0319 a * had greater involvement of both gm and wm in the left middle temporal gyrus as compared with kiaa0319 gg ( figure 3 and table e-3 ) . ( a ) gray matter ( gm ) atrophy in patients with primary progressive aphasia ( ppa ) carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( b ) correlation analysis between the gm volume of the middle temporal gyrus and semantic fluency / short story . ( c ) white matter ( wm ) atrophy in patients with ppa carrying kiaa0319 a * vs kiaa0319 gg ( a*<gg ) . ( d ) correlation analysis between the wm volume of the middle temporal gyrus and semantic fluency . structural correlation analysis to assess interhemispheric and intrahemispheric connectivity according to kiaa0319 genotype was carried out . in patients with ppa carrying at - risk kiaa0319 a * genotype , decreased structural gm correlation between the left middle temporal gyrus ( peak mni coordinates x , y , z : 56 , 2 , 20 ) and other both interhemispheric and intrahemispheric brain regions was highlighted , as compared with patients with ppa carrying kiaa0319 gg . the same pattern was seen for wm density ( figure 4 and table e-4 ) . pattern of gray matter ( a ) and white matter ( c ) structural correlation of the middle temporal gyrus with other regions of the brain in primary progressive aphasia ( ppa ) kiaa0319 a * vs ppa kiaa0319 gg . linear correlation of decreased gray matter ( b ) and white matter ( d ) structural association between brain regions in ppa kiaa0319 a * ( dashed lines ) vs ppa kiaa0319 gg ( continuous lines ) . in the present study , we tested whether genetic variations in specific dyslexia - related genes were involved in modulating the clinical phenotypes in an extensively characterized cohort of patients with ftd . dyslexia is known to run in families and has been associated with a number of susceptibility genes . it might be hypothesized that in some cases , such genetic predisposition may interfere with initial development of language ; in others , the effect may remain dormant for decades but resurface as ppa or as ftd with language disturbances in patients with an ongoing neurodegenerative disease . this is even supported by the evidence that different neuropathologic mechanisms can cause ppa , and on the other hand , that an identical neuropathologic entity may lead to ppa , bvftd , or , in other cases , amnestic dementia . thus , susceptibility factors interact with the neurodegenerative process to determine the anatomical location . in our sample , the majority of patients with ppa were women , which suggests the presence of sex - related vulnerability , as already reported in previous studies in which a male predominance in the semantic subtype and female prevalence in the nonfluent subtype were observed . moreover , grn mutations were overrepresented in patients with language variants , which reflects a peculiar role of the grn gene in language networks . to further explore genetic susceptibility to language impairment in ftd , functional genetic variations within 3 genes involved in language disabilities , in particular kiaa0319 rs17243157 g / a , dcdc2 rs793842 a / g , and cntnap2 rs17236239 a / g , were analyzed with respect to their effect on brain structures and the correlation with cognitive performances . it is noteworthy that , of all 3 genes , only the kiaa0319 a * ( ga or aa ) genotype was demonstrated to influence brain damage in language areas , therefore supporting the specificity of the results . no specific patterns of greater atrophy were identified as associated with dcdc2 and cntnap2 polymorphisms , although we can not exclude the possibility that with a larger patient sample or different approaches , an effect of these genes could also be found in patients with ftd . kiaa0319 functional polymorphism was shown to determine gm atrophy in the left inferior and middle temporal gyri and wm involvement in the middle temporal gyrus and to be associated with the semantic fluency test and the short story scores . of note , these results were stronger in patients with ppa than in those with bvftd . furthermore , in patients with ppa carrying the at - risk kiaa0319 polymorphism , damage of the left temporal lobe showed an interhemispheric and intrahemispheric breakdown with the rest of the brain . the areas found to be affected by kiaa0319 genetic variation are known to be one of the hubs of phonological , articulatory planning , and syntactic processes and are most notably affected in patients with dyslexia . accordingly , several independent studies reported that kiaa0319 allelic variants were associated with different reading and spelling abilities , being able to reduce kiaa0319 gene expression , and affecting neuronal migration in the developing neocortex . in healthy controls , kiaa0319 polymorphism was shown to influence asymmetric functional activation of superior temporal sulcus , suggesting its role in modulation and lateralization of phonemic process . our results further contribute to understanding of the effect of kiaa0319 on language networks in patients with ftd and have major relevance for those who present prominent language deficits at disease onset . indeed , the results we reported are not necessarily about ppa caused by a specific neuropathologic substrate , and further studies could explore whether the dyslexia genes may also influence brain atrophy in other neurodegenerative conditions , including lvppa and alzheimer dementia . unfortunately , we have no reliable information on learning disabilities in these patients , in part because the diagnosis of learning disabilities is relatively recently recognized . these findings shed light on genetic predisposition in defining how a disease process becomes distributed asymmetrically and how it targets language networks . indeed , the identification of a possible genetic risk factor for language deficits in ftd represents a possible molecular target for the development of disease biomarkers or future therapeutic strategies . all authors made substantial contributions to conception and design , and/or acquisition of data , and/or analysis and interpretation of data . donata paternic : first draft of the manuscript , imaging analysis , and interpretation of data . antonella alberici : first draft of the manuscript , patient cohort , and analysis and interpretation of data . barbara borroni : first draft of the manuscript , patient cohort , analysis and interpretation of data , and study conceptualization and design .

objective : in this study , we evaluated whether variations within genes specifically associated with dyslexia , namely kiaa0319 , dcdc2 , and cntnap2 , were associated with greater damage of language - related regions in patients with frontotemporal dementia ( ftd ) and primary progressive aphasia ( ppa ) in particular.methods:a total of 118 patients with ftd , 84 with the behavioral variant of ftd ( bvftd ) and 34 with ppa , underwent neuropsychological examination , genetic analyses , and brain mri . kiaa0319 rs17243157 g / a , dcdc2 rs793842 a / g , and cntnap2 rs17236239 a / g genetic variations were assessed . patients were grouped according to clinical phenotype and genotype status ( ga / aa or gg ) . gray matter ( gm ) and white matter ( wm ) differences were assessed by voxel - based morphometry and structural intercorrelation pattern analyses.results:patients carrying kiaa0319 a * ( ga or aa ) showed greater gm and wm atrophy in the left middle and inferior temporal gyri , as compared with kiaa0319 gg ( p < 0.001 ) . the effect of kiaa0319 polymorphism was mainly reported in patients with ppa . in patients with ppa carrying at - risk polymorphism , temporal damage led to loss of interhemispheric and intrahemispheric gm and wm structural association . no effect of dcdc2 and cntnap2 was found.conclusions:genes involved in dyslexia susceptibility , such as kiaa0319 , result in language network vulnerability in ftd , and in ppa in particular .

METHODS Setting and participants. Standard protocol approvals, registrations, and patient consents. Genetic analyses. MRI data acquisition and preprocessing. MRI data analyses. Statistical analysis. RESULTS Patients. MRI analyses in patients with FTD. MRI analyses in PPAs. DISCUSSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

PMC5384618

ovarian cancer is the most lethal gynecologic malignancy among women , with an estimated 150 000 annual deaths worldwide . however , due to the unspecific and inconspicuous symptoms in the early stage of ovarian cancer , there are no effective and accurate detection methods for this disease . moreover , although the initial response to surgical resection and platinum - based chemotherapy is relatively high , most patients still have recurrence and chemo - resistance to ovarian cancer . as a result , diagnosis of most ovarian cancer cases occurs at advanced stages , which is associated with poor prognosis , as shown by the low 5-year overall survival rate . therefore , there is an urgent need for further investigation of potential markers for prognosis , mechanisms of ovarian carcinogenesis , and therapeutic targets for novel alternative treatments . obesity provides a unique adipose tissue microenvironment in which several cytokines , including adiponectin , are secreted exclusively by differentiated adipocytes . adiponectin is a 25-kda adipocytokine composed of 244 amino acids and involved in insulin sensitivity and lipid metabolism . in adults , recently , adiponectin has been reported to act as anti - tumor factor through inhibiting cancer cell growth , proliferation , and migration , as well as promoting apoptosis [ 1113 ] . binding its receptors by adiponectin could activate a variety downstream signaling pathways , such as 5-amp - activated protein kinase ( ampk ) and wnt signaling . on the other hand , recent studies reported that adipor1 is positively correlated with the risk of obesity - associated malignancies such as endometrial , pancreatic , renal , colon , and breast carcinomas [ 1619 ] . however , little is known about expression of adipor1 in epithelial ovarian cancer ( eoc ) and its pathological implications . in this study , we analyzed the expression of adipor1 in human cancerous epithelial ovarian tissues by immunohistochemical assay . the pathological features , progression - free survival ( pfs ) , and overall survival ( os ) were further analyzed to investigate the relationship between adipor1 expression and prognostic outcomes of patients with eoc . this was a retrospective study enrolled 73 patients diagnosed with eoc in the second affiliated hospital , dalian medical university from july 2008 to december 2014 . all participating patients were formally informed for the purpose of this study and a letter of consent was signed by every individual involved . all patients had been confirmed to have eoc and had received 46 rounds of postoperative platinum - based chemotherapy according to nccn guidelines . patients ages ranged from 26 to 77 years , and the average age was 59.9610.19 years . among all patients , 67 ( 91.78% ) had serous adenocarcinoma , 3 ( 4.11% ) had endometrioid adenocarcinoma , 2 ( 2.73% ) had clear cell adenocarcinoma , and 2 ( 2.73% ) had mucous adenocarcinoma . the 73 eoc patients were divided into 2 groups : the low - to - medium differentiation group ( 28 with low differentiation and 29 with low - to - medium differentiation ) and the medium - to - high differentiation group ( 14 with medium differentiation , 1 with medium - to - high differentiation , and 1 with high differentiation ) . the expression level of adipor1 in ovarian cancer tissues was analyzed by immunohistochemical staining . all surgically obtained specimens were preserved in the pathology center of the second affiliated hospital , dalian medical university for further analysis . samples were fixed in formalin , embedded in paraffin , and cut into 4-m sections . for antigen retrieval , tissue sections were treated in a microwave oven with citrate buffer solution ( ph 6.0 ) for 20 min and blocked with normal goat serum for 1 h. after rinsing in pbs 3 times , the slides were incubated for 1 h with anti - human adipor1 antibody ( ab126611 , 1 : 100 ; abcam , cambridge , uk ) as the primary antibody . a hrp - conjugated goat anti - rabbit igg h&l ( ab6721 , 1 : 500 ; abcam , cambridge , uk ) all immunohistochemical - stained slides were independently evaluated by 2 pathologists who were blinded to the patients medical records . positivity of the samples was determined by the presence of brown or tan staining within the cytoplasmic membrane and/or cytoplasm . sections that contained more than 5% stained cells were considered as adipor1-positive samples , otherwise slides were considered as negative samples . for the semi - quantitative analysis of immunohistochemical staining for adipor1 expression , a h - score based analysis was conducted , as previously described . all statistical analyses were performed using the spss 13.0 software package ( spss inc . , this was a retrospective study enrolled 73 patients diagnosed with eoc in the second affiliated hospital , dalian medical university from july 2008 to december 2014 . all participating patients were formally informed for the purpose of this study and a letter of consent was signed by every individual involved . all patients had been confirmed to have eoc and had received 46 rounds of postoperative platinum - based chemotherapy according to nccn guidelines . patients ages ranged from 26 to 77 years , and the average age was 59.9610.19 years . among all patients , 67 ( 91.78% ) had serous adenocarcinoma , 3 ( 4.11% ) had endometrioid adenocarcinoma , 2 ( 2.73% ) had clear cell adenocarcinoma , and 2 ( 2.73% ) had mucous adenocarcinoma . the 73 eoc patients were divided into 2 groups : the low - to - medium differentiation group ( 28 with low differentiation and 29 with low - to - medium differentiation ) and the medium - to - high differentiation group ( 14 with medium differentiation , 1 with medium - to - high differentiation , and 1 with high differentiation ) . all surgically obtained specimens were preserved in the pathology center of the second affiliated hospital , dalian medical university for further analysis . samples were fixed in formalin , embedded in paraffin , and cut into 4-m sections . for antigen retrieval , tissue sections were treated in a microwave oven with citrate buffer solution ( ph 6.0 ) for 20 min and blocked with normal goat serum for 1 h. after rinsing in pbs 3 times , the slides were incubated for 1 h with anti - human adipor1 antibody ( ab126611 , 1 : 100 ; abcam , cambridge , uk ) as the primary antibody . a hrp - conjugated goat anti - rabbit igg h&l ( ab6721 , 1 : 500 ; abcam , cambridge , uk ) all immunohistochemical - stained slides were independently evaluated by 2 pathologists who were blinded to the patients medical records . positivity of the samples was determined by the presence of brown or tan staining within the cytoplasmic membrane and/or cytoplasm . sections that contained more than 5% stained cells were considered as adipor1-positive samples , otherwise slides were considered as negative samples . for the semi - quantitative analysis of immunohistochemical staining for adipor1 expression , a h - score based analysis was conducted , as previously described . all statistical analyses were performed using the spss 13.0 software package ( spss inc . , chicago , il ) . out of the 15 normal ovarian tissue samples , adipor1expression was observed in 11 samples ( 73.33% ) . in the 73 eoc patients , the expression of adipor1 the frequency of adipor1 expression in normal ovarian tissues was higher than in eoc tissues ; however , no significant difference between the 2 groups was observed ( p=0.966 ) ( table 1 , figure 1 ) . two independent - samples nonparametric tests indicated that expression of adipor1 was negatively correlated with patient classification as being in the terminal stage of disease according to the international federation of gynecology and obstetrics ( figo ) stages and degree of differentiation of ovarian tissue ( p<0.05 ) . however , adipor1 expression was not significantly associated with other pathologic features , including pathological types , ascites during initial manifestation , diabetes , preoperative or postoperative body mass index ( bmi ) , platelet count , or pfi of less than 6 months ( table 2 ) . univariate analysis indicated the median pfs of patients with positive expression of adipor1 was significantly higher than in patients with adipor1-negative expression ( 26.6 months vs. 7.4 months , p<0.001 ) . the median pfs of patients within stage i to ii and stage iii to iv was 31.0 months and 12.1 months , respectively , and the difference was statistically significant ( p<0.001 ) . moreover , significant differences were observed in median pfs of patients with vs. without ascites ( 10.3 months vs. 33.2 months , p<0.001 ) , presenting of diabetes ( 10.3 months in the diabetes group vs. 17.6 months in the non - diabetes group , p=0.019 ) , and platelet count ( 11.9 months in patients with platelet count higher than 35010/l vs. 26.6 months in patients with platelet count less than 35010/l , p=0.026 ) . other clinicopathological feathers , including different pathological types , tissue differentiation degree , and preoperative and postoperative bmi , were not associated with pfs in univariate analysis ( p>0.05 , all ) ( table 3 , figure 2 ) . cox proportional hazards regression modeling was used to evaluate the effects of various clinicopathological characteristics , including adipor1 expression , figo stage , ascites , diabetes , and platelet count on pfs of patients with eoc . the multivariate analyses identified adipor1 expression as an independent positive prognostic indicator of pfs . other pathologic features , such as figo stages and ascites , also had a significant effect on pfs ( table 4 ) . our data indicated that adipor1 expression , figo stage , ascites , and diabetes were correlated with os of eoc patients ( table 5 ) . the median os of adipor1-positive eoc patients was 60.2 months , which was significantly higher than the 23.5 months found in the adipor1-negative group ( p<0.001 ) . moreover , the median os of patients with ascites as initial manifestation was 29.7 months , which was significantly shorter than in patients without ascites ( 60.2 months ) . furthermore , presenting of diabetes also reduced the os from 57.7 months to 29.7 months ( p=0.022 ) . however , other clinicopathological variables , including different pathological types , tissue differentiation degree , preoperative and postoperative bmi , and platelet count , had no correlation with os of patients with eoc in univariate analysis ( p>0.05 , all ) ( figure 3 , table 6 ) . taken together , the multivariate analyses revealed that adipor1 expression could be an independent positive prognostic indicator of os of eoc patients ( table 6 ) . out of the 15 normal ovarian tissue samples , adipor1expression was observed in 11 samples ( 73.33% ) . in the 73 eoc patients , the expression of adipor1 the frequency of adipor1 expression in normal ovarian tissues was higher than in eoc tissues ; however , no significant difference between the 2 groups was observed ( p=0.966 ) ( table 1 , figure 1 ) . two independent - samples nonparametric tests indicated that expression of adipor1 was negatively correlated with patient classification as being in the terminal stage of disease according to the international federation of gynecology and obstetrics ( figo ) stages and degree of differentiation of ovarian tissue ( p<0.05 ) . however , adipor1 expression was not significantly associated with other pathologic features , including pathological types , ascites during initial manifestation , diabetes , preoperative or postoperative body mass index ( bmi ) , platelet count , or pfi of less than 6 months ( table 2 ) . univariate analysis indicated the median pfs of patients with positive expression of adipor1 was significantly higher than in patients with adipor1-negative expression ( 26.6 months vs. 7.4 months , p<0.001 ) . the median pfs of patients within stage i to ii and stage iii to iv was 31.0 months and 12.1 months , respectively , and the difference was statistically significant ( p<0.001 ) . moreover , significant differences were observed in median pfs of patients with vs. without ascites ( 10.3 months vs. 33.2 months , p<0.001 ) , presenting of diabetes ( 10.3 months in the diabetes group vs. 17.6 months in the non - diabetes group , p=0.019 ) , and platelet count ( 11.9 months in patients with platelet count higher than 35010/l vs. 26.6 months in patients with platelet count less than 35010/l , p=0.026 ) . other clinicopathological feathers , including different pathological types , tissue differentiation degree , and preoperative and postoperative bmi , were not associated with pfs in univariate analysis ( p>0.05 , all ) ( table 3 , figure 2 ) . cox proportional hazards regression modeling was used to evaluate the effects of various clinicopathological characteristics , including adipor1 expression , figo stage , ascites , diabetes , and platelet count on pfs of patients with eoc . other pathologic features , such as figo stages and ascites , also had a significant effect on pfs ( table 4 ) . our data indicated that adipor1 expression , figo stage , ascites , and diabetes were correlated with os of eoc patients ( table 5 ) . the median os of adipor1-positive eoc patients was 60.2 months , which was significantly higher than the 23.5 months found in the adipor1-negative group ( p<0.001 ) . moreover , the median os of patients with ascites as initial manifestation was 29.7 months , which was significantly shorter than in patients without ascites ( 60.2 months ) . furthermore , presenting of diabetes also reduced the os from 57.7 months to 29.7 months ( p=0.022 ) . however , other clinicopathological variables , including different pathological types , tissue differentiation degree , preoperative and postoperative bmi , and platelet count , had no correlation with os of patients with eoc in univariate analysis ( p>0.05 , all ) ( figure 3 , table 6 ) . taken together , the multivariate analyses revealed that adipor1 expression could be an independent positive prognostic indicator of os of eoc patients ( table 6 ) . obesity is currently regarded as a risk factor for ovarian cancer , but it is still unclear how obesity contributes to ovarian carcinogenesis . adiponectin , a protein hormone that regulates many metabolic processes , has been recently reported to be a crucial mediator during the progression of several malignancies associated with obesity , and adiponectin receptor 1 ( adipor1 ) expression in several cancers has been found to be associated with favorable prognosis for patients . however , the role of adipor1 in carcinogenesis is controversial because expression level of adipor1 varies greatly among different cancers . compared to normal tissue samples , there was a significant increase in the expression of adipor1 in pancreatic cancer and oesophageal adenocarcinoma ; in contrast , the percentage of adipor1-positive cells was significantly lower in endometrial carcinoma . moreover , in a recent study , the mrna levels of adiponectin and adipor1 were significantly lower in cancerous ovaries than in normal ovaries , which is consistent with our observation . in our study , adipor1 immunostaining tended to be less intense in cancerous epithelial ovarian tissues compared with normal ovarian tissue , but the difference was not significant , suggesting that adipor1 is involved in the carcinogenesis and progression of ovarian cancer . a previous report suggested that adiponectin and its receptors can decrease neoplasm metastasis and repress angiogenesis through activation of mitogen - activated protein kinase ( mapk ) . an in vitro study based on 6 gastric cancer cell lines showed that adiponectin treatment can induce apoptosis and inhibit the proliferation of gastric cancer cell lines . moreover , sirna knock - down of adipor1 suppressed the growth inhibitory effects of adiponectin in gastric cancer cell lines . taken together , these data show the potential role adipor1 plays in the development and prognosis of cancer , which may also apply to ovarian carcinogenesis . tiwari et al . reported that the expression of adiponectin and adipor1 was detectable in eoc tissues and ovarian cancer - derived cell lines . to investigate the relationship between adipor1 and ovarian cancer , we evaluated the correlation between adipor1 expression and clinicopathological features in ovarian cancer patients . advance figo stage , ascites , and short platinum - free intervals were confirmed to be correlated with adipor1 expression in eoc patients . a retrospective study revealed that figo stage is one of the most reliable predictors of the prognosis of patients with eoc . our data showed that adipor1-positive staining in eoc tissues was negatively correlated with advanced figo stage in eoc patients , which is in agreement with previous reports . moreover , data from colorectal cancer also showed that adipor1expresstion was negatively associated with tumor grade , and patients with negative expression of adipor1 were shown to be more prone to occurrence of extrathyroidal invasion and multicentricity in thyroid cancer . presentation of ascites is frequently associated with carcinogenesis of ovarian cancer at the end - stage of disease , and affects the progression of neoangiogenesis . however , we found no correlation between adipor1 expression and ascites in our study , indicating that expression of adipor1 is of great importance in delaying carcinogenesis in eoc patients . however , the details of the mechanisms involved need further investigation in larger prospective studies . recent studies indicated that adipor1-positive expression significantly contributed to improvement of prognosis in different cancers [ 3032 ] . accordingly , adipor1 was proposed as a potential quantitative biomarker for clinical outcomes of eoc patients . moreover , due to the important role adipor1 plays in mediating the carcinogenesis and progression of eoc , it might be as a promising anti - tumor therapeutic target . more detailed research on the cellular and molecular functions of adipor1 in eoc is needed . moreover , in survival analysis , eoc patients with adipor1-positive expression had significantly longer survival than those with adipor1-negative expression . accumulating data demonstrate that pfi is a reliable predictor of response and survival after platinum - based chemotherapy for patients with recurrent carcinomas . patients with more than 12 months of pfi had significantly longer pfs and os compared to patients with less than 6 months of pfi . however , no significant correlation between adipor1 expression and pfi was observed in patients in our study . on the other hand , the enrolled patients had disproportional representation of cancer types and figo stages : there were fewer cases of low - to - medium vs. medium - to - high differentiation and figo stages i ii vs. iii iv cases , which is a limitation of our study . it was reported that epithelial ovarian cancers account for 8590% of ovarian cancers , with a subset of epithelial ovarian cancers and high - grade serous ovarian cancers ( hgsocs ) representing nearly 70% of all ovarian cancer cases . moreover , most patients are not diagnosed until the disease is at an advanced stage . therefore , there were more serous ovarian cancers and figo iii iv cases in our study . to further confirm our observation , a large - scale investigation with equal distribution of cases based on differentiation and figo stage is needed . our study results suggest that the expression of adipor1 is an independent prognostic indicator in patients with eoc and is associated with longer pfs and os .

backgroundadiponectin receptor-1 ( adipor1 ) has been reported to be associated with the risk of obesity - associated malignancies , including epithelial ovarian cancer ( eoc ) . the aim of this study was to determine if adipor1 could serve as a prognosis indicator for patients with eoc.material/methodsin this study , expression of adipor1 in 73 eoc patients consecutively admitted to our hospital was detected by immunohistochemical staining . univariate and multivariate analyses were performed to assess the relationship between adipor1 expression level and progression - free survival ( pfs ) and overall survival ( os ) rates in patients.resultsa relatively lower expression of adipor1 in the cancerous tissues was detected compared to normal ovarian tissues , but the difference was not significant ( p>0.05 ) . adipor1 expression level in eoc patients was negatively correlated with advanced figo stages in patients and tumor differentiation , but had no correlation with pathological types , presenting of ascites , shorter platinum - free interval ( pfi ) , diabetes , preoperative and postoperative body mass index ( bmi ) , or platelet counts ( p>0.05 ) . moreover , patients with adipor1 expression had a significantly longer pfs and os compared to the negative expression group ( p<0.001).conclusionsour findings suggest that adipor1 expression level in cancerous tissues might serve as an independent prognostic indicator in eoc patients and is associated with longer pfs and os .

Background Material and Methods Ethics statement and general information on patients Immunohistochemical staining Analysis of immunohistochemical staining Statistical analysis Result AdipoR1 expression in cancerous ovarian tissues Correlation between AdipoR1 expression and clinicopathological features Correlation between AdipoR1 expression and progression-free survival (PFS) Correlation between AdipoR1 expression and overall survival (OS) Discussion Conclusions

PMC4608087

we developed and followed a standardized protocol to do this meta - analysis in accordance with the preferred reporting items for systematic reviews and meta - analyses ( prisma ) guidelines.28 two investigators ( x.l . independently conducted literature searches of medline , embase , and the cochrane central register of controlled trials published from january 1965 ( index date ) to march 2014 , using keywords and medical subject headings ( table1 ) . all relevant studies and review articles ( including meta - analysis ) and the reference lists of the identified articles were checked manually . institutional review board approval is not applicable because the current study is a systematic review and meta - analysis , which is not considered research involving human subjects . search strategy for medline articles were included ( 1 ) if the study was an rct that assigned at least 1 group of participants to exercise training and 1 group to control and ( 2 ) if crf ( absolute and relative maximal oxygen uptake ) or circulating cvd biomarkers of lipid and lipoprotein metabolism , glucose intolerance and insulin resistance , systemic inflammation , or hemostasis were measured at baseline and at the end of the trial . all abstracts about rcts reporting the effect of exercise training on cvd - related biomarkers or crf we excluded studies ( 1 ) if the study design was not a rct ; ( 2 ) if the exercise intervention was acute ( 1 week ) , because we are interested in the effects of exercise interventions of moderate to long duration ; ( 3 ) if interventions were based on education or counseling rather than a structured exercise training assignment ; ( 4 ) if maximal oxygen consumption , or vo2max , was indirectly calculated through heart rate or fixed time testing and no other biomarkers of interest to this study were reported ; ( 5 ) if levels of circulating biomarkers were not directly measured ; ( 6 ) if values of outcome measures at the end of trials were not reported ; ( 7 ) if participants had severe chronic diseases ( preexisting cvd , liver or kidney diseases , or cancers ) , any other conditions that could potentially compromise participants capacity to exercise ( disability , frailty , declined activities of daily living , or wheelchair dependency ) , or any mental conditions ( depression , anxiety , schizophrenia , bipolar disorder , parkinson s disease , or alzheimer s disease ) ; ( 8) if participants were identified as trained professionals , athletes , or soldiers ; ( 9 ) if participants were infants , children , or adolescents ; or ( 10 ) if participants were pregnant , postpartum , nursing , had recent surgery , or were undergoing rehabilitation exercise . if multiple articles were published based on the same trial , data were retrieved as 1 independent trial . if there were duplicate results from the same trial , the most updated and comprehensive ones were extracted . in total the following information was extracted from all eligible studies : general information ( first author s name , article title , and country of origin ) , study characteristics ( study design , eligibility criteria , randomization , blinding , cointervention , dropout rate , and reason for dropping out ) , participant characteristics ( age , sex , ethnicity , body mass index , life style , health status , and number of participants in each group ) , intervention and setting ( exercise type , duration , intensity , and supervision ) , and outcome measures ( definition of outcomes , statistical techniques , pre- and postintervention means , standard deviation , sample size of each arm , and adverse events ) . maximal oxygen uptake vo2max was measured directly and determined based on the highest vo2 obtained prior to volitional fatigue . in this meta - analysis , we focused on biomarkers in blood samples , including plasma , serum , and whole blood . all samples for fasting glucose and insulin measurement in the studies were collected after > 10 hours of fasting . ) using the cochrane collaboration s tool for assessing risk of bias.29 this included random sequence generation , allocation concealment , blinding of participants and personnel , blinding of outcome assessment , incomplete outcome data , selective reporting , and other sources of bias . for each trial , the risk of bias was reported as low risk , unclear risk , or high risk . the criteria for classifying exercise interventions as moderate exercise or vigorous exercise are summarized in table2 . if the intensity measures were not reported in individual studies , maximum heart rate , maximum heart rate percentage , speed of running , metabolic equivalent , oxygen uptake , or relative metabolic rate were used to classify exercise intensity . to maintain independence , the most vigorous intervention and the control group in each trial were included in the primary analysis if multiple training groups of different intensities were compared with a single control group . sensitivity analyses were performed by conducting separate analyses of all eligible comparisons for moderate and vigorous exercise interventions , respectively . criteria used for exercise intensity classification mean levels and standard deviations of crf and cvd biomarkers after the exercise interventions from individual trials were used to calculate weighted mean differences ( wmds ) and 95% cis using dersimonian and laird random - effects models.30 between - study heterogeneity was examined using q statistics and i statistics.31,32 i 25% , 50% , and 75% is suggestive , respectively , of low , medium , and high heterogeneity . egger s tests were used to formally test publication bias.33 if there was any evidence of publication bias , the trim and fill method was used to evaluate the impact of publication bias.34 all eligible trials were analyzed in subgroup analyses conducted within the strata of the predetermined potential modifiers , including age ( mean or median < 50 versus 50 years ) , sex ( women versus men ) , body mass index ( obese versus nonobese ) , lifestyle ( active versus sedentary ) , health status ( having at least 1 of the following comorbidities : type 2 diabetes , hypertension , hyperlipidemia , and metabolic syndrome versus none ) , and trial duration ( 16 versus < 16 weeks ) . obesity was defined as body mass index 30 kg / m . two - sided p0.05 was used as the significance level except for the q statistic and the egger s tests ( p=0.10).35 all statistical analyses were performed with stata statistical software version 12 ( stata corp ) . we developed and followed a standardized protocol to do this meta - analysis in accordance with the preferred reporting items for systematic reviews and meta - analyses ( prisma ) guidelines.28 two investigators ( x.l . independently conducted literature searches of medline , embase , and the cochrane central register of controlled trials published from january 1965 ( index date ) to march 2014 , using keywords and medical subject headings ( table1 ) . all relevant studies and review articles ( including meta - analysis ) and the reference lists of the identified articles were checked manually . institutional review board approval is not applicable because the current study is a systematic review and meta - analysis , which is not considered research involving human subjects . articles were included ( 1 ) if the study was an rct that assigned at least 1 group of participants to exercise training and 1 group to control and ( 2 ) if crf ( absolute and relative maximal oxygen uptake ) or circulating cvd biomarkers of lipid and lipoprotein metabolism , glucose intolerance and insulin resistance , systemic inflammation , or hemostasis were measured at baseline and at the end of the trial . all abstracts about rcts reporting the effect of exercise training on cvd - related biomarkers or crf we excluded studies ( 1 ) if the study design was not a rct ; ( 2 ) if the exercise intervention was acute ( 1 week ) , because we are interested in the effects of exercise interventions of moderate to long duration ; ( 3 ) if interventions were based on education or counseling rather than a structured exercise training assignment ; ( 4 ) if maximal oxygen consumption , or vo2max , was indirectly calculated through heart rate or fixed time testing and no other biomarkers of interest to this study were reported ; ( 5 ) if levels of circulating biomarkers were not directly measured ; ( 6 ) if values of outcome measures at the end of trials were not reported ; ( 7 ) if participants had severe chronic diseases ( preexisting cvd , liver or kidney diseases , or cancers ) , any other conditions that could potentially compromise participants capacity to exercise ( disability , frailty , declined activities of daily living , or wheelchair dependency ) , or any mental conditions ( depression , anxiety , schizophrenia , bipolar disorder , parkinson s disease , or alzheimer s disease ) ; ( 8) if participants were identified as trained professionals , athletes , or soldiers ; ( 9 ) if participants were infants , children , or adolescents ; or ( 10 ) if participants were pregnant , postpartum , nursing , had recent surgery , or were undergoing rehabilitation exercise . if multiple articles were published based on the same trial , data were retrieved as 1 independent trial . if there were duplicate results from the same trial , the most updated and comprehensive ones were extracted . the following information was extracted from all eligible studies : general information ( first author s name , article title , and country of origin ) , study characteristics ( study design , eligibility criteria , randomization , blinding , cointervention , dropout rate , and reason for dropping out ) , participant characteristics ( age , sex , ethnicity , body mass index , life style , health status , and number of participants in each group ) , intervention and setting ( exercise type , duration , intensity , and supervision ) , and outcome measures ( definition of outcomes , statistical techniques , pre- and postintervention means , standard deviation , sample size of each arm , and adverse events ) . maximal oxygen uptake vo2max was measured directly and determined based on the highest vo2 obtained prior to volitional fatigue . in this meta - analysis , we focused on biomarkers in blood samples , including plasma , serum , and whole blood . all samples for fasting glucose and insulin measurement in the studies were collected after > 10 hours of fasting . methodological quality was assessed by 2 investigators ( x.l . , x.z . ) using the cochrane collaboration s tool for assessing risk of bias.29 this included random sequence generation , allocation concealment , blinding of participants and personnel , blinding of outcome assessment , incomplete outcome data , selective reporting , and other sources of bias . for each trial , the risk of bias was reported as low risk , unclear risk , or high risk . the criteria for classifying exercise interventions as moderate exercise or vigorous exercise are summarized in table2 . if the intensity measures were not reported in individual studies , maximum heart rate , maximum heart rate percentage , speed of running , metabolic equivalent , oxygen uptake , or relative metabolic rate were used to classify exercise intensity . to maintain independence , the most vigorous intervention and the control group in each trial were included in the primary analysis if multiple training groups of different intensities were compared with a single control group . sensitivity analyses were performed by conducting separate analyses of all eligible comparisons for moderate and vigorous exercise interventions , respectively . criteria used for exercise intensity classification mean levels and standard deviations of crf and cvd biomarkers after the exercise interventions from individual trials were used to calculate weighted mean differences ( wmds ) and 95% cis using dersimonian and laird random - effects models.30 between - study heterogeneity was examined using q statistics and i statistics.31,32 i 25% , 50% , and 75% is suggestive , respectively , of low , medium , and high heterogeneity . egger s tests were used to formally test publication bias.33 if there was any evidence of publication bias , the trim and fill method was used to evaluate the impact of publication bias.34 all eligible trials were analyzed in subgroup analyses conducted within the strata of the predetermined potential modifiers , including age ( mean or median < 50 versus 50 years ) , sex ( women versus men ) , body mass index ( obese versus nonobese ) , lifestyle ( active versus sedentary ) , health status ( having at least 1 of the following comorbidities : type 2 diabetes , hypertension , hyperlipidemia , and metabolic syndrome versus none ) , and trial duration ( 16 versus < 16 weeks ) . two - sided p0.05 was used as the significance level except for the q statistic and the egger s tests ( p=0.10).35 all statistical analyses were performed with stata statistical software version 12 ( stata corp ) . a total of 7487 participants aged between 18 and 90 years , from 169 articles based on 160 rcts , were included in the meta - analysis . characteristics of eligible studies are summarized in table3 . among all participants , 4276 ( 57.1% ) were women ; 3211 ( 42.9% ) were men ; 5845 ( 78.1% ) were free of type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome ; and 1640 ( 21.9% ) had at least 1 of those conditions . the median duration of trials was 12 weeks ( range : 2 weeks to 2 years ) . characteristics of the trials included in the meta - analysis apo ai indicates apolipoprotein a1 ; apo aii , apolipoprotein a2 ; apo b , apolipoprotein b ; bmi , body mass index ; c , control group ; crf , cardiorespiratory fitness ; crp , c - reactive protein ; ffa , free fatty acid ; hba1c , glycosylated hemoglobin a1c ; hdl - c , high - density lipoprotein cholesterol ; homa - ir , homeostatic model assessment insulin resistance ; icam-1 , intercellular adhesion molecule 1 ; igf-1 , insulin - like growth factor 1 ; igf - bp , insulin - like growth factor binding protein ; il , interleukin ; ldl - c , low - density lipoprotein cholesterol ; nr , not reported ; pai-1 , plasminogen activator inhibitor-1 ; t , training group ; tc , total cholesterol ; tnf , tumor necrosis factor ; vcam-1 , vascular cell adhesion molecule 1 ; vldl - c , very low - density lipoprotein cholesterol . summary of study selection process . in total , 6135 articles were retrieved from the literature search that evaluated the effect of exercise interventions on crf or cardiometabolic biomarkers . after exclusion , 160 rcts reported in 169 articles were included in the meta - analysis . apo ai indicates apolipoprotein a1 ; apo aii , apolipoprotein a2 ; apo b , apolipoprotein b ; crf , cardiorespiratory fitness ; crp , c - reactive protein ; ffa , free fatty acid ; hba1c , glycosylated hemoglobin a1c ; hdl - c , high - density lipoprotein cholesterol ; home - b , homeostatic model assessment - beta cell function ; homa - ir , homeostatic model assessment insulin resistance ; homa - s , homeostatic model assessment - insulin sensitivity ; icam-1 , intercellular adhesion molecule 1 ; igf-1 , insulin - like growth factor 1 ; igf - bp , insulin - like growth factor binding protein ; il , interleukin ; ldl - c , low - density lipoprotein cholesterol ; lp(a ) , lipoprotein(a ) ; pai-1 , plasminogen activator inhibitor-1 ; rcts , randomized controlled trials ; tc , total cholesterol ; tg , triglycerides ; tnf- , tumor necrosis factor ; vcam-1 , vascular cell adhesion molecule 1 ; vldl - c , very low - density lipoprotein cholesterol . random sequence generation was reported in 50 trials , and allocation concealment was reported in 20 trials ; only 1 of these trials showed a high probability of selection bias because the random allocation was not concealed . the risk of potential performance bias was high in all trials because it was not possible to blind participants and trainers in exercise interventions . among 26 trials reporting the blinding of outcome assessment , the risk of detection bias was high in only 1 trial . the risk of other bias was high in 46 trials because of poor compliance , the use of intention - to - treat analysis , limited sample sizes , or limitations discussed in individual articles . a total of 67 and 123 independent comparisons were included in the primary analysis for absolute and relative crf , respectively ( table4 ) . both measures were significantly raised by exercise interventions ( both p<0.001 ) . the wmds comparing exercise groups and control groups were 0.28 l / min ( 95% ci 0.23 to 0.33 ; i=93.7% ; p<0.001 for heterogeneity ) for absolute crf and 3.90 ml / kg per minute ( 95% ci 3.45 to 4.35 ; i=91.4% ; p<0.001 for heterogeneity ) for relative crf . the egger s tests showed evidence of publication bias in both instances ( p<0.05 ) . when applying the trim and fill method , the conclusion regarding the associations between exercise training and crf did not change ( filled analysis for absolute crf : wmd 0.14 l / min , 95% ci 0.20 to 5.28 , p<0.001 ; filled analysis for relative crf : wmd 2.56 ml / kg per minute , 95% ci 3.06 to 10.16 , p<0.001 ) . wmds in cardiorespiratory fitness and circulating concentrations of biomarkers between exercise groups and control groups apo ai indicates apolipoprotein a1 ; apo aii , apolipoprotein a2 ; apo b , apolipoprotein b ; crp , c - reactive protein ; ffa , free fatty acid ; hba1c , glycosylated hemoglobin a1c ; hdl - c , high - density lipoprotein cholesterol ; homa - ir , homeostatic model assessment insulin resistance ; igf-1 , insulin - like growth factor 1 ; igf - bp3 , insulin - like growth factor binding protein 3 ; il , interleukin ; ldl - c , low - density lipoprotein cholesterol ; tc , total cholesterol ; tg , triglycerides ; tnf- , tumor necrosis factor ; vldl - c , very low - density lipoprotein cholesterol ; wmds , weighted mean differences . number of eligible independent comparisons . exercise training significantly lowered the levels of triglycerides ( p=0.02 ) and increased the levels of high - density lipoprotein cholesterol ( hdl - c ; p<0.001 ) and apolipoprotein a1 ( p<0.001 ) . the wmds were 5.31 mg / dl ( 95% ci 10.63 to 0.89 ; i=71.8% ; p<0.001 for heterogeneity ) for triglycerides , 2.32 mg / dl ( 95% ci 1.16 to 3.87 ; i=87.5% ; p<0.001 for heterogeneity ) for hdl - c , and 0.03 g / l ( 95% ci 0.02 to 0.04 ; i=0.0% ; p=0.81 for heterogeneity ) for apolipoprotein a1 . the p value of the egger s test for hdl - c was 0.03 , suggesting possible publication bias ; however , the results from the trim and fill analysis did not show substantial impact of publication bias on the estimates or the statistics ( filled analysis : wmd 2.32 mg / dl , 95% ci 1.16 to 3.87 , p<0.001 ) . significant associations were found for interleukin-18 ( wmd 18.3 pg / ml ; 95% ci 0.10 to 36.6 ; i=0.0% ; p=0.95 for heterogeneity ) but not for c - reactive protein , interleukin-6 , or tumor necrosis factor in the primary analysis ( table4 ) . although there was no effect on adiponectin , exercise training was significantly associated with reduced levels of leptin ( wmd 2.72 ng / ml ; 95% ci 4.03 to 1.42 ; i=82.10% ; p<0.001 for heterogeneity ) ( table4 ) . table4 also shows the effects of exercise training on markers of glucose intolerance and insulin resistance . fasting insulin levels ; homeostatic model assessment insulin resistance , or homa - ir ; and glycosylated hemoglobin a1c were significantly lowered in exercise groups compared with control groups ( p=0.002 , p=0.002 , and p<0.001 ) ( table4 ) . the wmds between exercise groups and control groups were 1.03 iu / ml ( 95% ci 1.69 to 0.37 ; i=79.8% ; p<0.001 for heterogeneity ) for fasting insulin . the wmd for homa - ir was 0.30 ( 95% ci 0.49 to 0.11 ; i=77.5% ; p<0.001 for heterogeneity ) , whereas the wmd for hemoglobin a1c was 0.28% ( 95% ci 0.42 to 0.14 ; i=80.1% ; p<0.001 for heterogeneity ) . the egger s tests for fasting glucose and insulin were not suggestive of substantial publication bias ( p=0.18 and p=0.24 , respectively ) . the results from the trim and fill analysis suggested that there was no substantial impact of publication bias on the results for homa - ir or hemoglobin a1c ( filled analysis for homa - ir : wmd 0.30 , 95% ci 0.49 to 0.11 , p=0.002 ; filled analysis for hemoglobin a1c : wmd 0.28% , 95% ci 0.42 to 0.14 , p<0.001 ) . the primary analysis examined 3 hemostatic factors : fibrinogen , endothelin-1 , and angiotensin ii ( table4 ) . on average , the levels of fibrinogen and angiotensin ii were 0.39 g / l ( 95% ci 0.03 to 0.75 ; i=45.00% ; p=0.18 for heterogeneity ) and 1.32 pg / ml ( 95% ci 0.54 to 2.11 ; i=0.00% ; p=0.71 for heterogeneity ) lower in exercise groups than in control groups . our metaregression results suggest that the differences in crf between exercise and control groups were modified by age and sex ( absolute crf : p=0.008 and p<0.001 for age and sex , respectively ; relative crf : p=0.003 and p=0.001 for age and sex , respectively ) ( table5 , figure3 ) . in addition , the effects of exercise on levels of total cholesterol ( p=0.04 ) , low - density lipoprotein cholesterol ( ldl - c ; p=0.06 ) , and fasting insulin ( p=0.05 ) were modified by the presence of at least 1 of the following comorbidities : type 2 diabetes , hypertension , hyperlipidemia , and metabolic syndrome ( tables6 and 7 , figure3 ) . sex differences in the effects of exercise were also found for fasting insulin ( p=0.04 ) . wmds in absolute and relative cardiorespiratory fitness comparing exercise intervention groups to control groups by specific modifiers bmi indicates body mass index ; crf , cardiorespiratory fitness ; wmds , weighted mean differences , p values for the impact of potential modifiers on the exercise effects . health status : participants having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome ( yes ) vs those with none of them ( none ) . wmds in lipid biomarkers comparing exercise intervention and control groups by specific modifiers bmi indicates body mass index ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol ; wmds , weighted mean differences . health status : participants having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome ( yes ) vs those with none of them ( none ) . wmds in biomarkers of glucose intolerance and insulin resistance comparing exercise intervention groups to control groups by specific modifiers bmi indicates body mass index ; na , not available due to the lack of comparisons reported for active participants ; wmds , weighted mean differences . health status : participants having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome ( yes ) vs those with none of them ( none ) . forest plot of effects of exercise interventions on cardiorespiratory fitness , tc , tg , hdl - c , ldl - c , fasting glucose , and fasting insulin within subgroups . the wmds ( diamonds ) and corresponding cis ( extended line ) between exercise groups and control groups are shown for each subgroup . crf indicates absolute cardiorespiratory fitness ; bmi , body mass index ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol ; rel . crf , relative cardiorespiratory fitness ; tc , total cholesterol ; tg , triglycerides ; wmds , weighted mean differences . after conducting metaregressions , analyses within subgroups were performed . consistent with the metaregression results , men seemed to have greater exercise - related improvement in crf , ldl - c , and fasting insulin than women did ( figure3 ) . exercise interventions appreciably improved the levels of total cholesterol , ldl - c , and fasting insulin ( p=0.004 , p=0.01 , and p=0.01 , respectively ) in people having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , and metabolic syndrome ( tables6 and 7 , figure3 ) ; no such improvements were observed among people without any of those health conditions ( p=0.44 , p=0.19 , and p=0.13 , respectively ) ( tables6 and 7 , figure3 ) . in light of the potential impact of exercise intensity , we conducted separate analyses of all eligible comparisons for moderate and vigorous exercise interventions , respectively . the 95% cis for moderate and vigorous interventions overlapped for both crf measures and for all biomarkers ( table8 ) . wmds in cardiorespiratory fitness and circulating concentrations of biomarkers comparing moderate and vigorous exercise intervention groups to control groups apo ai indicates apolipoprotein a1 ; apo aii , apolipoprotein a2 ; apo b , apolipoprotein b ; crp , c - reactive protein ; ffa , free fatty acid ; hba1c , glycosylated hemoglobin a1c ; hdl - c , high - density lipoprotein cholesterol ; homa - ir , homeostatic model assessment insulin resistance ; igf-1 , insulin - like growth factor 1 ; igf - bp3 , insulin - like growth factor binding protein 3 ; il , interleukin ; ldl - c , low - density lipoprotein cholesterol ; na , not available due to the lack of comparisons reported ; tc , total cholesterol ; tg , triglycerides ; tnf- , tumor necrosis factor ; vldl - c , very low - density lipoprotein cholesterol ; wmds , weighted mean differences . random sequence generation was reported in 50 trials , and allocation concealment was reported in 20 trials ; only 1 of these trials showed a high probability of selection bias because the random allocation was not concealed . the risk of potential performance bias was high in all trials because it was not possible to blind participants and trainers in exercise interventions . among 26 trials reporting the blinding of outcome assessment , the risk of detection bias was high in only 1 trial . the risk of other bias was high in 46 trials because of poor compliance , the use of intention - to - treat analysis , limited sample sizes , or limitations discussed in individual articles . a total of 67 and 123 independent comparisons were included in the primary analysis for absolute and relative crf , respectively ( table4 ) . both measures the wmds comparing exercise groups and control groups were 0.28 l / min ( 95% ci 0.23 to 0.33 ; i=93.7% ; p<0.001 for heterogeneity ) for absolute crf and 3.90 ml / kg per minute ( 95% ci 3.45 to 4.35 ; i=91.4% ; p<0.001 for heterogeneity ) for relative crf . the egger s tests showed evidence of publication bias in both instances ( p<0.05 ) . when applying the trim and fill method , the conclusion regarding the associations between exercise training and crf did not change ( filled analysis for absolute crf : wmd 0.14 l / min , 95% ci 0.20 to 5.28 , p<0.001 ; filled analysis for relative crf : wmd 2.56 ml / kg per minute , 95% ci 3.06 to 10.16 , p<0.001 ) . wmds in cardiorespiratory fitness and circulating concentrations of biomarkers between exercise groups and control groups apo ai indicates apolipoprotein a1 ; apo aii , apolipoprotein a2 ; apo b , apolipoprotein b ; crp , c - reactive protein ; ffa , free fatty acid ; hba1c , glycosylated hemoglobin a1c ; hdl - c , high - density lipoprotein cholesterol ; homa - ir , homeostatic model assessment insulin resistance ; igf-1 , insulin - like growth factor 1 ; igf - bp3 , insulin - like growth factor binding protein 3 ; il , interleukin ; ldl - c , low - density lipoprotein cholesterol ; tc , total cholesterol ; tg , triglycerides ; tnf- , tumor necrosis factor ; vldl - c , very low - density lipoprotein cholesterol ; wmds , weighted mean differences . exercise training significantly lowered the levels of triglycerides ( p=0.02 ) and increased the levels of high - density lipoprotein cholesterol ( hdl - c ; p<0.001 ) and apolipoprotein a1 ( p<0.001 ) . the wmds were 5.31 mg / dl ( 95% ci 10.63 to 0.89 ; i=71.8% ; p<0.001 for heterogeneity ) for triglycerides , 2.32 mg / dl ( 95% ci 1.16 to 3.87 ; i=87.5% ; p<0.001 for heterogeneity ) for hdl - c , and 0.03 g / l ( 95% ci 0.02 to 0.04 ; i=0.0% ; p=0.81 for heterogeneity ) for apolipoprotein a1 . the p value of the egger s test for hdl - c was 0.03 , suggesting possible publication bias ; however , the results from the trim and fill analysis did not show substantial impact of publication bias on the estimates or the statistics ( filled analysis : wmd 2.32 mg / dl , 95% ci 1.16 to 3.87 , p<0.001 ) . significant associations were found for interleukin-18 ( wmd 18.3 pg / ml ; 95% ci 0.10 to 36.6 ; i=0.0% ; p=0.95 for heterogeneity ) but not for c - reactive protein , interleukin-6 , or tumor necrosis factor in the primary analysis ( table4 ) . although there was no effect on adiponectin , exercise training was significantly associated with reduced levels of leptin ( wmd 2.72 ng / ml ; 95% ci 4.03 to 1.42 ; i=82.10% ; p<0.001 for heterogeneity ) ( table4 ) . table4 also shows the effects of exercise training on markers of glucose intolerance and insulin resistance . fasting insulin levels ; homeostatic model assessment insulin resistance , or homa - ir ; and glycosylated hemoglobin a1c were significantly lowered in exercise groups compared with control groups ( p=0.002 , p=0.002 , and p<0.001 ) ( table4 ) . the wmds between exercise groups and control groups were 1.03 iu / ml ( 95% ci 1.69 to 0.37 ; i=79.8% ; p<0.001 for heterogeneity ) for fasting insulin . the wmd for homa - ir was 0.30 ( 95% ci 0.49 to 0.11 ; i=77.5% ; p<0.001 for heterogeneity ) , whereas the wmd for hemoglobin a1c was 0.28% ( 95% ci 0.42 to 0.14 ; i=80.1% ; p<0.001 for heterogeneity ) . the egger s tests for fasting glucose and insulin were not suggestive of substantial publication bias ( p=0.18 and p=0.24 , respectively ) . the results from the trim and fill analysis suggested that there was no substantial impact of publication bias on the results for homa - ir or hemoglobin a1c ( filled analysis for homa - ir : wmd 0.30 , 95% ci 0.49 to 0.11 , p=0.002 ; filled analysis for hemoglobin a1c : wmd 0.28% , 95% ci 0.42 to 0.14 , p<0.001 ) . the primary analysis examined 3 hemostatic factors : fibrinogen , endothelin-1 , and angiotensin ii ( table4 ) . on average , the levels of fibrinogen and angiotensin ii were 0.39 g / l ( 95% ci 0.03 to 0.75 ; i=45.00% ; p=0.18 for heterogeneity ) and 1.32 pg / ml ( 95% ci 0.54 to 2.11 ; i=0.00% ; p=0.71 for heterogeneity ) lower in exercise groups than in control groups . our metaregression results suggest that the differences in crf between exercise and control groups were modified by age and sex ( absolute crf : p=0.008 and p<0.001 for age and sex , respectively ; relative crf : p=0.003 and p=0.001 for age and sex , respectively ) ( table5 , figure3 ) . in addition , the effects of exercise on levels of total cholesterol ( p=0.04 ) , low - density lipoprotein cholesterol ( ldl - c ; p=0.06 ) , and fasting insulin ( p=0.05 ) were modified by the presence of at least 1 of the following comorbidities : type 2 diabetes , hypertension , hyperlipidemia , and metabolic syndrome ( tables6 and 7 , figure3 ) . sex differences in the effects of exercise were also found for fasting insulin ( p=0.04 ) . wmds in absolute and relative cardiorespiratory fitness comparing exercise intervention groups to control groups by specific modifiers bmi indicates body mass index ; crf , cardiorespiratory fitness ; wmds , weighted mean differences , p values for the impact of potential modifiers on the exercise effects . health status : participants having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome ( yes ) vs those with none of them ( none ) . wmds in lipid biomarkers comparing exercise intervention and control groups by specific modifiers bmi indicates body mass index ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol ; wmds , weighted mean differences . health status : participants having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome ( yes ) vs those with none of them ( none ) . wmds in biomarkers of glucose intolerance and insulin resistance comparing exercise intervention groups to control groups by specific modifiers bmi indicates body mass index ; na , not available due to the lack of comparisons reported for active participants ; wmds , weighted mean differences . health status : participants having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome ( yes ) vs those with none of them ( none ) . forest plot of effects of exercise interventions on cardiorespiratory fitness , tc , tg , hdl - c , ldl - c , fasting glucose , and fasting insulin within subgroups . the wmds ( diamonds ) and corresponding cis ( extended line ) between exercise groups and control groups are shown for each subgroup . crf indicates absolute cardiorespiratory fitness ; bmi , body mass index ; hdl - c , high - density lipoprotein cholesterol ; ldl - c , low - density lipoprotein cholesterol ; rel . crf , relative cardiorespiratory fitness ; tc , total cholesterol ; tg , triglycerides ; wmds , weighted mean differences . after conducting metaregressions , analyses within subgroups were performed . compared with older people , those aged < consistent with the metaregression results , men seemed to have greater exercise - related improvement in crf , ldl - c , and fasting insulin than women did ( figure3 ) . exercise interventions appreciably improved the levels of total cholesterol , ldl - c , and fasting insulin ( p=0.004 , p=0.01 , and p=0.01 , respectively ) in people having at least 1 of type 2 diabetes , hypertension , hyperlipidemia , and metabolic syndrome ( tables6 and 7 , figure3 ) ; no such improvements were observed among people without any of those health conditions ( p=0.44 , p=0.19 , and p=0.13 , respectively ) ( tables6 and 7 , figure3 ) . in light of the potential impact of exercise intensity , we conducted separate analyses of all eligible comparisons for moderate and vigorous exercise interventions , respectively . the 95% cis for moderate and vigorous interventions overlapped for both crf measures and for all biomarkers ( table8 ) . wmds in cardiorespiratory fitness and circulating concentrations of biomarkers comparing moderate and vigorous exercise intervention groups to control groups apo ai indicates apolipoprotein a1 ; apo aii , apolipoprotein a2 ; apo b , apolipoprotein b ; crp , c - reactive protein ; ffa , free fatty acid ; hba1c , glycosylated hemoglobin a1c ; hdl - c , high - density lipoprotein cholesterol ; homa - ir , homeostatic model assessment insulin resistance ; igf-1 , insulin - like growth factor 1 ; igf - bp3 , insulin - like growth factor binding protein 3 ; il , interleukin ; ldl - c , low - density lipoprotein cholesterol ; na , not available due to the lack of comparisons reported ; tc , total cholesterol ; tg , triglycerides ; tnf- , tumor necrosis factor ; vldl - c , very low - density lipoprotein cholesterol ; wmds , weighted mean differences . this systematic review and meta - analysis of 160 rcts involving 7487 participants indicates that exercise training may significantly improve crf and cvd biomarkers of lipid and lipoprotein metabolism , glucose intolerance and insulin resistance , systemic inflammation , and hemostasis ( figure4 ) . in addition , we identified several important modifiers , including age , sex , and health status , that may partially modify the exercise effects on cardiovascular health . the current meta - analysis shows that exercise , with relatively low risk of side effects compared with medications , may be an effective way to prevent cvd through impact on various biomarkers . our results from the meta - analysis showed that exercise training significantly raised crf , which has been demonstrated to be an independent predictor of cvd risk , cvd mortality , and total mortality.200,201 lower levels of triglycerides and higher levels of hdl - c were observed in exercise groups . aside from conventional cvd biomarkers , our meta - analysis also examined the effects on biomarkers that have not been well studied in previous studies , including biomarkers of insulin resistance and hemostasis , adipokines , and novel lipid and inflammatory biomarkers . we found evidence supporting the favorable effects of exercise on apolipoprotein a1 , interleukin-18 , fasting insulin , homa - ir , and hemoglobin a1c . although the exact biological mechanisms are not clear , our findings indicate that exercise may exert cardioprotective effects by altering dyslipidemia , inflammation , insulin resistance , and hemostasis.19 as a major component of hdl , apolipoprotein a1 plays an important role in the cardioprotective effects of hdl - c.202204 our findings on apolipoprotein a1 strengthen the hypothesis that exercise may accelerate reverse cholesterol transport . another plausible mechanism by which exercise improves the lipid profile is by regulation of lipoprotein lipase . various studies have suggested that exercise may decrease the levels of triglycerides and increase the levels of hdl - c through its impact on lipoprotein lipase expression and activity , which were consistent with the results from our meta - analysis.205207 in addition , our analysis also confirmed that the proportion of cvd risk that could have been reduced by exercise via effects on total cholesterol and ldl - c is much lower than what has been observed previously.208,209 consequently , the results from our meta - analysis provide additional evidence in support of the notion that , in addition to modifying total cholesterol and ldl - c , exercise training may also affect cardiovascular health through other pathways . we found that people in exercise groups also had significantly lower levels of il-18 and several biomarkers of insulin resistance and hemostatic factors , indicating that exercise may exert its effects via pathways of inflammation - characterized atherothrombosis and insulin resistance . a recent review suggested that exercise training may regulate white adipose tissue mass and the expression of adipokines.210 obesity has become widely regarded as a chronic proinflammatory state , and substantial evidence indicates that chronic inflammation in adipose tissues , especially in white adipose tissue , could lead to insulin resistance.211,212 consequently , it is biologically plausible that by reducing the white adipose tissue mass and regulating the expression of adipokines , exercise could mitigate the chronic inflammation in adipose tissues , resulting in improved insulin sensitivity . consistent with previous evidence,213 both moderate and vigorous exercise training appeared to have favorable effects on cardiorespiratory fitness and cardiometabolic health . we found that the differences in cvd risk between exercise groups and control groups were not significantly modified by lifestyle , body mass index , or intervention duration . these findings suggest that exercise interventions may have similar effects on cardiovascular health in populations regardless of these factors . alternatively , the effectiveness of exercise training appeared to be different across strata of age , sex , and health status . the effects of exercise interventions on crf measures were modified by age , sex , and health status such that people aged < 50 years , men , and people with type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndrome appeared to benefit more from exercise interventions . we also observed significant modification of the effects on total cholesterol and ldl - c by preexisting medical conditions ( type 2 diabetes , hypertension , hyperlipidemia , or metabolic syndromes ) , and that may explain why we did not find significant effects of exercise on total cholesterol and ldl - c . this finding also suggests that exercise interventions may provide significant benefits for people with those preexisting conditions by lowering total cholesterol and ldl - c . strengths of this meta - analysis include the comprehensive and systematic review of both conventional and novel cvd biomarkers , detailed subgroup analyses for potential effect modifiers that have not been conducted previously , assessment of robustness with regard to exercise intensity , and evaluation of the risk of different bias . the 2008 physical activity guidelines advisory committee report included a number of comprehensively systematic reviews and meta - analyses based mostly on observational studies.214 the evidence from rcts has been relatively scarce , especially for novel cardiometabolic biomarkers . our study is the first that synthesized evidence from the rct setting and covered a comprehensive set of both traditional and novel biomarkers . our findings are corroborated by several previous meta - analyses of rcts,20,215 but the inclusion of both sexes , more studies , subgroup analyses , and sensitivity analyses allowed us to achieve higher precision in the estimates and to determine the effect modification in subgroups . first , the evidence for hemostatic factors is based on a limited number of available trials , and we were not able to synthesize evidence for some novel biomarkers , such as plasminogen activator inhibitor 1 , lipoprotein(a ) , and homocysteine due to sparse available data . second , subgroup analyses were restricted to outcomes with > 20 studies included , and cutoff points used for categorizing modifiers were arbitrarily selected . third , due to the heterogeneity of exercise training programs and the limited number of rcts that provided separate data , this meta - analysis can neither perform a dose - response analysis nor distinguish exercise types . we maximized the utility of data regarding exercise duration and intensity available from original rcts and found that exercise effects were not significantly different across subgroups defined by duration and intensity . our findings are consistent with previous evidence showing that both moderate and vigorous exercise training has similarly favorable effects on cardiometabolic health.213 the duration threshold at which exercise exerts its effects needs further investigation . fourth , to maintain independence , we selected 1 comparison from each trial with exercise groups of different intensities compared with 1 single control group . the results may potentially be subject to bias by excluding several eligible intervention groups with moderate intensity ; however , we found that the direction and magnitude of the effects on most of the outcome measures were quite similar between moderate and vigorous interventions ( table8 ) . finally , like any meta - analysis , our results may be prone to publication bias and inherent weaknesses of individual studies . in conclusion , this large meta - analysis of rcts clearly shows that exercise training significantly improved crf and some traditional and novel cvd biomarkers in adults without cvd , indicating the causal role of exercise in the primary prevention of cvd morbidity and mortality . indiana university school of medicine strategic research initiative grant ( zhang and song ) , r01dk09406 ( roberts ) and p50hl105188 ( roberts ) from the national institutes of health ( nih ) , and brown university . the nih , brown university , or indiana university had no role in the design and conduct of the study ; the collection , management , analysis , and interpretation of the data ; or the preparation , review , or approval of the manuscript .

backgroundguidelines recommend exercise for cardiovascular health , although evidence from trials linking exercise to cardiovascular health through intermediate biomarkers remains inconsistent . we performed a meta - analysis of randomized controlled trials to quantify the impact of exercise on cardiorespiratory fitness and a variety of conventional and novel cardiometabolic biomarkers in adults without cardiovascular disease.methods and resultstwo researchers selected 160 randomized controlled trials ( 7487 participants ) based on literature searches of medline , embase , and cochrane central ( january 1965 to march 2014 ) . data were extracted using a standardized protocol . a random - effects meta - analysis and systematic review was conducted to evaluate the effects of exercise interventions on cardiorespiratory fitness and circulating biomarkers . exercise significantly raised absolute and relative cardiorespiratory fitness . lipid profiles were improved in exercise groups , with lower levels of triglycerides and higher levels of high - density lipoprotein cholesterol and apolipoprotein a1 . lower levels of fasting insulin , homeostatic model assessment insulin resistance , and glycosylated hemoglobin a1c were found in exercise groups . compared with controls , exercise groups had higher levels of interleukin-18 and lower levels of leptin , fibrinogen , and angiotensin ii . in addition , we found that the exercise effects were modified by age , sex , and health status such that people aged < 50 years , men , and people with type 2 diabetes , hypertension , dyslipidemia , or metabolic syndrome appeared to benefit more.conclusionsthis meta - analysis showed that exercise significantly improved cardiorespiratory fitness and some cardiometabolic biomarkers . the effects of exercise were modified by age , sex , and health status . findings from this study have significant implications for future design of targeted lifestyle interventions .

Methods Data Sources and Searches Study Selection Data Extraction and Quality Assessment Data Synthesis and Analysis Results Description of Study Quality Cardiorespiratory Fitness Lipid and Lipoprotein Markers Adipokine and Inflammatory Markers Markers of Glucose Intolerance and Insulin Resistance Hemostatic Factors Subgroup Analyses Sensitivity Analyses Discussion Sources of Funding Disclosures

PMC3914278

exercise physiology and the salutatory effects on weight loss , fat reduction , and insulin sensitivity have been described in great detail . these beneficial effects are now considered to reflect , at least in part , the effect of exercise on the activation of amp - activated protein kinase ( ampk ) . in obese non - diabetics , exercise has been shown to reduce the risk of developing type 2 diabetes by up to 46% . physical training , consisting of 20 min cycling or running , 20 min swimming at submaximal heart rate , followed by 20 min of warm up / cool down three times per week for 4 wk , resulted in a significant reduction in body weight and percentage body fat , and this was associated with improved whole - body glucose uptake , decreased fasting insulin concentrations , and increased circulating adiponectin and mrna expression in muscle . among patients with type 2 diabetes mellitus , increasing exercise led to a reduction in fasting plasma glucose . the 24-hour energy expenditure , whether measured in a respiratory chamber or by using doubly labelled water , increases linearly with increasing body weight . multivariate analysis shows that fat - free mass is the major determinant of energy expenditure , with minor influences of fat mass , age , and gender . nevertheless , the considerable interindividual variation is currently unexplained . there is at the present time little evidence for major adaptations of energy expenditure during overfeeding , and changes in body weight and body composition appear to be the major factors that , by increasing energy expenditure , allow energy balance to be restored . the body - weight gain may therefore be seen as an adaptative change to overfeeding [ 6 , 7 ] . total energy expenditure can be subdivided into three main components : basal metabolic rate ( bmr ) ; the thermic effect of food , or diet - induced thermogenesis ; energy expended in physical activity ( pha ) . the first two components can be measured opportunely with reasonable accuracy and have been fully studied in lean and obese subjects . there is actually no clear evidence that a low bmr is a factor in the development of obesity , even if the issue continues to be debated [ 8 , 9 ] . the thermic effect of food , and more specifically the thermic effect of carbohydrate , has been shown to decrease in obese subjects [ 10 , 11 ] . this decrease may , nevertheless , be secondary to obesity - related insulin resistance and is of too small magnitude to account for a major weight gain . nonexercise activity thermogenesis ( neat)the energy expended for everything we do that is not sleeping , eating , or sports - like exercise and pha , that is crucial for weight control , may be important in the physiology of weight change . we review the current concepts about energy expenditure and evaluate the pha in the context of this knowledge and the available literature . during pha , mechanical work associated with muscle contractions clearly requires energy . as a result of the associated loss of energy as heat during atp synthesis in the mitochondria and atp hydrolysis during muscular contraction , pha may thus have a major impact on the total 24-hour energy expenditure and energy balance . based on the possible means by which alterations in pha can impact on 24-hour energy expenditure and energy balance , the following hypotheses can be formulated . this response would induce to a lower energy expenditure for any given work load and would probably contribute to the realization of a positive energy balance in affected subjects . it was further shown to be similar in both lean and post - obese women . there is then little reason to suppose that this factor might be involved in body - weight gain . , nevertheless , showed that a common polymorphism of uncoupling protein 2 , a protein with close homology to the uncoupling protein of brown adipose tissue ubiquitously expressed in man , was associated with a lower energetic efficiency of muscle contractions . therefore , brown adipose tissue , where uncoupling protein 1 ( ucp1 ) activity uncouples mitochondrial respiration , is an important site of facultative energy expenditure . ucp1 single nucleotide polymorphisms ( snps ) could represent thrifty factors that promote energy storage in prone subjects . these observations and the relationship between this polymorphism and body weight remain to be further evaluated . bmr is higher in trained subjects than in sedentary subjects of the same weight , but the difference can be principally ascribed to changes in body composition , with a lower fat mass and a higher fat - free mass , metabolically active body mass in trained athletes . when exercise training is associated with a substantial negative energy balance , bmr may better still decrease . the increase in energy expenditure induced by a -adrenergic stimulation , derived by mental stress , was also found to be unchanged in obese subjects after a 6-week period of physical training . third , the total energy expended in pha , that is , the period of time devoted daily to pha , may play an important role in determining energy balance . nevertheless , this component of daily energy expenditure is actually very difficult to assess accurately . the doubly labelled water method permits evaluation of total energy expenditure under conditions of everyday life over several days and , therefore , is likely to include habitual pha . the difference between the total energy expenditure and the basal energy expenditure , the suprabasal energy expenditure ( see ) , comprises both the thermic effect of food and pha . this finding strongly suggests that the amount of energy expended in pha is low in obese subjects . whether a low pha exists before obesity , or is merely a consequence of it , remains an open question at the present time . the conclusion from the substantial literature on this topic is that during an acute bout of exercise in untrained fed subjects , carbohydrate oxidation replaces the major portion of the extra energy expended . in endurance - trained subjects consequently , there is an increased ability to oxidize lipids , due to the upregulation of the enzyme amp - activated protein kinase in skeletal muscle . the outcomes are increased maximal o2 consumption and a higher proportion of fat oxidized during the exercise of low - to - moderate intensity . at high intensity , there is evidence that endurance - trained athletes consume a diet containing a high proportion of carbohydrate . due to the hierarchy in substrate oxidation mentioned earlier , these dietary habits perhaps impact on substrate oxidation in everyday conditions . it can be expected , therefore , that athletes who conserve a stable body composition while consuming such a diet will have a high 24-hour oxidation of carbohydrates . in healthy lean untrained subjects studied in a respiratory chamber , moderate pha increased total energy expenditure , essentially by increasing carbohydrate oxidation when the exercise took place after a meal . nevertheless , the same exercise performed in the fasting state , before breakfast , led to a marked increase in lipid oxidation . in fact , since the subjects were fed the same isoenergetic diet under both conditions , exercising before breakfast versus after breakfast led to a negative lipid balance and to a positive carbohydrate balance . it might , therefore , be hypothesized that exercise in the fasting state may preferably promote lipid utilization and favourably affect body composition by decreasing fat stores . this hypothetical scheme appears unlikely , nevertheless , if the diet consumed is not changed , since the positive carbohydrate balance will probably increase carbohydrate oxidation . is there an effect of food intake on pha ? it has been recognized for several decades that severe underfeeding leads to behavioural adaptations that result in a decreased spontaneous pha . more recently , there have been reports suggesting that overfeeding increases energy expenditure more than would be predicted on the basis of body weight and lean body mass changes [ 26 , 27 ] . furthermore , this increase in energy expenditure showed marked interindividual variations and was inversely correlated with body - weight gain . in fact , since changes in bmr and in the thermic effect of food were mainly accounted for by alterations in body composition and dietary intakes , this increase in suprabasal energy expenditure was attributed to a stimulation of spontaneous pha . such pha was unrelated to exercise and hence was termed nonexercise activity thermogenesis ( neat ) . neat is the energy expended for everything we do that is not sleeping , eating , or sports - like exercise . it includes the energy expended walking to work , typing , performing yard work , undertaking agricultural tasks , and fidgeting . the first approach is to measure or estimate total neat . here , total daily energy expenditure is measured and from it , the bmr plus thermic effect of food is subtracted . the second approach is the factorial approach whereby the components of neat are quantified and total neat calculated by summing these components . the amount of neat that humans perform represents the product of the amount and types of physical activities and the thermogenic cost of each activity [ 28 , 29 ] . the factors that impact a human 's neat are readily divisible in biological factors such as weight , gender , body composition , and environmental factors such occupation . the variability in neat might be viewed as random and unprogrammed but human data contradict this thesis . it appears that changes in neat accompany experimentally induced changes in energy balance and may be important in the physiology of weight change . it then becomes intriguing to dissect mechanistic studies that delineate how neat is regulated by neural , peripheral , and humoral factors . neat may be a carefully regulated tank of physical activity that is crucial for weight control . there is no much understanding of the mechanisms that may probably be responsible for the stimulation of neat during overfeeding at the present time . calorimetric studies show that 24-hour energy expenditure increases in healthy subjects after short - term carbohydrate , but not fat , overfeeding [ 30 , 31 ] . this effect was not correlated to the increase in plasma leptin concentrations observed after carbohydrate overfeeding . several animal studies show that the stimulation of the melanocortin-4 receptor ( mc4r ) is associated with an increase in spontaneous pha [ 32 , 33 ] . showed that new mc4r mutations in a large number of severely obese adults living in southern italy . these mutations , not present in normal - weight individuals , are further evidence that defects in the melanocortin pathway are related to severe obesity . how the intake of specific macronutrients impact on this pathway and the mechanisms involved remains to be clarified . the hypothesis that physical activity exerts beneficial effects on body - weight status by mechanisms not associated to an increase in energy expenditure should be considered . nevertheless , data on energy expenditure and body composition do allow some conclusions to be drawn . if an endurance - trained athlete consistently expends a substantial amount of energy in pha and at the same time maintains a constant weight and body composition , energy intake must have been increased appropriately , most probably spontaneously . the mechanisms responsible for this increase in energy intake have not been completely studied . as a result of the complex network in the central nervous system that regulates energy expenditure and the known effects of pha on neuroendocrine regulations , it is very likely that the relationship between energy expended in pha and food intake are is complex . nevertheless , endurance - trained athletes are characterized by a low fat mass , which may be the result of previous periods of negative energy and fat balances . they have low plasma leptin levels too , which are mainly ascribable to the low fat mass , since comparable leptin levels are observed in very lean sedentary subjects . several studies have searched for a direct inhibition of leptin release by acute or chronic exercise . except for a decrease in plasma leptin levels following particularly intense exercises such as an ultra - marathon , these researches have failed to identify a long - lasting reduction in leptin levels after exercise [ 3640 ] . it may specify that a substantial decrease in fat - free mass induced by exercise is required to decrease leptin secretion before low plasma leptin in turn increases food intake . intriguingly , this absence of effects of exercise contrasts with the effect of severe energy restriction , which quickly decreases plasma leptin levels before any marked changes in body composition occur . it may specify that exercise is more efficient than severe energy restriction in the promotion of weight loss without excessive rebound hyperphagia . the effects of pha on energy metabolism and body - weight control remain incompletely understood . there is ample evidence that physical training is associated with low body weight and low fat mass . this relationship unequivocally suggests that negative energy and fat balances are correlated with physical training . the negative energy balance is perhaps to be at once secondary to the quantity of energy expended while exercising , so there is no evidence that exercise influences other components of energy expenditure . the negative fat balance is probably secondary to this negative energy balance . in obese individuals the amount of energy expended in pha appears to be small , which certainly represents a factor that could prevent weight loss . it appears useful to focus on obese patients also in general practice in order to recognize sedentary life styles and encourage pha through individualized programs . some studies [ 43 , 44 ] show a mild and nonsignificant reduction of bmr in patients treated with an integrated dietetic plus physical exercise program and a significant reduction of bmr in surgically treated patients . it will be necessary to have a larger number of patients to confirm these findings . our knowledge of the pathological consequences of the lack of adequate exercise on adipose tissue , skeletal muscle , and the liver is improving , and this will help establish more specific guidelines for the proper exercise regimens that will improve underlying metabolic pathways . neat and physical activity , that is crucial for weight control , may be important in the physiology of weight change . two issues that remain to be resolved are whether pre - obese individuals have a low pha level that contributes to weight gain and , if so , what are the biological determinants of this low pha .

we review the current concepts about energy expenditure and evaluate the physical activity ( pha ) in the context of this knowledge and the available literature . regular pha is correlated with low body weight and low body fat mass . the negative fat balance is probably secondary to this negative energy balance . nonexercise activity thermogenesis ( neat ) and physical activity , that is crucial for weight control , may be important in the physiology of weight change . an intriguing doubt that remains unresolved is whether changes in nutrient intake or body composition secondarily affect the spontaneous physical activity .

1. Introduction 2. Physical Activity and Body Weight 3. Physical Activity and Fat Oxidation 4. Physical Activity and Spontaneous Food Intake 5. Conclusive Remarks

PMC3806382

large scale trials with insecticide treated bed nets ( itns ) have been shown to have a profound impact on reducing malaria transmission in experimental trials in sub - saharan africa and were recommended for large scale operations . however , this tool did not become practical before the first long lasting insecticide nets ( llins ) were marketed and recommended by world health organization . preliminary who recommendations of llin are based on short term studies on vector impact and wash resistance . however , in sub - saharan african settings , loss of insecticide is not primarily due to washing , but due to handling and evaporation . dabire indicated that , though llins showed good efficacy on mosquitoes under controlled conditions , their effectiveness in the field conditions with respect to actual duration of insecticide protection in the field did not last for five years as indicated on the bed nets . recent research has shown that wear and tear may be more important for bed net durability than wash removal of insecticide . this has led to the development of several hole indexes , and who recently modified the model it recommend . since holes can only be measured on nets still present , it is the most meaningful when attrition rate can be followed , which means that cohorts of nets must be followed . however , the effect of gradually declining insecticide in the net , increasing the number of holes , and net disappearing has not been combined with observations of malaria incidence in field studies , and the effect is thus uncertain . this study was set up to see how an llin with a preliminary who recommendation performs in the field as a vector control tool , combining field observations of mosquito density to net parameters as insecticide load , bioassay performance , and hole index , and to see how the expected decline in efficacy impact malaria incidens . netprotect bed nets were reportedly developed on the advantage of the first two long lasting bed nets using a fine mesh - like polyester net but having the strength and incorporation technology of a polyethylene nets . the bed net is made out of polyethylene mixture in which the insecticide ( deltamethrin ) is incorporated directly into the fabric at the rate of 1.8 g / kg or 60 mg / m . kanyaboli ( figure 1 ) is one of the villages clustered around the dominion rice farm at the north shores of victoria lake around the yala swamp , about 70 km west of kisumu town in western kenya . the area encompasses about 18.5 km and is adjacent to an oxbow lake of kanyaboli . yala swamp is one of the most important flood plain wetlands around kanyaboli lake and one of the largest swamps in kenya . the swamp forms the mouth of two rivers yala and nzoia and is a freshwater deltaic wetland arising from backflow of water from victoria lake as well as the rivers flood water . the area covers 17500 hectares and contains three freshwater lakes , kanyaboli , sare , and namboyo . part of yala swamp covering about 2300 hectares has been reclaimed for rice production by the dominion group of companies . the total annual rainfall in this region averages 1400 mm with the first peak between march and april and second peak between november and december . most of the inhabitants of kanyaboli village live in traditional houses with mud walls and grass thatched roofs . the eaves of most houses are open allowing for unimpeded entry and exit of mosquitoes , which bite the unprotected humans sleeping in these houses . family compounds , consisting of one or more houses are separated from each other by farmland . apart from working in the rice fields under contract at the dominion farms , the inhabitants also practice subsistence agriculture , growing crops such as maize , millet , and cassava . fishing is carried out on small scale in the lake to be eaten as a source of protein and sold to supplement for monthly family income . the domion farm has cooperated with malaria programs to gradually cover the villages around with bed nets over a time frame of 3 years at the expense of the farm . in cooperation with the net producer , we offered our bed nets at cost price to two steps of this campaign , for the intervention village at start and for the control village 6 months later . cdc in kisumu had previously been interested in this area and found that the malaria vectors in the area are anopheles arabiensis and funestus with a year round transmission recorded at a local clinic ( o. skovmand , pers . the dominion farm had established the clinic in a village near the farm building complex and paid the staff and other costs . it receives patients from all the villages around the farm area and the farm staff . our co - operation with the clinic thus allowed for passive detection of malaria cases . a sample size of 150 houses in 150 compounds matched in size was selected through simple random sampling by lottery at the intervention village . long lasting treated mosquito bed nets netprotect were hung in the selected houses in december 2007 . two five llins were put up by the study group per household for full coverage of all sleeping places . a compound consisted often of a main house and one or more small cottages for teenagers or elder people . typically , there would be two sleeping places in the main house and one or more in each of the cottages . only the nets in the main house were followed after the first month . to cater for mosquito flight range , another 150 houses were randomly selected 2 km away in the adjacent area to act as control arm where untreated bed nets were distributed . after 6 months , the households of the control area received around 450 llins of the same type . the trial thus fit with the local plan for a gradual expansion of bed net coverage in the villages around the swamp . houses for sampling were selected by two - stage cluster sampling each month upon the consent of their owners . the second stage was random selection of one house and the nearest ten neighboring houses with probability proportional to size within each cluster in the village were then included in the sample . indoor resting adult mosquitoes were monitored with a pyrethrum spray catch ( psc ) between 7:00 am and 10:00 am . two collectors , one inside the house and one outside , sprayed the selected houses with 0.025% pyrethrum with 0.1% piperonyl butoxide emulsified concentrate . the collector outside sprayed around the eaves , while the one inside the house sprayed the roof and the walls . the house was then closed for 10 minutes , after which knocked - down mosquitoes were collected from the white sheets indoor and put on moist filter paper in petri dishes . each petri dish was labeled as per house identification number and packed in a cool box . the collected mosquitoes were transferred to the laboratory at kenya medical research institute ( kemri ) in kisumu . funestus were then sorted out based on the abdominal status and characterized as fed , unfed , gravid , or half gravid then stored in vials containing anhydrous calcium sulphate . a record sheet was completed detailing gonotrophic stages , sex , and species identification . all blood fed mosquitoes from each collection were preserved in labeled vials containing anhydrous calcium sulphate . samples of blood fed mosquitoes were cut transversely between the thorax and the abdomen for all posterior portions containing the blood meal . the abdomen of each mosquito was ground in 50 l of phosphate - buffered saline ( pbs ) ph 7.2 with subsequent addition of 950 l of pbs and then stored at 20c in the refrigerator . blood meals were identified by a direct enzyme - linked immunosorbent assay ( elisa ) at kemri in kisumu , using anti - host igg conjugates ( kirkegaard and perry , gaithersburg , md ) against human , bovine , chicken , and goat according to a protocol by beier et al . . 397 blood fed anopheles mosquitoes were tested by elisa technique for the origin of their blood meal . individual mosquito specimens from field collections were prepared for identification by removing a leg with sterile forceps and placing it into one well of a 96-well pcr tray . each well contained 100 l of grinding buffer ( 0.10 m nacl , 0.20 m sucrose , 0.30 m trizma base 0.01 m edta , and 100 ml sterile water ph 8.0 ) . trays were covered securely with sterile adhesive foil and placed in water in a sonicator bath ( bransonic ultrasonic cleaner , shelton , ct , usa ) at 65c for 2040 minutes . to these trays 14 l of 8 m potassium acetate was added , and the mixture was placed in cool ice to precipitate the protein . the mixtures were microfuged , top speed , for 30 minutes , and the resulting supernatants were transferred to new tubes . 200 l cold 95% ethanol was added to samples and placed in a freezer for at least 20 minutes to precipitate the dna . samples were washed with 200 l 70% etoh followed by 200 l 95% etoh , and tubes inverted for complete drying ( about 1 hour ) . 1 l dna samples and controls for an . 14 l master mix ( water , taq enzyme specific mosquito primers mgcl2 , and 10x buffer ) was added to all wells followed by 4 drops of heavy mineral oil to overlay the reaction mixture . the reaction program had an initial step of 80c for 1 min , followed by 30 cycles of denaturation at 94c for 30 seconds , annealing at 50c for 30 seconds , and extension at 72c for 30 seconds , with a final extension at 72c for 4 min . the pcr products were separated by electrophoresis on 2% agarose tbe gels and stained with ethidium bromide . malaria cases were detected passively at the village clinic established by the nearby dominion farm . the two villages of our study were about 2 km away from the clinic . people seeking treatment were compared to the list of people participating in the study either in intervention group or control group but with status blind to the clinic personnel . no other records of malaria cases were used , and self - treatment in households was not followed . malaria was identified using clinical manifestation , and we added rapid diagnostic testing after training the staff . rapid diagnostic tests ( rdt : paracheck pf tests kits ) were carried out according to the manufacturer 's instructions . children under five years who were clinically identified as malaria positive were treated , irrespective of the results for paracheck test in accordance with the national guidelines . children older than five years and adults were first examined and scored clinically then tested with paracheck but only treated according to moh guidelines in 2007 amodiaquine ( 10 mg / day ) for 3 days if a paracheck result was positive for malaria . the field counting of nets took place 4 days , one month , and 35 months after nets hanging . net hanging above a bed or told to be used were counted as nets in use . for the months from 1 to 9 , bioassays were made on nets in the field by placing cones with lab reared , transported mosquitoes on the nets as they were hanging . the collections of nets after years 1 , 2 , and 3 actually took place after 18 , 28 , and 38 months , where 3438 nets were collected and brought back to the laboratory for further evaluation . all the nets brought back to the laboratory were examined for damages ( see below ) . removed nets were replaced with new nets , same size but in a different color . for some or all of these , 5 swatches of 30 30 cm were cut according to who guidelines ( from the four sides and the roof ) and used for biological and/or chemical assays . because bioassay capacity in the laboratory in kisumu showed to be limited , the 18-month samples were sent to a larger bioassay laboratory in chiang mai , thailand . samples collected after two years were sent for chemical analysis only . among the 30 nets collected after 3 years , 10 bed nets were randomly sampled square pieces measuring 30 30 cm were cut from the sides and tested in bioassays in kisumu . chemical analyses were made on 2 of the 5 samples of the nets collected this year . the rest of the samples were stored at room temperature for 3 years and analyzed again in the same laboratory . in 2009 , iic established in - house chemical analysis facilities in hanoi , vietnam , that were used for the 3-year samples . standard who cone bioassays were performed on random samples of netprotect monthly from month 3 to 9 after installation using a laboratory colony of susceptible strain of an . a bioassay cone was attached to one side of hanging bed nets in the houses . one hundred mosquitoes in replicates of ten were exposed to each bed net in ten randomly chosen houses using standardized procedures . after 3 minutes of exposure , mosquitoes were transferred to 200 ml labeled paper cups , provided with 10% sucrose solution , brought back to the laboratory , and maintained at 28c 2 , rh 80% 10% . knocked down at 60 minutes , kd60 were observed in the field and percentage mortalities after 24 hours were recorded in the lab . the nets swatches from 18 months were sent for bioassay at the university of chiang mai that made standard who cone test with 3 min exposure on 2 of these swatches per net , randomly chosen among nets , but with known position . 50 females , 24 days old anopheles cracens ( former dirus b ) , were used for these bioassays per sample , procedure as described above . kisumu female mosquitoes were exposed to net swatches from year 3 , following the procedure described above ; the lower number due to the limited test capacity in kisumu . two criteria for mortality were applied , absolute mortality where the mosquito does not move when touched , and functional morality , where it has lost at least 3 legs or one wing , and therefore will fall to the ground in a house and be eaten by ants . the nets were delivered by the producer bestnet a / s with a chemical analysis made by the singapore laboratory also used by crown agent and unicef . in our analyses , the net swatches analyzed per net were cut to pieces , and a subsample was drawn for extraction in xylen under reflux following the who protocol for deltamethrin incorporated pe nets . hanoi university used the hplc method according to cipac 333/ln , whereas the iic laboratory used gc fid to analyze extracts of the net as recommended by the cipac / who reference laboratory in gembloux , iso 17025 . this method is mostly the same as the cipac method except that for the gc fid the xylen is not evaporated followed by a dissolve of the insecticide in injection solvent ; the xylen solution is injected directly . procedures for this method are very close to these of gc-ecd recently published by the same laboratory . 4 days after the initial distribution , 1 month and 36 months later , all households in the first distribution area were revisited to see if the nets were still hanging . if not hanging , the owners were kindly asked where the nets were and if they were still in use . after years 1 , 2 , and 3 , around 60 households were visited , and net presence and state ( holes , dirtiness ) were recorded . 34 to 38 of these nets were randomly picked ( by tossing a coin before entering the house ) from the first village and brought back to the laboratory . however , it was found that especially smaller holes were underestimated in the field and only data from the nets collected and brought back to the laboratory are presented below . for each of these houses , the following information was taken : the net user , number of rooms in the house , whether the kitchen was in the same room as the net taken ( fire damage ) , and how often the net was reported to be washed . the nets were suspended on a tube frame borrowed from cdc in kisumu , and holes were counted and sizes estimated in 5 categories : below 1 cm , 1 - 2 cm , 35 cm , 510 cm , and above 10 cm . holes were divided into tear hole and burn holes ; the latter is recognized from the borders of the holes . this scale is different from the one recently introduced guidelines from who on long lasting insecticidal nets since our data were collected before this scale was published . in the who hole index , holes below 0.5 cm are discarded , category n1 holes are 0.52 cm , category n2 are 210 cm , n3 are 1025 cm , and n4 are holes above 25 cm . the number of holes per category is multiplied with an index factor ( 1 , 23 , 196 , and 576 , resp . ) , and the sum is divided by the numbers of nets . since we had above 10 cm as the largest group , we arbitrarily divided these holes into two groups , ( 1/2 ) 1025 cm and ( 1/2 ) above 25 cm . the who index is used for relative comparison between products tested and provides no categories for the values found . the net height was measured in the 4 corners of the net suspended on the frame and averaged per net . comparison of proportions between categorical variables was performed by chi - square test at 95% confidence level using sas statistical software version 9 . repeated poisson regression using the genmod procedure in sas was used to analyze the effects of the bed nets on human feeding success on blood meal for the anopheles mosquitoes found resting indoors . for each model , the percentage reduction was calculated as one minus the relative risk as estimated by poisson regression . odds ratio was also used to determine the difference between densities of the anopheles mosquito species collected in treatment and control villages . general linear variance analysis was used to estimate parameters influencing efficacy measured in bioassays after 2 years using statistix , version 9 . linear regression analysis was used to estimate the impact of reported number washes on deltamethrin loss , epimerization , and net height . the netprotect project was reviewed and approved by kemri and kenyatta university graduate school board . this was done after the explanation of objectives and collection methods through individual discussion and group meetings . a total of 807 indoor resting female adult anopheles mosquitoes were collected from 80 randomly sampled houses during the first 6 months of study . out of these , 82.5% of the mosquitoes collected were from the control houses while 17.5% from the intervention ones . arabiensis found in the control houses compared to the intervention houses ( = 22.6 , df = 1 , and p < 0.001 ) . the probability of finding an . arabiensis in the control area was 2.6 times higher than that in intervention area ( or = 2.6 , ci : 1.93.9 ) . funestus were collected ; 13.3% of them were from the intervention houses and 86.7% from the control houses . funestus between the two areas ( = 22.63 , df = 1 , and p < 0.0001 ) , and there were 6.5 more chances to catch an an . arabiensis ( = 2.99 , df = 1 , p = 0.084 ) collected from control than from intervention areas ( table 2 ) . funestus were significantly higher in control area compared to the intervention area ( = 17.44 , df = 1 , and p < 0.0001 ) . funestus in the control area was four times more than that of finding a fed an . funestus in the intervention area ( or = 4 : 95% ; ci : 2.07.2 ) . a total of 515 blood fed anopheles mosquitoes were tested by elisa technique for the origin of their blood meal . arabiensis ( table 3 ) . in the intervention area , 53% of the an . funestus were positive for bovine antibodies , 29% for human antibodies ( igg ) , and 2% had mixed blood meals human / bovine . in the control area , 46% of an . funestus were engorged on human blood meal , 29% were positive for bovine blood meal , and 2.2% engorged on mixed blood meals of either chicken / human or bovine / human , and 23% , blood meals could not be identified with our methods . funestus is highly significant ( = 30.7 , df = 1 , and p < 0.001 ) . arabiensis sampled from houses with llins fed on bovine host , to a lesser extent on mixed human / bovine ( 5.7% ) , and none ( 0% ) was positive for human blood meal . in the control villages , 42.3% of the an . arabiensis were positive for bovine blood meal and 4% for human blood meal , 7.7% had mixed blood meal of human / bovine , and 1% had mixed blood meal of bovine / chicken ( table 3 ) . bioassay results on 10 randomly sampled bed nets showed a knockdown rate and absolute mortality rate of 80% and 100% respectively , up till seven months of use . however , after 9 months of use , 2 sampled nets showed 24 hr mortality below 80% . around 50% of the mosquitoes exposed to these 2 bed nets were still alive 24 hours later but had lost three or four of their legs , respectively , thus were functionally dead . after 18 months , 38 nets were brought to the laboratory for damage estimates ( hole counts ) , and from 22 of these , 2 among 5 swatches were analyzed in bioassays . using a threshold of 50% mortality as done by lindblade et al . , 13 nets passed in both samples , 6 passed in one and not the other , and 3 failed in both . general linear variance analysis showed that net code was a significant parameter for knockdown for 1 hr ( df = 21 , f = 3.2 , and p = 0.008 ) , whereas swatch position was not significant ( df = 4 , p = 0.85 ) . for 24 hr mortality , net code was significant ( p = 0.006 ) , whereas position was not significant . mean kd1hr was 86% , median was 96% , and mean mortality was 68% with median 67% . three nets ( 6 swatches ) were very dirty when tested and gave lowest mortality recorded , 10 to 40% . the general aov analysis showed that very dirty was a highly significant parameter ( p < 0,001 ) for bioefficacy . these swatches were washed and tested again after 3 days . for 2 of these , this reduced the efficacy to near 0 , but for the third , it increased from 10% to 96% mortality . if these very dirty nets were removed from the mean values , kd1hr became 91% and mort 24 hr 76% . after 3 years , bioassays were performed on 10 nets randomly picked from the 34 nets taken to the laboratory and tested for bio - efficacy . eight of them provided 100% killing efficacy , one 85% , and one 76% ; however , kd1hr was 100% for these two nets , so the 10 nets passed . start concentration of deltamethrin was determined by the provider , bestnet a / s , before the nets were sent to kenya and determined to be 1.90 g deltamethrin / kg net . 5 net samples taken after 9 months were sent for chemical analysis after the bioassay . three of these induced mortality samples were between 95 , and 100% and two below 80% . the three with high mortality contained 0.999 , 1.191 , and 1.529 g deltamethrin / kg , whereas the two that gave low mortality had 1.164 and 0.976 twenty - four and 34 nets were sampled for chemical analysis at the end of year , 2 and 3 , respectively . these nets were among the nets taken for hole analysis , so a description of each net ( general state including loss of color , holes , and dirt ) and the chemical analysis are known as the comments of the owners on the nets , including informed date of last washing . average deltamethrin was 0.77 g / kg plus 0.30 g r - isomer / kg net . among the 34 nets taken after 3 years of use , 3 nets had no insecticide content . one of these had no color at all indicating the use as fishing net or outdoor use for long time . one looked as brand new ( shiny blue ) , had no label , and was probably not a netprotect . the data show that deltamethrin content had declined to mean 0.73 g deltamethrin / kg net plus 26% isomer after 2 years use and 0.49 g deltamethrin / kg net with 49% isomer after 3 years ( table 4 ) . one net with a value of 0.11 g deltamethrin / kg net had the comment of the owner that then mosquitoes were biting him the fact despite that he used the net . to estimate the impact of net storage after sampling , net swatches from 15 nets collected in year 3 were analyzed 3 years later . a pairwise analysis ( analyses of swatches from same nets paired ) showed that storage at room temperature for 3 years did not provide a significant decline in insecticide content . the mean content after 3 years of use and 3 years in storage was 0.33 g deltamethrin per kg net compared to 0.44 g from the same nets determined shortly after collection . the frequency of net washing was reported from two questions : how often this net is washed and when this net was washed last time . from the responses , the number of washes over 3 years was estimated per net . among the 34 nets taken to the laboratory for closer examination , 3 came from houses where the response was do not know , therefore , chemical data from these houses are omitted in the correlation study to wash frequency . reported frequency of washes at the end of the third year was contrasted to chemical content and net height , two parameters that might be expected to change in a polyethylene net with the number of washes . number of washes varied from 36 to 0 over the 3 years , with a mean of 5.8 and median 3 , meaning once per year . there was no correlation between deltamethrin content and number of washes ( p = 0.22 ) . however , when data for r - isomer content was correlated to number of washes , the correlation was highly significant ( p = 0.009 ) , though the correlation coefficient was moderate ( r = 0.51 ) . nets taken back from the field were inspected for holes when hanging on a frame . the size of holes and their position from the border were noted as summarized in table 5 . around 50% of holes were below 1 cm and were caused by burst of a few yarns or by sparkles . including all hole sizes , the number of holes per net varied from 5.48 in the first year , to 4.09 in the second year , and 11.06 in the third year . the number of holes on the lower 30 cm increased from 45% in the first year to around 70% in the second and third year . mean hole index as defined according to the new who bed net hole index was 333 , 114 and 381 for the 3 years , respectively , but the standard deviations are much larger than the means . the number of nets with no holes present was nearly constant around 15% ( year 1 : 6/38 , year 2 : 6/34 , year 3 : 5/33 , excluding the new looking , hole free , and probably not netprotect net ) . we examined if burn holes were determined by the number of rooms of the house , based on the hypothesis that , in a one - room house , the net would be together with the kitchen fire . however , this association was not significant ( p = 0.30 ) . therefore , in the year 3 it was tabulated if the kitchen fire was in the same room as the net taken back to the laboratory for holes examination . again , this analysis did not reveal the impact of this coexistence on any burn hole found . finally , using the who index for burn holes only and pairing this index per net with the presence of kitchen fire in the same room all households were revisited 4 days after the net hanging campaign and again one month later . after 4 days , the houses of 398 nets were found ; one house with two nets could not be found . twenty nets could not be inspected because the owners were absent . among the rest ( 378 net ) the second most common reason was that the roof leaked where we had hung the net , so it was taken down . after 1 month , 73% of all nets were seen hanging , but we had no access to observe 37 nets because the owners were absent . among 35 nets missing , 21 had been given away or sold , and 5 had moved with the net user ; 4 of these were kids that had gone to a boarding school . after 35 months , the study population was 398 minus the 35 missing after one month . we had removed 38 nets for inspection after 18 months and 34 after 26 months . the remaining nets were thus 291 at the last counting in november 2009 . among these , nine had moved with user , 4 with kids going to boarding schools , and 26 were not traceable , either discarded or used for other purposes . attrition rate including nets moved with users was thus ( 35 + 35)/(398 38 34 ) or 21 % , excluding nets moved with users , 16% . the average net height after 3 years of use was 140 cm ( nets purchased for the study were 150 cm ) with a variation from 120 cm ( one net ) to 150 cm , but there was no correlation to the number of washes . the total number of people living in the compounds was counted as a part of the study preparation . 1350 living in compounds were used for the study in the intervention area , and 1622 in the compounds in the control area of included in this study , total 2972 . 1234 inhabitants living in compounds of the study or the control areas came to the clinic and had the diagnosis , malaria based on clinical manifestations between january and july in 2007 . from the intervention area , 220 had malaria diagnosis , and 67 of these were confirmed by rapid test kit ( rdt , paracheck ) . from the control area , 670 patients had diagnosis of malaria ; 277 of these were confirmed by rdt . malaria incidence among people seeking the clinic thus was 41.3% in the control area and 16.3% in the intervention area during this period . the overall effect of netprotect on malaria prevalence was significant between the two areas before the bed nets were also distributed in the control area ( = 8.28 , p = 0.004 ) . the peak outbreak of malaria cases in the control area was recorded in april in the larger rainy season with a total 88 cases ( figure 2 ) . however , malaria cases in intervention area remained stable at a low level with a small peak in july . netprotect bed nets were then distributed to the control area at end of july 2007 and malaria incidences followed passively at the local clinic for 2 years . the peak transmission could no longer be seen the second year in any of the areas . in the third year , the clinic became involved in the distribution of nets to pregnant women , and incidence could no longer be attributed to netprotect only . when comparing malaria identification from clinical manifestations and from rapid test kits ( paracheck ) , fewer were confirmed by the rdt in both areas , and the ratio ( confirmed by rdt / confirmed by clinic ) was the same for the two areas and constant over time . for the first 6 months , clinical manifestation revealed 3.1 more cases in the control area than in the intervention area , rdt 3.7 times more . these differences between intervention and control areas are significant for both methods ( p < 0.05 ) . further , overestimation of malaria incidence from clinical manifestations was not influenced by incidence level . the results showed that netprotect had a significant impact on densities of indoor resting an . the number of indoor resting anopheles mosquitoes was significantly lower in the intervention houses compared to control houses ( table 1 ) . funestus in the control area was 6.5-fold higher than that in the intervention area , and the probability of collecting an . arabiensis in the intervention area , however , was 2.6 times greater compared to that of the control area . this conforms the results reported in gambia , sierra leone , and kenyan coast studies on itns and curtains which demonstrated decreased indoor resting densities when pyrethrum spray catch ( psc ) was used as the collection method for anopheles mosquitoes . however , while itns exhibit rapid loss of efficacy unless retreatment is adhered to every six months , this study shows that netprotect efficacy on anopheles mosquito was high without the necessity for re - treatment during the project period . was found to be the predominant vector resting indoors in this area constituting approximately 70% of the total mosquitoes collected indoors ; an . funestus was strongly affected by the net campaign ( reduction of 65.5% , p < 0.001 ) compared to an . similarly , studies conducted by gillies and coetzee demonstrated that this species was highly susceptible to chemical control measures and was slow in recolonizing an area from which it has been controlled . there was increased degree of zoophily in houses where netprotect was in use ( table 3 ) . arabiensis is known to be zoophilic but also bite humans and rest indoor , whereas an . funestus is considered very anthropophilic , so this effect is a warning about its flexibility at high bed net coverage . alternatively , a fraction of the an . funestus population feeds on cattle and is not much influenced by the use of bed nets . funestus is a complex species , but it was not tested if the bovine feeding was different from that feeding on humans . three minutes exposure of bioassays on netprotect showed a mortality of 100% during the first seven months of net use and the failure of a few . chemical analysis of the nets that failed to those that did not did not reveal a dose difference . further , bioassays on two samples per nets with known positions and taken from 30 nets did not reveal an effect of net position , not even from the roof . the general idea that the roof is less exposed to handling and that handling is a major reason for insecticide loss can therefore not be confirmed from this study . results after three years of ten randomly sampled bed nets indicated that they were still highly efficacious . bioassay data from the three years were not made with the same anopheles species , and data produced using an . gambiae strain kisumu gave higher mortality than those obtained with an cracens independent of net age . when running the test for insecticide resistance , who use a different thresholds for different species of anopheles ; however , when running these 3 min exposures , the known difference in sensitivity is not considered . for comparison , we sent a net to the who reference laboratory lin in montpellier in 2012 that gave 60% mortality with an cracens , and this gave 100% mortality at lin with an gambiae strain kisumu ( data not shown ) . since the lin is the reference laboratory for these evaluations , the data generated with an gambiae strain kisumu are the most relevant . however , the large amount of bioassays with an cracens relating effect to the position on the net is still valid information . loss of efficacy on other llins has also been cited to be due to external factors such as dirt and fume accumulation on the net fabric . three of 24 nets tested after 9 months were extremely dirty and showed low mortality . they were washed and tested after 3 days , and still two failed , but one jumped to 96% from 10% mortality . impact of dirt is thus not always the same , neither the effect of washing dirty nets . chemical analysis of 30 nets after 2 and 3 years use revealed a decline in deltamethrin from the original 1.9 g deltamethrin to 0.49 g / kg net after 3 years ( table 4 ) . loss in deltamethrin was not depending on the number of washes reported but deltamethrin r - isomer content was . the reason might be that the main reason for loss of deltamethrin is evaporation , but the washing process or the drying in the sun after washing causes the epimerization . five series of nets from a test program of test recipes showed that the amount of r - isomer was constant measured after 0 , 5 , 10 , 15 , and 20 washes ( o. skovmand , prs communication ) . the reported number of washings is not off course a very solid parameter , but the significant correlation to the degree of epimerization indicates that it has value . a single complaint of net failure from a user showed the net content to be 0.11 g deltamethrin more data are needed to know if this represents the whereabouts of a threshold for field failure . bed net use rate was measured for all 400 nets in the first intervention village after 4 days , one month ( table 6 ) , and 3 years . the major reason for not seeing a net hanging was that we could not enter the house where it was supposed to be . if the use rate among these people is estimated to be the same as that in these houses , then 95% of all nets were hanging after 4 days and 90% after a month . the second most common reason for not finding a net was that it was given away or sold , and the third ( after 1 month ) was that the net had moved with the owner . if these latter nets are supposed to be in use , the use rate after one month is 91% . net gone was explained as the net being sold to generate supplementary income or donated to relatives from other regions . after 35 months , further 35 nets were gone , 9 moved with the owner and 26 were missing , either discarded or used for other purposes . again anticipating that the 14 nets moved with owners were in use with the same frequency as those remaining , around 87% of the nets were still in use after 3 years , which is a high percentage . it should be mentioned that this collection was in november therefore in the smaller rainy season . the number of holes did not increase significantly between years one and two ( table 5 ) but did increase dramatically after three years of use . we examined if burn holes could be correlated to the presence of kitchen fire in the same room , but such a correlation was not found . the use of small open fire oil can lamps is thus probably a better explanation . tear holes were mostly on the lower 30 cm are that mostly tucked under the mattress . tear holes from the lower 30 centimeters increased dramatically the third year as an indication of daily use . the hole index was calculated per net and mean ; standard deviation and median were calculated according to who guidelines ( table 5 ) . however , these values were not normal distributed and the standard deviation per year was always much bigger than the mean . bigger holes have a multiplication factor of 576 , so a single hole of that size in one net has a dominant effect on the mean . neither of the median values seems to reflect an evolution since it went from 24 to 3 to 61 for the 3 years . contrary to this complex model , simply counting the number of holes showed an increase from year 1 to year 3 . that number of holes increased , and the who hole index did not show that nets with big holes were discarded preferentially , which off course make sense . but it may also show that the new who hole index is too sensitive to big holes and shows random variation in values caused by these rarely found nets . however , in the third year , an antenatal program of distributing another polyethylene net started , and the mass effect could no longer be ascribed to the polyethylene net of this study . the study collected morbidity results on passive and clinic - based cases and showed a significant difference in malaria cases during the six months of trials between the original intervention area and the original control area . the incidences rate was higher in the control village ( 41.3% ) compared to intervention area ( 16.3% ) . when bed nets were used in both areas , the incidences of malaria cases in the two areas were no longer different . in the second year , malaria incidence as measured here remained low in both areas ( figure 2 ) . of interest , the peaks of malaria incidence nearly disappear and what remains is year round background malaria not influenced by the bed nets . preintervention data were not collected from the two parts of the village where the study was carried out . however , during the first 6 months of the trial , no llins were distributed to the control area , and 450 nets were distributed after these 6 months . we thus have pre - intervention data from the control area . when llins were also distributed in the control area after 6 months , the mosquito density and malaria prevalence mirrored these of the of intervention area ( figure 2 ) . further , the villages were in the same environment around a swamp with no observed differences in breeding sites and with approximately the same distance of 2 km from the clinic . there was lower human blood feeding index in intervention area compared to the control area . malaria prevalence in the intervention area was at a low , stable level despite seasonal malaria variation observed in the control area . the disappearance of the seasonal peak was observed in both the areas the following year where the llins covered both areas . after 3 years , 84% of nets were still found hanging or being washed , and a further 3.5% had moved with the owner . gambiae kisumu strains showed nets to be effective with kd1hr at 100% mortality after 3 years , use , but a large series of bioassays with an cracens after year 1 showed that 1 net out of 19 nets failed in bioassays from both of two samples collected per net on a 50% mortality acceptance criteria . gambiae strain kisumu , however , is the fully sustainable strain used for whopes evaluation of long lasting nets . chemical analysis showed a decline from 1.9 g to 0.49 g deltamethrin / kg over 3 years of use .

we studied the effect on malaria incidence , mosquito abundance , net efficacy , net use rate , chemical analysis , and holes of a long lasting insecticide treated bed net ( netprotect ) in western kenya , 20072010 . nets were hung in 150 households 6 months before they were hung in a second , 2 km away . indoor resting densities were monitored by pyrethrum spray catch and malaria cases by passive detection using clinical manifestations and rapid diagnostic test . the probability of finding an . arabiensis in the control area was 2.6 times higher than that in intervention area during the first 6 months . human blood feeding index of anopheles funestus declined 17% . after bed nets were hung in the second area , malaria incidence declined 25% down to the level in the first area . incidence remained at this low level for 2 years . 90% of collected nets were efficacious after 3-year use . deltamethrin dosage declined from 1.9 to 0.5 g / kg over 3 years . attrition rate after 3 years was 21% . who hole index changed from 333 to 114 to 381 over the three years . this index summarizes the numbers of holes in size categories and multiplies with the mean hole area per category . it is very sensitive to the impact of big holes in a few nets .

1. Introduction 2. Materials and Method 3. Results 4. Discussion 5. Conclusions

PMC4809626

a flow diagram of the algorithm is shown in figure 1 . to implement this procedure for a given model it is necessary to define bounds for input parameters and model outputs ( e.g. , steady states or dynamic behavior ) . if bounds can not be defined empirically , feasible ranges of parameter values can be asserted from physiological knowledge or theoretical considerations . for example , the tissue concentration of a species may not be known , but typical weight and water content of that tissue may be known , which allows us to put an upper limit on the species concentration . overview of algorithm for efficient generation and prevalencebased selection of virtual patients . to generate virtual patients from a model , the prior information ( green boxes ) a virtual population is constructed by selecting from this population with probability proportional to the prevalence in the real population relative to the prevalence in the plausible population . this selection is optimized to produce the best virtual population given the patients in the plausible population . we have provided a detailed description of terminology , definitions , and the derivation of this algorithm in table 1 and the supplementary material . we define these patients as a parameter set for which every component of the model ( whether it be the parameter values themselves , computed species concentrations , or combinations of these that are experimentally measurable ) falls into a biologically plausible range . from this plausible population we can then select the virtual population such that it matches the empirical distribution of interest . this is achieved by calculating a probability of inclusion of a plausible patient into the virtual population . this probability is computed from both the empirical distribution and the density of plausible patients ( see supplementary materials for more details ) . an overview of the terminology used in this article an important prerequisite to this approach is the ability to generate a large number of plausible patients within the region of the empirical data . to accelerate this process we take an initial parameter guess ( within the predefined bounds ) and optimize this choice until the required outputs are within physiologically plausible ranges . rather than optimize to specific points , it is more efficient to be agnostic as to where in the plausible ranges the optimization routine ends . to implement this we shift the typical cost function f(p ) we would use optimizing a model to a new function , g(p ) , where we consider both as purely dependent on the parameter if we constrain parameters using a number of model outputs mi(p ) , with data di then f ( in the simplest , unweighted case ) would be : f(p ) = (mi(p)di)2 . to generate plausible patients , we modify this sumofsquared errors expression to : g(p ) = imax[(mi(p)li+ui2)2(ui2li2)2,0]where ui and li are the predefined plausible upper and lower bounds , respectively , for mi(p ) . this expression ensures that if mi(p ) is in the plausible range then the contribution of the corresponding term in the expression is zero . the effect of replacing f(p ) with g(p ) is visualized in 2d in figure 2 . outputs of the model contribute to the cost function to be minimized by considering the sum of squared errors ( sse ) from an associated experimental observation . for each observation we define a physiologically plausible range ( arrows in a , b ) and shift the sse associated with that observation so that it is zero if the model output is in this range ( a , b ) . combining these transformations in each dimension leads to a broader cost function that is minimized by many points , rather than one ( black rectangle in c ) . to test this approach we used a previously published model of cholesterol metabolism.17 we chose this model because we could use publicly available data from the nhanes database16 to establish the empirical multivariate distribution for ldl cholesterol , hdl cholesterol , and total cholesterol ( ldlc , hdlc , and tc , respectively ) . note that the distribution of these variables is well approximated by a multivariate lognormal distribution ( supplementary figure 1 ) . for the remainder of the article we will describe these variables , either as model outputs or from nhanes , in log units ( prior to taking the logarithm , units are mg / dl for cholesterol measures ) . the published version of this model does not explicitly calculate ldlc or tc ; instead , the outputs are hdlc and nonhdlc . from these two quantities tc is easily calculated . for full comparison with the nhanes data we introduced a new parameter to the model , input_ranges.m gives details on parameter and output ranges for the van de pas model . also in the supplementary material is the code used in this case , which is easily modifiable for application to other models . as expected , the initial plausible population does not match the populationlevel statistics of the nhanes data ( figure 3 ) but covers the empirical distribution ( i.e. , where there are likely to be empirical observations there are plausible patients ) . comparison of the initial plausible population ( n = 300,000 ) with nhanes multivariate distribution ( ( ac ) black dotted lines estimated pdf , supplementary figure 1ac . ( df ) 2d projection of the 95% confidence surface of the estimated probability density function ) . once calculated , we established that most of the plausible patients are highly unlikely to be in the final distribution ( figure 4 ) . this is due to the relative density of the plausible population to the empirical distribution . with these probabilities , only 2% of the plausible population was selected to be in the virtual population ( inset , figure 4 ) . based on examining goodnessoffit of the distributions it appears , in this case , there is no further value in increasing the size of the plausible population ( supplementary figure 2 ) . the red histogram ( main figure , and figure inset ) is a virtual population that matches nhanes data , and is selected from the plausible population ( blue histogram ) based on displayed probability . the distribution of an example selection fits the nhanes data well ( figure 5 ) . the 1d histograms ( when normalized for comparison with the nhanes probability density function ) are indistinguishable from the data ( figure 5 a c ) and the correlations between variables also match the data based on visual predictive check . the virtual population ( red dots and red histogram ) matches the mean , variance , and covariance of the multivariate experimental distribution ( ( ac ) black dotted lines estimated probability density function , supplementary figure 1ac . ( df ) 2d projection of the 95% confidence surface of the estimated pdf ) . when selecting a subset of vps from a larger population , one concern is that the selected subset of vps does not reflect the variability of the original ensemble , which was generated from the biologically plausible range of the parameters . analyzing the final fitted population , we found little change in either the distribution of parameters or the correlation structure between the parameters ( figure 6 and supplementary figure 3 ) . this also shows that despite the virtual population being constrained against the nhanes data the parameter values of the virtual population ( figure 6 b ) are only slightly better constrained than those of the plausible population . furthermore , correlations between parameters are only slightly increased in the virtual population ( figure 6 d ) vs. the plausible population ( figure 6 c ) . at least in this case , constraining all outputs into realistic ranges is a more stringent constraint than selection of a virtual population . violin plots of the plausible and virtual populations ( a , b , respectively ) parameter values ( normalized to each parameter 's upper and lower bounds ) and correlation matrix of the plausible and virtual population ( c , d , respectively ) . one of the primary uses for qsp models is to prospectively simulate the effects of a dynamic perturbation ( pharmacological or otherwise ) in populations of interest . due to the underconstrained nature of these models it would be difficult to have confidence in the simulation results if we simulated a single parameterization of that model , even if that set of parameters is an excellent fit to the available data . for example , imagine creating a single hypercholesterolemia vp to simulate the effect of various anticholesterol therapies . for the baseline characteristics of the vpop , we have good data for the expected mean ldl and hdl ( e.g. , a prior clinical cohort ) , but we could still choose to mechanistically represent hypercholesterolemia several ways using the same model . we could increase cholesterol production , decrease clearance , or apply some combination of both . our choice of how to parameterize that vp could have significant consequences for the sensitivity of the followon therapy simulations . having impaired production vs. clearance of ldl could lead to differential responses to statins ( production ) vs. antiproprotein convertase subtillisin / kexin type 9 ( clearance ) . a better approach is to explore the underconstrained nature of these models and sample the biological uncertainty in the creation of plausible patients by varying mechanistic parameters , such as production and clearance rates , within biologically reasonable ranges . simultaneously , we need to constrain the higherlevel observables of the model based on known population distributions ( e.g. , the baseline characteristics of a clinical trial cohort ) . an appealing aspect of the approach we outline is that , since the algorithm is probabilistic , once the plausible patients are generated any number of subpopulations can be selected as long as the generated patients reasonably cover the full range of the ( sub)population . additionally , for any particular population , any number of vpops can be reselected to bootstrap the sensitivity of the model predictions to the choice of vpop . achieving an acceptable fit to the data distribution however , if we have higherorder density functions , we will likely require additional gains in efficiency , above and beyond what is presented here , in methods for generating sufficient plausible patients . one potential avenue , for a future iteration of this algorithm , may be to use methods that follow a directed search through the parameter space , such as using markov chain monte carlo ( mcmc ) algorithms.18 , 19 , 20 however , it should be noted that the method presented here is in fact a hybrid approach because the simulated annealing step , used to generate a plausible patient is essentially an mcmc method . the advantage of this approach is it generates plausible patients ( and hence virtual patients ) independently which is critical for the purpose of making a virtual population . also , once the plausible population is established new virtual populations , suitable for new applications , can be selected . however , a limitation of our approach is the computational cost of generating large plausible populations , which may make this method unfeasible in models that are slow to simulate . it should be noted that prior methods13 , 14 generated smaller virtual populations in larger models , partly due to the computational cost of running the models . nevertheless , the computational efficiency of a model does not alter the necessity for exploring the parameter space ( which in this context equates to larger virtual populations ) . we therefore advocate for the optimization of large models for speed , such that a fuller exploration of parameter uncertainty is possible ( either via the method presented here or alternatives ) . it is important to remark that the larger the model ( specifically , in terms of the number of poorly constrained parameters ) the greater the necessity for larger virtual populations to attempt to account for the uncertainty inherent in such a model . an advantage of our technique over prior approaches is that it is relatively unbiased , while still leveraging all available information on possible parameter values . the approach by klinke may overweight spurious model solutions , whereas the approach by schmidt et al . requires binning of parameter values into mechanistic axis which , for axes containing more than one parameter , requires an assumption about the correlation between parameters in the population of interest that may not be supported by available data . as an introduction to this algorithm , we demonstrated how to generate a vpop that matches the baseline characteristics of a population or clinical cohort ; however , in practice , a dynamic model should be constrained additionally against as many inscope perturbation experiments as possible ( determined by available data ) . for this example , simulating changes in ldlc and tc to standardofcare lipid therapies , such as statins and ezetimibe , would likely be an important step before using the model to predict the response to a novel mechanism . ideally , information would be available detailing the distribution of the data before and after the application of therapy ( i.e. , not just summary statistics ) . the therapeutic response can be treated as a baseline constraint for vp selection , just as we used hdlc and tc at baseline . we have applied this approach , without major adaptation , to an unpublished model of chronic kidney disease ( 42 ordinary differential equations ( odes ) , 200 parameters ) and to a model of body weight change6 ( 8 odes , 50 parameters ) ; we have made a brief summary of the results in these cases available.21 one challenge that we foresee , for virtual populations in general ( i.e. , not specific to this approach ) , is development of efficient ways to combine populations when merging distinct models . we believe that for two models that have large validated virtual populations , navely combining every possibility will be computationally daunting . quantitative systems pharmacology models are becoming established as a valuable component of the drug discovery and development process . communicating their complexity and uncertainty to an interdisciplinary project team is a critical but challenging component of their utility . virtual populations are one tool that we have found to be successful in exploring mechanistic and parametric uncertainty in an intuitive framework that is easily understandable by most audiences . however , despite their widespread use , there are very few published methods for generating virtual patients and forming virtual populations . here we have contributed an approach to efficiently generate virtual patients and construct a virtual population for which each patient is weighted equally . plausible populations , made up of plausible patients each of which is a candidate to become a virtual patient . because a large plausible population is necessary , models that are slow to integrate ( for example , with dynamics across multiple timescales ) may not be good candidates for this approach . however , in cases where quantitative predictions are required and the model is amenable to thorough examination of parameter space , we have found this method to be an improvement over previous approaches . supporting information click here for additional data file . supporting information click here for additional data file . supporting information click here for additional data file . supporting information click here for additional data file .

quantitative systems pharmacology models mechanistically describe a biological system and the effect of drug treatment on system behavior . because these models rarely are identifiable from the available data , the uncertainty in physiological parameters may be sampled to create alternative parameterizations of the model , sometimes termed virtual patients . in order to reproduce the statistics of a clinical population , virtual patients are often weighted to form a virtual population that reflects the baseline characteristics of the clinical cohort . here we introduce a novel technique to efficiently generate virtual patients and , from this ensemble , demonstrate how to select a virtual population that matches the observed data without the need for weighting . this approach improves confidence in model predictions by mitigating the risk that spurious virtual patients become overrepresented in virtual populations .

METHODS RESULTS DISCUSSION Supporting information

PMC4540125

although chemical substances are a very important factor in the realization of current industrialization , because they were used indiscriminately without a full understanding of the damage they could cause , issues have arisen on a continual basis , such as global environment changes like ozone depletion and global warming , and chemical damage cases like minamata disease and itai - itai disease , so that the importance of safe chemical use has emerged . consequently , the major developed countries and international society agreed in 1972 , at the united nations ( un ) conference on the human environment held for safe use and control of chemicals , to the need for a specialized agency affiliated to the un to deal with global environmental problems ; the the un environment programme was thus established through the 27th un general assembly . further efforts to ensure the safer use of commercial chemicals and for reduction in the use of hazardous chemicals have included the stockholm convention on persistent organic pollutants for restriction of volatile organic compounds ( vocs ) and the rotterdam convention to regulate the international trade in hazardous substances . p. t. anastas of the us environmental protection agency ( epa ) proposed green chemistry theory in 1998 , arguing for a systemic reduction in the use of hazardous chemicals and the safer use and control of chemicals . the theory is composed of 12 principles to reduce or remove the harmfulness of chemicals throughout their entire life - cycle , from raw material step to the waste disposal step , via manufacturing and production to safer use of hazardous chemicals . risk assessment studies of chemicals and research into the development of alternative chemicals have been actively carried out , in recognition of their value as a practical alternative for the establishment of an infrastructure for green chemistry ; such investigations have been led by the major developed countries . in addition , national support for the establishment of an infrastructure for green chemistry has been provided , along with award programs , to encourage the active participation of industry . the green chemistry market of south korea ( hereafter korea ) is about us$ 96.4 billion , placing it at 13th in the world , after major developed countries such as the us , china , and japan , and accounting for about 1.8% of the entire green chemistry market , alongside canada . the global green chemistry market is expected to grow continuously to about us$3000 billion by 2020 , and efforts to monopolize the green chemistry market have swiftly centered on the major developed countries . since it is necessary for korean industries , which occupy only 1.8% of the global green chemistry market , to enter into overseas markets , various environmental changes , along with growth of the green market , may provide a chance but also a crisis for korean industries , so that a response from the korean government is very crucial ( table 1 ) . t. anastas , the epa has maintained various policies to spread green chemistry , including environmental assessments of hazardous chemicals by the federal government ; relevant research and education ; support for environment - related industries , such as prevention programs and energy saving ; the presidential green chemistry challenge awards ; and support for the reduction in use and generation of hazardous substances . the epa also developed and distributed a tool to provide information about the results of the policies related to green chemistry , including green chemistry expert system ( gces ) and green chemistry assistant ( gca ) . gces is composed of five modules that enable industries to identify information for evaluation and prediction , leading to the selection of safer green chemistry alternative chemicals . first , the synthetic methodology assessment for reduction techniques module receives inputs such as information about chemicals , number of reactions , and yield of reactions ; identifies properties of the chemical reaction process ; and estimates and assesses the amounts of products and wastes . second , the green synthetic reaction module makes it possible to identify reaction information to synthesize chemicals in a safer and less hazardous way , with the input of basic information about chemicals . third , the designing safer chemicals module proposes compound designs and analyzes toxic mechanisms , which supports the prediction of structures that can reduce toxicity . fourth , the green solvent / reaction condition module makes it possible to identify information on the physical - chemical properties of about 600 different solvents , as well as the reaction conditions for some alternative chemicals that have been confirmed . fifth , the green chemistry reference module has been designed to search and identify reports and data related to studies on green chemistry . a web - based program developed collaboratively by the epa and st . olaf college , gca has functions to calculate the theoretical yields of reactions , using chemical reaction equations , and the appropriate amounts of chemicals that are required for reactions ; this avoids the need to use chemicals during design . in addition , it provides information on about 60000 chemicals and about 600 solvents through a search function linked with gces database . state governments and a number of colleges in the us have made various attempts to establish an infrastructure for green chemistry , alongside the us federal government . massachusetts state has established and operates the toxics use reduction institute , in collaboration with industries and government agencies , in line with the toxics use reduction act for research and public relations on the reduction in the use of hazardous chemicals . to reduce hazardous chemicals , the institute has been running various environmental protection programs , including continuous education for industry and the general public , reduction technology , funding , a chemical fact sheet provided by the toxic use reduction science advisory board , and evaluation of research on alternatives to hazardous chemicals like hexavalent chrome and diethylhexyl phthalate . the institute has also developed pollution prevention options assessment system , an excel - based program that has the function of calculating the harmfulness scores of chemicals , based on the collected toxicity information , in order to help users to understand objectively and easily and to allow them to evaluate comparatively . massachusetts institute of technology of the us has formed a network of research organizations , academics , and research institutes related to green chemistry . this network has constructed a database focusing on data from major journals and has been running the green alternatives purchasing wizard , which provides information about hazardous chemicals . this is part of an eco - friendly purchasing policy to minimize waste and prevent pollution , in line with the resource conservation and recovery act . the wizard is a web - based program that was developed for cost reduction purposes achieved through reduction in hazardous wastes and environmental loads from research institutes . it also provides information about alternative chemicals , their advantages and disadvantages , and sources ; it is linked to a purchasing system called systems applications and products to facilitate purchases ( figure 1 ) . clean production action ( cpa ) is a project run by the tides center , a non - profit profit organization that financially supports innovative and creative groups in the us . in order to provide green chemistry solutions to the design and production of sustainable eco - friendly products , for the safety of products and materials , and for rapid and effective methods of toxicity evaluation , cpa has produced green screen , composed of four categories environmental fate , eco - toxicity , human health , and physical- chemical properties and 17 detailed evaluation items , presented in four types , as benchmark 1 to 4 , based on the evaluation results . the epa of denmark developed a web - based program in 2003 to search for hazardous chemicals and alternatives , based on a case report called a catalogue of example of substitution of hazardous chemicals , which in 1989 provided information on alternatives to 162 hazardous chemicals and actual cases that utilized alternatives . the program provides information , collected from danish businesses , about alternative chemicals and technologies , the occupational health service , and the danish working environment authority . the program provides information on 311 actual cases in 83 companies where alternative chemicals were used and encourage more active use of alternative chemicals in industry . in 2009 , the ministry of environment of korea formed a forum composed of representatives from industry , academia , and research institutes in recognition of the strengthening international regulation of hazard and risk controls of chemicals ; the ministry proposed a measure for the advance of chemical management to move toward a green chemistry system in order to improve the chemical management system in korea and to support the chemical industry . accordingly , the green shift to move toward green chemistry has ultimately been pursued in the direction of advancing management of the risks of hazardous chemicals ; of safety management , people s health , and environmental protection from chemicals and chemical products ; of advances in the management of chemical information ; of the construction of mutual communication and cooperation systems between interested parties;and of the attainment of two goals : health and environmental protection and improvement of the competitiveness of the chemical industry . as a part of this project , the ministry of environment of korea has run the next - generation eco - innovation ( ei ) project since 2011 , with the goals of constructing the infrastructure for green chemistry in industrial facilities in korea and of improving foreign trade competitiveness . the project also had the research group on green chemistry management techniques along with four sub - organizations , led by the korea environment corporation , in order to construct the green chemistry policy / application support system . in particular , the project has been developing a search program for related alternative chemicals for small and medium - sized businesses that have difficulty being competitive because of difficulties in accessing information about the regulation of chemicals and the regulation of alternative chemicals in korea and other countries . the program has enabled them to identify and search for information about reliable alternative chemicals and rapidly changing international chemical regulations . the project has collected information about alternative chemicals based on domestic and international journals , research data , and particularly patent data . it has analyzed this information , verified it with the help of experts in the field to secure the reliability of data , and then presented it . in addition , successful cases of the application of alternative chemicals to actual processes were focused on in the process of collection and presented in order to increase the applicability of alternative chemicals . at present , information on a total of 341 alternative chemicals by use and 75 application cases have been collected and provided . the program can be downloaded from the next - generation ei research group homepage and the homepage of to21 co. , ltd . ; it is distributed to companies free of charge ( figure 2 ) . t. anastas , the epa has maintained various policies to spread green chemistry , including environmental assessments of hazardous chemicals by the federal government ; relevant research and education ; support for environment - related industries , such as prevention programs and energy saving ; the presidential green chemistry challenge awards ; and support for the reduction in use and generation of hazardous substances . the epa also developed and distributed a tool to provide information about the results of the policies related to green chemistry , including green chemistry expert system ( gces ) and green chemistry assistant ( gca ) . gces is composed of five modules that enable industries to identify information for evaluation and prediction , leading to the selection of safer green chemistry alternative chemicals . first , the synthetic methodology assessment for reduction techniques module receives inputs such as information about chemicals , number of reactions , and yield of reactions ; identifies properties of the chemical reaction process ; and estimates and assesses the amounts of products and wastes . second , the green synthetic reaction module makes it possible to identify reaction information to synthesize chemicals in a safer and less hazardous way , with the input of basic information about chemicals . third , the designing safer chemicals module proposes compound designs and analyzes toxic mechanisms , which supports the prediction of structures that can reduce toxicity . fourth , the green solvent / reaction condition module makes it possible to identify information on the physical - chemical properties of about 600 different solvents , as well as the reaction conditions for some alternative chemicals that have been confirmed . fifth , the green chemistry reference module has been designed to search and identify reports and data related to studies on green chemistry . a web - based program developed collaboratively by the epa and st . olaf college , gca has functions to calculate the theoretical yields of reactions , using chemical reaction equations , and the appropriate amounts of chemicals that are required for reactions ; this avoids the need to use chemicals during design . in addition , it provides information on about 60000 chemicals and about 600 solvents through a search function linked with gces database . state governments and a number of colleges in the us have made various attempts to establish an infrastructure for green chemistry , alongside the us federal government . massachusetts state has established and operates the toxics use reduction institute , in collaboration with industries and government agencies , in line with the toxics use reduction act for research and public relations on the reduction in the use of hazardous chemicals . to reduce hazardous chemicals , the institute has been running various environmental protection programs , including continuous education for industry and the general public , reduction technology , funding , a chemical fact sheet provided by the toxic use reduction science advisory board , and evaluation of research on alternatives to hazardous chemicals like hexavalent chrome and diethylhexyl phthalate . the institute has also developed pollution prevention options assessment system , an excel - based program that has the function of calculating the harmfulness scores of chemicals , based on the collected toxicity information , in order to help users to understand objectively and easily and to allow them to evaluate comparatively . massachusetts institute of technology of the us has formed a network of research organizations , academics , and research institutes related to green chemistry . this network has constructed a database focusing on data from major journals and has been running the green alternatives purchasing wizard , which provides information about hazardous chemicals . this is part of an eco - friendly purchasing policy to minimize waste and prevent pollution , in line with the resource conservation and recovery act . the wizard is a web - based program that was developed for cost reduction purposes achieved through reduction in hazardous wastes and environmental loads from research institutes . it also provides information about alternative chemicals , their advantages and disadvantages , and sources ; it is linked to a purchasing system called systems applications and products to facilitate purchases ( figure 1 ) . clean production action ( cpa ) is a project run by the tides center , a non - profit profit organization that financially supports innovative and creative groups in the us . in order to provide green chemistry solutions to the design and production of sustainable eco - friendly products , for the safety of products and materials , and for rapid and effective methods of toxicity evaluation , cpa has produced green screen , composed of four categories environmental fate , eco - toxicity , human health , and physical- chemical properties and 17 detailed evaluation items , presented in four types , as benchmark 1 to 4 , based on the evaluation results . the epa of denmark developed a web - based program in 2003 to search for hazardous chemicals and alternatives , based on a case report called a catalogue of example of substitution of hazardous chemicals , which in 1989 provided information on alternatives to 162 hazardous chemicals and actual cases that utilized alternatives . the program provides information , collected from danish businesses , about alternative chemicals and technologies , the occupational health service , and the danish working environment authority . the program provides information on 311 actual cases in 83 companies where alternative chemicals were used and encourage more active use of alternative chemicals in industry . in 2009 , the ministry of environment of korea formed a forum composed of representatives from industry , academia , and research institutes in recognition of the strengthening international regulation of hazard and risk controls of chemicals ; the ministry proposed a measure for the advance of chemical management to move toward a green chemistry system in order to improve the chemical management system in korea and to support the chemical industry . accordingly , the green shift to move toward green chemistry has ultimately been pursued in the direction of advancing management of the risks of hazardous chemicals ; of safety management , people s health , and environmental protection from chemicals and chemical products ; of advances in the management of chemical information ; of the construction of mutual communication and cooperation systems between interested parties;and of the attainment of two goals : health and environmental protection and improvement of the competitiveness of the chemical industry . as a part of this project , the ministry of environment of korea has run the next - generation eco - innovation ( ei ) project since 2011 , with the goals of constructing the infrastructure for green chemistry in industrial facilities in korea and of improving foreign trade competitiveness . the project also had the research group on green chemistry management techniques along with four sub - organizations , led by the korea environment corporation , in order to construct the green chemistry policy / application support system . in particular , the project has been developing a search program for related alternative chemicals for small and medium - sized businesses that have difficulty being competitive because of difficulties in accessing information about the regulation of chemicals and the regulation of alternative chemicals in korea and other countries . the program has enabled them to identify and search for information about reliable alternative chemicals and rapidly changing international chemical regulations . the project has collected information about alternative chemicals based on domestic and international journals , research data , and particularly patent data . it has analyzed this information , verified it with the help of experts in the field to secure the reliability of data , and then presented it . in addition , successful cases of the application of alternative chemicals to actual processes were focused on in the process of collection and presented in order to increase the applicability of alternative chemicals . at present , information on a total of 341 alternative chemicals by use and 75 application cases have been collected and provided . the program can be downloaded from the next - generation ei research group homepage and the homepage of to21 co. , ltd . ; it is distributed to companies free of charge ( figure 2 ) . in recent international trade , the free trade agreement has promoted an increase in trade between countries by removing or reducing the existing trade barriers , such as customs or import restrictions . as such , major developed countries have utilized technology regulations called technical barriers to trade ( tbt ) as new non - tariff barriers , including energy saving , a reduction of carbon emissions , and environment - related standards , in order to protect the environment but also the trade of their own countries . according to a report from the world trade organization , the number of tbt notice letters increased from 638 cases in 2004 to 1491 in 2013 . in addition , there have been various efforts to develop alternative chemicals and alternative process technologies to meet stronger technology regulations . accordingly , various environment - related projects , including the next - generation ei project , have been implemented in collaboration with industry , academia , and research institutes in korea since 2011 , in order to improve the international competitiveness of korean businesses ; through the project , various forms of content were developed and provided to companies . in addition , the existing hazardous chemical control act has been completely changed , becoming the act on the registration and evaluation , etc . of chemical substances and the chemical control act , based on the obtained data , to protect the korean environment and businesses , and through which korea has endeavored more complete chemical control . previously , the chemical industry was evaluated by its ability to synthesize a higher amount of chemicals and to develop new chemicals . however , conversion to green chemistry , which uses chemicals that are safer and less harmful , will be an important force for national growth in the future , and it may provide a good opportunity to secure the competitiveness of korean industrial facilities in the international markets .

objectivesdespite the great contribution made by chemical substances to the development of modern civilization , their indiscriminate use has caused various kinds of damage to the global environment and human beings . accordingly , the major developed countries and international society have tried to ensure the safe use of chemicals and a reduction in the use of hazardous chemicals through the establishment of the united nations environment programme and various international agreements . in this reason , we tried to introduce about green chemistry progress at the present in worldwide and korea.methodswe checked and analyzed relative journals , reports using keyword as like green chemistry , alternative chemicals , eco - friendly etc . and major country s government homepage search.resultsgreen chemistry theory , which argues for the reduction or removal of harmfulness in chemicals throughout their entire life - cycle , has been spreading , and major developed countries , such as the us and denmark , have developed and operate programs to provide reliable chemical information to help replace hazardous chemicals . korea has also been conducting studies as like eco - innovation project . through this project the alternative chemical search program , has been developed , distributed , and operated since 2011 to provide reliable information to small and medium - sized businesses that have difficulties collecting information to ensure conformity to international regulations . the program provides information that includes the regulations of major countries and korea , information on 340 alternative chemicals , 70 application cases , and 1:1 consulting.conclusionsthe alternative chemical search program is expected to contribute to the establishment of response systems for regulation of korean small and medium - sized businesses , and it also will be used to provide basic data for korean hazardous chemical regulation , together with the act on the registration and evaluation , etc . of chemical substances and the chemical control act , making it possible to establish an infrastructure for green chemistry in korea and to increase national competitiveness .

Introduction Research Conditions for Green Chemistry in Foreign Countries US Environmental Protection Agency Toxics Use Reduction Institute (US) Massachusetts Institute of Technology (US) Clean Production Action (US) Catalogue of Example of Substitution of Hazardous Chemicals (Denmark) Current Support Situations for Green Chemistry in Korea Conclusion

PMC4506206

down syndrome ( ds ) is one of the most frequent congenital disorders in humans . many of them develop symptoms of alzheimer s disease ( ad ) at a relatively young age , and a high proportion of ds individuals develop dementia at a later stage . ds is caused by a trisomy of chromosome 21 as a consequence of nondisjunction during meiotic cell division . although the mechanisms leading from trisomy 21 to the clinical disease pattern are not yet understood , the 30 genes located in the so - called down syndrome critical region ( dscr ; 21q22.122.3 ) of chromosome 21 are highly likely to be associated with the disease . there is mounting evidence that overexpression of dyrk1a ( dual - specificity tyrosine phosphorylation - regulated kinase 1a ) , a protein kinase encoded by a gene located within dscr , contributes to mental retardation in ds . since dyrk1a is also connected to neurological disorders such as ad , the early onset of ad in ds individuals has been related to dyrk1a overexpression . two histopathological features are found in the brains of ad patients : extracellular -amyloid plaques and intracellular tangles consisting of hyperphosphorylated tau protein . hyperphosphorylation of tau by dyrk1a diminishes its microtubule - stabilizing effects and increases aggregation . the neurological impairment of mice with modified dyrk1a expression has also been attributed to deregulated splicing , leading to an imbalance of 3r - tau and 4r - tau isoforms . the increased 3r - tau concentration ( by a factor of up to 4 ) observed in ds has been associated with changes of the neuronal cytoskeleton and neurofibrillary degeneration . dyrk1a - overexpressing mice also exhibit increased production of a peptide , which has been attributed to phosphorylation of the amyloid precursor protein ( app ) and presenilin 1 by dyrk1a . dyrk1a is a member of the dyrk protein kinases ( dyrk1a , dyrk1b , dyrk2 , dyrk3 , dyrk4 ) , which are part of the cmgc superfamily and share structural similarity of the catalytic domain and a small sequence nearby , the so - called dyrk homology box ( dh - box ) . although dyrks are serine / threonine kinases , autophosphorylation occurs at a conserved tyrosine residue of the activation loop . this autophosphorylation is constitutive and seems to be not related to regulation . in the light of its involvement in ds and other neurodegenerative disorders , the inhibition of dyrk1a with small chemical inhibitors has been suggested as a therapeutic strategy . selective dyrk1a inhibitors would also be valuable tools for the investigation of the role of dyrk1a in physiological and pathobiochemical processes . to date , most protein kinase inhibitors exert their activity by competing with atp in the binding pocket of the kinase . since atp binding sites of protein kinases share a common structure with subtle differences , selectivity with regard to closely related kinases is not easily achieved . when selective dyrk1a inhibitors are to be developed , special attention should therefore be devoted to other members of the cmgc superfamily , consisting of cdks , mapkinases ( such as erks ) , gsk-3 , and cdc - like kinases . the structures and properties of dyrk1a inhibitors have been summarized in recent reviews . a well established dyrk1a inhibitor is the -carboline alkaloid harmine ( 1 , chart 1 ) , which however also shows high affinity for serotonine and tryptamine receptor binding sites , acts as monoamine oxidase a ( mao a ) inhibitor , and also inhibits clks and therefore is inappropriate for use as a cellular chemical probe . epigallocatechin 3-gallate ( egcg ) is a polyphenolic constituent of green tea reported to inhibit dyrk1a in a non - atp - competitive manner , but it is chemically reactive and also interacts with numerous intracellular signaling pathways by other mechanisms . the benzothiazole derivative indy showed potent inhibition of dyrk1a and related kinases ( dyrk1b , dyrk2 , dyrk3 , clk1 , clk4 , ck1 , and pim1 ) . a prodrug of this compound , proindy ( 2 ) , has been shown to protect xenopus tadpoles that overexpress dyrk1a against head malformation during development . leucettine l41 ( 3 ) , derived from the marine natural product leucettamine b , is an atp - competitive inhibitor of dyrks and clks that also interacts with gsk-3 and ck2 to a lower extent . it modulates pre - mrna splicing , protects ht22 hippocampal cells from glutamate - induced cell death , induces autophagy , and inhibits phosphorylation of tau on thr212 . similar to the agents mentioned before , the recently reported chemical dyrk1a inhibitors comprising meridianines , indirubin 5-carboxylates , thiazolo[5,4-f]quinazolines , pyrido[2,3-d]pyrimidines , 3,5-diaryl-7-azaindoles ( dandys ) , kh - cb19 , 2,4-bisubstituted thiophenes , and hydroxybenzothiophenes either show limited selectivity against structurally closely related dyrk and clk kinase isoforms or were not tested on these enzymes . in this regard , a dyrk1a inhibitor with high selectivity over other cmgc kinases would be useful for biochemical and cellular studies and as lead motif for the development of new pharmaceuticals targeting neurodegenerative diseases . to identify novel hit matter , we tested a small diverse in - house compound library against dyrk1a and the following cmgc protein kinases : cdk1/cyclin b , cdk2/cylin a , cdk5/p25 , ck1 , gsk-3 , and erk2 . the only compound showing a moderate selective dyrk1a inhibition ( ic50 = 2.6 m ) was the 11h - indolo[3,2-c]quinoline-6-carboxylic acid 5a ( table 1 ) . experimental dyrk1a inhibitors described so far have suffered from poor selectivity toward other closely related protein kinases , namely , other dyrks and members of the clk family . we have developed two 10-iodo-11h - indolo[3,2-c]quinoline-6-carboxylic acids 5j and 5o as the first selective inhibitors of dyrk1a . docking studies and x - ray structure analyses revealed a nonclassical binding mode in which inhibitors were oriented via the 10-halogen substituent toward the hinge region of the kinase . although the 10-substituent obviously was of central importance for the hinge inhibitor attraction , the nature of this interaction is not yet clear . the title compounds demonstrate that within the cmgc family of protein kinases selective inhibition of dyrk1a is possible . with the aim of developing compounds that are better suited for cellular assays or animal disease models , further studies in this compound class will be directed to increase solubility and cell permeability . starting materials were purchased from acros organics ( geel , belgium ) or sigma - aldrich ( steinheim , germany ) . solvents were used as commercially available grades for synthesis , with the exceptions of toluene , thf , ch2cl2 , and diethyl ether , which were dried and purified by published methods.5b , 5c , and 5f were prepared as reported previously . synthetic procedures and structure characterization data for the following compounds are available in the supporting information : 5a , 5d e , 5g n , 5p w , 6 , 7 , 10n , s , t . melting points ( mp ) were determined on an electric variable heater ( electrothermal ia 9100 , barnstedt international , southend - on - sea , u.k . ) in open glass capillaries . ir spectra were recorded as kbr discs on a thermo nicolet ft - ir 200 . h nmr spectra and c nmr spectra were recorded on the following instruments : bruker avance drx-400 , bruker avance iii-400 , and bruker avance ii-600 ( bruker , billerica , ma , usa ) ; internal standard tetramethylsilane ; signals in ppm ( scale ) . signals in c spectra were assigned based on the results of c dept135 experiments ( nmr laboratories of the chemical institutes of the technische universitt braunschweig ) . elemental analyses were determined on a ce instruments flashea 1112 elemental analyzer ( thermo quest ) . mass spectra were recorded on a finnigan - mat 95 ( thermo finnigan mat , bremen , germany ) . accurate measurements were conducted according to the peak match method using perfluorokerosene ( pfk ) as an internal mass reference . ( ei ) ms : ionization energy 70 ev ( department of mass spectrometry of the chemical institutes of the technische universitt braunschweig ) . tlc : polygram sil g / uv254 , macherey - nagel , 40 mm 80 mm , visualization by uv illumination ( 254 and 366 nm ) . isocratic elution : elite lachrom ( merck / hitachi ) , pump l-2130 , autosampler l-2200 , diode array detector l-2450 , organizer box l-2000 ; column , merck lichrocart 1254 , lichrosphere 100 , rp 18 , 5 m ; flow rate 1.000 ml / min ; volume of injection , 10 l ; detection ( dad ) at 254 and 280 nm ; auc , % method ; time of detection 15 min , net retention time ( tn ) , dead time ( tm ) related to dmso . gradient elution : elite lachrom ( merck / hitachi ) , pump l-2130 , autosampler l-2200 , uv detector l-2400 , organizer box l-2000 ; column , merck lichrocart 125 - 4 , lichrosphere 100 , rp 18 , 5 m ; flow rate 1.000 ml / min ; volume of injection , 10 l ; detection at 254 nm ; auc , % method ; net retention time ( tn ) , dead time ( tm ) related to dmso . for all gradient runs , mixtures of acn and water or aqueous formic or trifluoroacetic acid were used as specified for particular compounds . preparation of h2o + ( et3nh)2so4 buffer ( ph 2.6 ) for isocratic hplc : triethylamine ( 20.0 ml ) and sodium hydroxide ( 242 mg ) are dissolved in water to 1 l. the solution is adjusted to ph 2.6 by addition of sulfuric acid . all compounds that were biologically tested were of > 95% purity with the exception of 5h ( hplc , 93.6% at 254 nm and 94.8% at 280 nm detection wavelength ) and 5w ( hplc , 95.1% at 254 nm and 94.2% at 280 nm detection wavelength ) . absorption maxima ( max ) were extracted from the spectra recorded by the dad in the hplc peak maxima in isocratic runs ( software , ez chrom elite client / server , version 3.1.3 . ) . an appropriate substituted 3,4-dihydro-1h-1-benzazepine-2,5-dione ( 1 equiv ) and an appropriately substituted phenylhydrazine hydrochloride ( 1.21.5 equiv ) and sodium acetate ( 1.21.5 equiv ) are stirred in acetic acid ( 10 ml ) at 70 c for the indicated time . sulfuric acid ( 0.1 ml ) is added , and stirring at 70 c is continued for the indicated time . after cooling to room temperature , the mixture is poured into 5% aqueous sodium acetate solution ( 20 ml ) and kept at 8 c for 2 h. a precipitate is formed , which is filtered off with suction , washed with water and petrol ether . the appropriate paullone 4 ( 1 equiv ) , nhpi ( 2 equiv ) , and cobalt(ii ) acetate tetrahydrate ( 0.25 equiv ) are dissolved in the given volume of dmf . oxygen is bubbled through the stirred mixture for the given time at the indicated temperature . if no precipitate appears , water ( 5 ml ) is added and the resulting precipitate is separated and purified by centrifugation , decanting , and washing with water . the resulting solid is washed with a small amount of acetone . the predried product is finally dried at 130 c in vacuo . ethanol ( 100 ml ) is added to a solution of an appropriate 11h - indolo[3,2-c]quinoline-6-carboxylic acid 5 in dmso ( 5 ml ) . hydrogen chloride gas is bubbled through the solution for 30 min at room temperature . after removal of the ethanol by evaporation , water ( 100 ml ) is added and the mixture is kept at 8 c for 1 h. the resulting precipitate is filtered off , washed with water , and dissolved in acetone ( 5 ml ) . after addition of silica gel ( 2 g ) , the mixture is evaporated . the product is subsequently eluted from the silica gel by flash chromatography using the indicated eluent . synthesis was according to general procedure a from 3,4-dihydro-1h-1-benzazepine-2,5-dione ( 88 mg , 0.50 mmol ) , 2-iodophenylhydrazine hydrochloride ( 203 mg , 0.750 mmol ) , and sodium acetate ( 62 mg , 0.75 mmol ) . reaction times were 1 h before and 4 h after addition of sulfuric acid , respectively . formic acid 5:5:1 ) yielded a dark yellow powder ( 51 mg , 28% ) ; mp ( dec ) starting at 288 c . ir ( kbr ) : 3194 cm ( nh ) , 1666 cm ( c = o ) . h nmr ( dmso - d6 , 400.4 mhz ) : ( ppm ) = 3.47 ( s , 2h ) , 6.90 ( t , 1h , j = 7.6 hz ) , 7.227.31 ( m , 2h ) , 7.40 ( ddd , 1h , j = 7.9/7.4/1.6 hz ) , 7.59 ( dd , 1h , j = 7.5/0.9 hz ) , 7.71 ( d , 1h , j = 7.9 hz ) , 7.91 ( dd , 1h , j = 7.8/1.5 hz ) , 10.11 ( s , 1h , lactam nh ) , 11.29 ( s , 1h , indole nh ) . c nmr ( dmso - d6 , 100.7 mhz ) : ( ppm ) = 31.6 ( ch2 ) ; 118.0 , 121.1 , 122.1 , 123.3 , 128.3 , 128.5 , 131.5 ( ch ) ; 76.7 , 109.4 , 122.4 , 127.1 , 133.6 , 135.6 , 139.4 ( c ) ; 171.7 ( c = o ) . c16h11in2o ( 374.18 ) . ms ( ei ) : m / z ( % ) = 374 [ m ] ( 100 ) , 373 [ m h ] ( 35 ) , 345 [ m cho ] ( 49 ) . hrms ( ei ) : m / z [ m ] calcd 373.991 06 , found 373.990 84 . hplc ( isocr ) : 96.1% at 254 nm and 92.9% at 280 nm , tn = 4.7 min , tm = 1.0 min ( acn / h2o 50:50 ) ; max : 230 and 313 nm . synthesis was according to general procedure a from 8-methoxy-3,4-dihydro-1h-1-benzazepine-2,5-dione ( 500 mg , 2.44 mmol ) , 2-iodophenylhydrazine hydrochloride ( 993 mg , 3.67 mmol ) , and sodium acetate ( 301 mg , 3.67 mmol ) . reaction times were 3 h before and 16 h after addition of sulfuric acid , respectively . purification by column chromatography ( toluene ethyl acetate formic acid 5:5:1 ) yielded a dark yellow powder ( 340 mg , 34% ) ; mp ( dec ) starting at 290 c . ir ( kbr ) : 3205 cm ( nh ) , 1680 cm ( c = o ) . h nmr ( dmso - d6 , 600.1 mhz ) : ( ppm ) = 3.47 ( s , 2h ) , 3.81 ( s , 3h ) , 6.82 ( d , 1h , j = 2.6 hz , ) , 6.88 ( t , 1h , j = 7.7 hz ) , 6.91 ( dd , 1h , j = 8.7/2.6 hz ) , 7.55 ( dd , 1h , j = 7.5/0.9 hz ) , 7.67 ( d , 1h , j = 7.8 hz ) , 7.85 ( d , 1h , j = 8.7 hz ) , 10.04 ( s , 1h , lactam nh ) , 11.19 ( s , 1h , indole nh ) . c nmr ( dmso - d6 , 150.9 mhz ) : ( ppm ) = 55.2 ( ch3 ) , 31.6 ( ch2 ) , 106.6 , 109.9 , 117.6 , 121.0 , 129.6 , 130.9 ( ch ) , 76.5 ( c(11 ) ) , 107.6 , 115.3 , 127.1 , 133.8 , 136.9 , 139.0 , 159.2 ( c ) . c 50.51 , h 3.24 , n 6.93 , found c 50.38 , h 3.29 , n 6.72 . ms ( ei ) : m / z ( % ) = 404 [ m ] ( 100 ) , 403 [ m h ] ( 36 ) , 375 [ m cho ] ( 42 ) . hrms ( ei ) : m / z [ m ] calcd 404.001 62 , found 404.001 61 . hplc ( isocr ) : 96.0% at 254 nm and 95.2% at 280 nm , tn = 5.2 min , tm = 1.0 min ( acn / h2o 50:50 ) . synthesis was according to general procedure b1 from 11-iodo-7,12-dihydroindolo[3,2-d]benzazepin-6(5h)-one ( 4j , 43 mg , 0.11 mmol ) , nhpi ( 36 mg , 0.22 mmol ) , and cobalt(ii ) acetate tetrahydrate ( 7 mg , 0.03 mmol ) in dmf ( 2 ml ) . oxygen was bubbled through the reaction mixture at room temperature for 4 h. a yellow powder was formed , which was purified by washing with acetone ( 34 mg , 82% ) ; mp ( dec ) 358359 c . ir ( kbr ) : 3224 cm ( nh ) , 1659 cm ( c = o ) . h nmr ( dmso - d6 , 400.4 mhz ) : ( ppm ) = 7.10 ( t , 1h , j = 7.7 hz ) , 7.357.42 ( m , 1h ) , 7.567.62 ( m , 2h ) , 7.82 ( dd , 1h , j = 7.7/1.0 hz ) , 8.30 ( dd , 1h , j = 8.0/1.0 hz ) , 8.38 ( d , 1h , j = 8.0 hz ) , 11.54 ( s , 1h , nh ) , 11.95 ( s , 1h , cooh ) . c nmr ( dmso - d6 , 100.7 mhz ) : ( ppm ) = 121.2 ( 2c ) , 123.4 , 124.4 , 129.2 , 131.1 , 134.8 ( ch ) ; 76.7 ( c(10 ) ) , 115.5 , 117.2 , 127.3 , 135.2 , 139.3 , 142.5 , 161.8 ( c ) ; 176.3 ( c = o ) . ms ( ei ) : m / z ( % ) = 388 [ m ] ( 37 ) , 360 [ m co ] ( 100 ) . hrms ( ei ) : m / z [ m ] calcd 387.970 32 , found 387.968 99 . hplc ( isocr ) : 96.7% at 254 nm and 98.6% at 280 nm , tn = 2.8 min , tm = 1.0 min ( acn / h2o + ( et3n)2so4 50:50 ) . hplc ( grad ) : 97.4% at 254 nm , tn = 10.0 min , tm = 1.1 min ( acn / h2o ; 0 min , 10/90 13 min , 90/10 ( linear ) ; 20 min , 90/10 ) . synthesis was according to general procedure b1 from 11-iodo-3-methoxy-7,12-dihydroindolo[3,2-d]benzazepin-6(5h)-one ( 4o , 40 mg , 0.10 mmol ) , nhpi ( 33 mg , 0.20 mmol ) , and cobalt(ii ) acetate tetrahydrate ( 7 mg , 0.03 mmol ) in dmf ( 2 ml ) . oxygen was bubbled through the reaction mixture for 1 h. for the purification by column chromatography , impurities were removed initially by elution with toluene ethyl acetate diethylamine ( 1:1:1 ) prior to elution by toluene ethyl acetate yellow powder ( 20 mg , 48% ) ; mp ( dec ) 346348 c . ir ( kbr ) : 3200 cm ( nh ) , 1669 cm ( c = o ) . h nmr ( dmso - d6 , 600.1 mhz ) : ( ppm ) = 3.86 ( s , 3h ) , 7.01 ( dd , 1h , j = 9.0/2.6 hz ) , 7.07 ( t , 1h , j = 7.7 hz ) , 7.17 ( d , 1h , j = 2.5 hz ) , 7.78 ( dd , 1h , j = 7.5/0.9 hz ) , 8.31 ( dd , 1h , j = 7.9/0.7 hz ) , 8.36 ( d , 1h , j = 9.0 hz ) , 11.44 ( s , 1h , nh ) , 11.76 ( s , 1h , cooh ) . c nmr ( dmso - d6 , 150.9 mhz ) : ( ppm ) = 55.5 ( ch3 ) ; 104.9 , 110.6 , 121.1 , 124.2 , 130.8 , 134.4 ( ch2 ) ; 76.3 , 110.3 , 114.2 , 127.5 , 137.0 , 139.1 , 143.0 , 161.1 , 161.5 ( c ) ; 175.5 ( c = o ) . ms ( ei ) : m / z ( % ) = 418 [ m ] ( 47 ) , 390 [ m co ] ( 100 ) , 375 [ m 43 ] ( 24 ) , 347 [ m 71 ] ( 22 ) . hrms ( ei ) : m / z [ m ] calcd 417.980 89 , found 417.981 26 . hplc ( isocr ) : 96.3% at 254 nm and 97.7% at 280 nm , tn = 3.2 min , tm = 1.0 min ( acn / h2o + ( et3n)2so4 50:50 ) . hplc ( grad ) : 99.4% at 254 nm , tn = 10.8 min , tm = 1.2 min ( gradient , acn / h2o ; 0 min , 10/90 13 min , 90/10 ( linear ) ; 20 min , 90/10 ) . synthesis was according to general procedure c from 10-iodo-3-methoxy-11h - indolo[3,2-c]quinoline-6-carboxylic acid ( 5o , 120 mg , 0.300 mmol ) . diethylamine 2:2:1 ) yielded pale yellow crystals ( 100 mg , 75% ) ; mp 214217 c . ir ( kbr ) : 3384 ( nh ) , 1726 cm ( c = o ) . h nmr ( dmso - d6 , 400.4 mhz ) : ( ppm ) = 1.45 ( t , 3h , j = 7.1 hz ) , 3.99 ( s , 3h ) , 4.59 ( q , 2h , j = 7.1 hz ) , 7.14 ( t , 1h , j = 7.8 hz ) , 7.46 ( dd , 1h , j = 9.1/2.6 hz ) , 7.63 ( d , 1h , j = 2.6 hz ) , 7.93 ( dd , 1h , j = 7.6/1.0 hz ) , 8.33 ( dd , 1h , j = 8.1/0.9 hz ) , 9.05 ( d , 1h , j = 9.1 hz ) , 12.34 ( s , 1h , nh ) . c nmr ( dmso - d6 , 100.7 mhz ) : ( ppm ) = 14.2 ( ch2ch3 ) ; 55.5 ( och3 ) ; 61.7 ( ch2 ) ; 108.7 , 119.1 , 122.0 , 122.5 , 124.7 , 135.1 ( ch ) ; 76.8 , 111.2 , 111.8 , 121.4 , 141.1 , 142.0 , 145.1 146.0 , 160.1 ( c ) ; 166.7 ( c = o ) . c19h15in2o3 ( 446.24 ) ; calcd c 51.14 , h 3.39 , n 6.28 , found c 51.21 , h 3.41 , n 6.01 . ms ( ei ) : m / z ( % ) = 446 [ m ] ( 22 ) , 374 [ m c3h4o2 ] ( 100 ) . hplc ( isocr ) : 99.7% at 254 nm and 99.8% at 280 nm , tn = 7.6 min , tm = 1.0 min ( acn / h2o 80:20 ) . hplc ( grad ) : 99.7% at 254 nm , tn = 14.1 min , tm = 1.1 min ( gradient , acn / h2o ; 0 min , 10/90 13 min , 90/10 ( linear ) ; 20 min , 90/10 ) . crystals for x - ray structure analysis were prepared by crystallization from ethanol ( 96% ) . crystal data for 10oh2o at 100 k : orthorhombic , p212121 , a = 6.36362(12 ) , b = 15.0401(3 ) , c = 18.3209(4 ) , v = 1753.48 , z = 4 . a yellow irregular crystal 0.3 mm 0.2 mm 0.15 mm was used to record 76097 intensities , 5096 independent ( rint = 0.036 ) on an oxford diffraction xcalibur e diffractometer using mo k radiation ( = 0.710 73 , 2max = 60 ) . the structure was refined anisotropically on f using the program shelxl-97 to wr2 = 0.049 , r1 = 0.020 for 249 parameters ; s = 1.12 , max . crystallographic data have been deposited with the cambridge crystallographic data centre as supplementary publication no . buffer a consisted of 10 mm mgcl2 , 1 mm egta , 1 mm dtt , 25 mm tris - hcl , ph 7.5 , 50 g heparin / ml . buffer c consisted of 60 mm -glycerophosphate , 15 mm p - nitrophenylphosphate , 25 mm mops ( ph 7.2 ) , 15 mm egta , 15 mm mgcl2 , 1 mm dtt , 1 mm sodium vanadate , 1 mm phenylphosphate . kinase activities were assayed in buffer a or c at 30 c at a final atp concentration of 15 mol / l . blank values were subtracted , and activities were expressed in percent of the maximal activity , i.e. , in the absence of inhibitors . the gs-1 , cks , cdk7/9 tide , and rs peptide substrates were obtained from proteogenix ( oberhausbergen , france ) . cdk1/cyclin b ( m phase starfish oocytes , native ) , cdk2/cyclin a , and cdk5/p25 ( human , recombinant ) were prepared as previously described . their kinase activity was assayed in buffer a , with 1 mg of histone h1/ml , in the presence of 15 mol / l [ -33p ] atp ( 3000 ci / mmol ; 10 mci / ml ) in a final volume of 30 l . after a 30 min incubation at 30 c , the reaction was stopped by harvesting onto p81 phosphocellulose supernatant ( whatman ) using a filtermate harvester ( packard ) and washing in 1% phosphoric acid . cdk9/cyclin t ( human , recombinant , expressed in insect cells ) was assayed as described for cdk1/cyclin b but using cdk7/9 tide ( ysptspsysptspsysptspskkkk ) ( 8.1 g / assay ) as a substrate . gsk-3 ( porcine brain , native , affinity purified on axin1-sepharose beads ) was assayed , as described for cdk1 , with 0.5 mg of bsa / ml + 1 mm dtt and using a gsk-3 specific substrate ( gs-1 , yrraavppspslsrhssphqspedeee ) ( ps stands for phosphorylated serine ) . ck1/ ( porcine brain , native , affinity purified on axin2-sepharose beads ) was assayed as described for cdk1 but in buffer c and using 25 m cks peptide ( rrkhaaigpsaysita ) , a ck1-specific substrate . clk1 , -2 , -3 , and -4 ( mouse , recombinant , expressed in e. coli as gst fusion proteins ) were assayed as described for cdk1/cyclin b with 0.5 mg bsa / ml + 1 mm dtt and rs peptide ( grsrsrsrsrsr ) ( 1 g / assay ) as a substrate . dyrk1a , -1b , -2 , -3 ( human , recombinant , expressed in e. coli as gst fusion proteins ) and clk1 , -2 , -3 , and -4 ( mouse , recombinant , expressed in e. coli as gst fusion proteins ) were assayed in buffer a ( supplemented extemporaneously with 0.15 mg of bsa / ml + 1 mm dtt ) with 1 g of rs peptide ( grsrsrsrsrsr ) as a substrate . response curves were recorded in triplicate . typically , the standard deviation of single data points was below 10% . the assay for inhibition of sf3b1 phosphorylation was performed as described previously . for the assay of tau phosphorylation , we used a hek293 subclone with regulatable expression of gfp - dyrk1a and constitutive expression of gfp - tau ( hek293-tau - dyrk1a ) that was kindly provided by dr . matthias engel ( department of pharmaceutical and medicinal chemistry , saarland university , saarbrcken , germany ) . cells were grown overnight in six - well plates before expression of gfp - dyrk1a was induced with 2 g / ml doxycyclin . the inhibitors were then added from stock solutions in dmso to the desired final concentration and cells were further incubated for 20 h. cells were lysed in sds lysis buffer ( 20 mm tris - hcl , ph 7.4 , 1% sds ) . samples were sonicated and cleared by centrifugation before sds page and immunoblotting with a goat antibody for gfp ( no . 600 - 101 - 215 , rockland immunochemicals , gilbertsville , pa , usa ) and a phosphorylation state specific antibody directed against pthr212 in the tau protein ( no . immunoreactivities were detected by enhanced chemiluminescence using hrp - coupled secondary antibodies and quantified using the aida image analyzer 5.0 program ( raytest , straubenhardt , germany ) . relative tau phosphorylation was calculated by normalization to total tau expression , as determined from gfp immunoreactivity . to calculate relative dyrk1a activity , the basal pt212 signal in control cells not treated with doxycyclin was subtracted from all values , and the phosphorylation in dyrk1a expressing cells not treated with inhibitors was set to 100% . curve fitting for ic50 determination was done with the help of the graphpad prism 5.0 program ( graphpad software , la jolla , ca , usa ) . recombinant dyrk1a was purified as previously described and was treated with tev protease to remove the n - terminal his6 tag . the kinase at 1315 mg / ml in 50 mm hepes , ph 7.5 , 500 mm nacl and 5 mm dtt was incubated with the inhibitors at 1 mm prior to crystallization . crystals were obtained using the sitting drop vapor diffusion method at 4 c using either 2 m ammonium sulfate , 0.2 m na / k tartrate , 0.1 m citrate , ph 5.6 ( for 5s and 5 t ) , or 37% peg 400 , 0.2 m lithium sulfate , 0.1 m tris , ph 8.8 ( for 5j ) , as the reservoir solutions . diffraction data collected at diamond light source , beamline i04 - 1 , were processed with xds or mosflm and subsequently scaled with scala from ccp4 suite . structures were determined by molecular replacement method using phaser and the coordinates of dyrk1a structure as a search model . iterative cycles of manual model building in coot alternated with refinement using refmac , and a tls model calculated from tlsmd server was performed . dyrk1a - ligand complexes ( pdb i d ) : dyrk1a-5j ( 4ylj ) ; dyrk1a-5s ( 4ylk ) ; dyrk1a-5 t ( 4yll ) . molecular docking was performed using gold , version 4.0 , running under linux ubuntu dapper drake . the poses obtained with gold 4.0 were later reproduced using gold , version 5.1 . the pdb file 2wo6 was downloaded from the protein data bank . only the b chain present in the structure hydrogen atoms were added , and the protonation / tautomerization status of amino acid side chains within the atp binding site was checked and adjusted manually , if necessary . a zone of 10 around the original ligand was defined as relevant for binding . the options flip side chains of asn / gln and alter tautomers of his were switched off . ligands were constructed with moe , energy minimized , and saved as mol2 files . the option redocking of the originally bound ligand djm2005 into chain b of the protein reproduced the original pose of the inhibitor , albeit not as highest ranked pose .

the protein kinase dyrk1a has been suggested to act as one of the intracellular regulators contributing to neurological alterations found in individuals with down syndrome . for an assessment of the role of dyrk1a , selective synthetic inhibitors are valuable pharmacological tools . however , the dyrk1a inhibitors described in the literature so far either are not sufficiently selective or have not been tested against closely related kinases from the dyrk and the clk protein kinase families . the aim of this study was the identification of dyrk1a inhibitors exhibiting selectivity versus the structurally and functionally closely related dyrk and clk isoforms . structure modification of the screening hit 11h - indolo[3,2-c]quinoline-6-carboxylic acid revealed structure activity relationships for kinase inhibition and enabled the design of 10-iodo - substituted derivatives as very potent dyrk1a inhibitors with considerable selectivity against clks . x - ray structure determination of three 11h - indolo[3,2-c]quinoline-6-carboxylic acids cocrystallized with dyrk1a confirmed the predicted binding mode within the atp binding site .

Introduction Conclusion Experimental Procedures

PMC2850978

daytime running lights ( drls ) are a safety feature intended to reduce crashes by increasing the contrast between vehicles and the background . currently , finland , sweden , norway , canada , denmark , hungary , and iceland all require vehicle lights during daytime hours . finland was the first to institute drl legislation in rural areas , and literature reports a 27% crash rate reduction . in 1977 , sweden started requiring the use of daytime vehicle lights on all roads , and reduction of crash rates from 9 to 21% were reported by andersson and nilsson . norway began to require installation of drls in all new cars beginning in 1985 and use of daytime lights on all vehicles by 1988 . a 15% crash rate reduction for crashes involving more than one vehicle was later reported by elvik . lastly , denmark has required use of drls on all roads since 1990 , with a statistically significant 37% rate reduction for crashes involving a left turn in a study by hansen . a 1995 paper by theeuwes and riemersma criticized the odds ratio methodology of all these early studies . in response , a meta - analysis of 17 studies by elvik estimated a decrease in crash rate of 1015% for multi - vehicle crashes and total crash reduction of 312% . the first studies of drls in north america were done on fleet vehicles . in a study by stein , corporate fleet vehicles in the usa equipped with drls had 7% fewer relevant crashes compared to the group of fleet vehicles without drls during 19831984 . sparks et al . reported 15% crash reduction in government fleet vehicles in canada equipped with drls . by december 1989 all newly manufactured vehicles in canada were required to be equipped with drls , and within 4 years , arora et al . reported a statistically significant 8% reduction in relevant collisions . drls in non - fleet passenger vehicles have been introduced more recently in the usa . in 1995 , volvo and saab were first to install drls on all their new cars sold in the usa . by 1997 , all new suzuki , volkswagen , and general motors models included drls . yet a decade later , only a few studies and reports have been published regarding the use of daytime headlights in the usa . farmer and williams used a case - control method to analyze multiple vehicle daytime crashes in nine states for a group of vehicles equipped with drls . the national highway traffic safety administration ( nhtsa ) reported a preliminary assessment in june 2000 . using the fatality analysis reporting system ( fars ) , they analyzed fatal crashes in four states from 1995 to 1997 . they found no significant difference in risk of two vehicle opposite - direction crashes comparing vehicles with drls to vehicles without drls . however , using the state data system ( sds ) from florida , maryland , missouri , and pennsylvania , a statistically significant 7% reduction in risk for relevant ( including crash subtypes presumably affected by drls , such as opposite - direction ) nonfatal crashes was identified , and drl - equipped vehicles were associated with 28% fewer pedestrian fatalities . in this study , we tested the hypothesis that passenger vehicles in the usa equipped with drls are associated with decreased crash rates compared to those without drls under high test weather ( daylight and optimal visibility ) and road ( dry ) conditions . this was a retrospective study using the minnesota department of transportation ( mndot ) crash database from 1995 to 2002 . vehicle crashes , for which police reports were filed , were cross - verified and matched against the nhtsa archival registry maintained for research purposes . definitions of crash and fatality were based on the terminology referenced by mndot traffic accident report ( form version : ps-32003 - 10 ) as documented by police authorities at the time of the actual accident . specifically , fatalities recorded were for any scene deaths immediately related to the motor vehicle collision . crash reports included in the analyses were limited to crashes involving automobiles , pickups , and vans and crashes that occurred under high test weather and road conditions all defined a priori . the high test conditions included : ( 1 ) temporal limitations to daylight , defined as dawn to dusk , ( 2 ) optimal visibility , defined as clear or cloudy , and ( 3 ) road surface identified as dry . studied vehicles were also limited to models 1995 and newer , since prior models did not have drls . the vehicle identification number ( vin ) of vehicles involved in crashes was used to determine the specific make , model , and year . this information was cross - referenced with a nhtsa table of manufacturer listed drl conditions to determine each vehicle drl status . crash rates for vehicles with standard drl and without drl feature were calculated as relative to the number of all registered vehicles in minnesota with or without the drl feature , respectively . the number of registered vehicles in minnesota was determined from the mndot vehicle registration file obtained in 2004 for models 19952002 . in 2004 , the number of these vehicles , with and without standard drls , was 788,840 and 1,763,134 , respectively . therefore , the only total number of vehicles which can be obtained is a number in real time . use of this single - year denominator assumes that the proportion of vehicles with and without the standard drl feature was constant over the years of this study . although the rates will be overestimated since the denominators represent a single year , the rate ratios will be appropriate if the previous assumption holds . ninety - five percent confidence intervals ( ci ) for the rates were constructed using a poisson error distribution . the two rates were compared using a two - sided f test for the ratio of two poisson random variants . during the 7-year study period , 184,637 vehicles ( 1995 or newer ) had identifiable vins and were involved in accidents that occurred under the specified test conditions . of these vehicles , 37,909 were determined to have standard drls and 146,728 were determined to be models without drls ( fig . 1 ) . the standard drl group had a higher percentage of automobiles vs pickups and vans ( 78.5% ) than the group without standard drls ( 66.3% ) . other accident characteristics were similar between the standard vs nonstandard drl groups ( table 1 ) . drl , year 1995 + , n = 37,909 ( % ) no std . drl , year 1995 + , n = 146,728 ( % ) vehicle typeautomobile29,750 ( 78.5)97,317 ( 66.3)pickup5,600 ( 14.8)30,959 ( 21.1)van2,559 ( 6.8)18,452 ( 12.6)type of accidentcollision with vehicle34,475 ( 90.9)133,892 ( 91.3)collision with train15 ( < 0.1)30 ( < 0.1)collision with bike358 ( 0.9)1,379 ( 0.9)collision with pedestrian230 ( 0.6)911 ( 0.6)diagramrear end13,721 ( 36.2)52,700 ( 35.9)sideswipe passing2,396 ( 6.3)9,379 ( 6.4)left turn into oncoming2,412 ( 6.4)9,723 ( 6.6)ran off road , left side539 ( 1.4)2,145 ( 1.5)right angle7,979 ( 21)30,347 ( 20.7)right turn into cross traffic218 ( 0.6)814 ( 0.6)ran off road , right side728 ( 1.9)3,019 ( 2.1)head on499 ( 1.3)2,147 ( 1.5)sideswipe opposing406 ( 1.1)1,612 ( 1.1)road descriptionfreeway ( including ramps)5,701 ( 15)21,698 ( 14.8)other divided highway6,054 ( 16)22,640 ( 15.4)one - way street793 ( 2.1)3,409 ( 2.3)46 lane undivided , 23 each7,063 ( 18.6)27,380 ( 18.7)3 lanes undivided482 ( 1.3)1,735 ( 1.2)2 lanes , 1 each way11,894 ( 31.4)45,577 ( 31.1)alley , driveway149 ( 0.4)603 ( 0.4)private property153 ( 0.4)486 ( 0.3)functional classrural6,717 ( 17.7)23,685 ( 16.1)urban30,152 ( 79.5)118,558 ( 80.8)where percentages do not equal 100 , categories labeled not applicable and other were incomplete and therefore not included in the data set summary of vehicles identified for analysis vehicle characteristics by drl status where percentages do not equal 100 , categories labeled not applicable and other were incomplete and therefore not included in the data set the crash rate per 10,000 vehicles among vehicles with standard drls was 481 ( 37,909/788,840 ; 95% ci : 476485 ) . for vehicles without standard drls the crash rate per 10,000 was 832 ( 146,728/1,763,134 ; 95% ci : 828836 ) . 0.001 ) ( table 2 ) . table 2crash rate ratios : vehicles with drls versus vehicles without drlsall vehicles1.74 vehicles involved in fatal crashes1.48 vehicles involved : in collisions with other vehicles1.74 in collisions with pedestrians1.77 in collisions with bicycles1.72 crash rate ratios : vehicles with drls versus vehicles without drls crashes were also analyzed based on whether a fatality was reported . the rate of fatal vehicle crashes for vehicles with standard drls in minnesota between 1995 and 2002 was 2.0 per 10,000 ( 158/788,840 ; 95% ci : 1.72.3 ) . the rate of fatal vehicle crashes for vehicles without standard drls was 3.0 per 10,000 ( 521/1,763,134 ; 95% ci : 2.73.2 ) . vehicle crashes were divided by the type of collision , including collisions with other vehicles , pedestrians , and bicycles . ( table 1 ) . of the 37,909 vehicles with standard drls involved in accidents , this is a crash rate of 437 per 10,000 vehicles ( 95% ci : 432442 ) . of the 146,728 vehicles without standard drls involved in accidents , this is a crash rate of 759 per 10,000 vehicles ( 95% ci : 755764 ) . the rate ratio for vehicles involved in collisions with other vehicles was 1.74 ( 95% ci : 1.721.76 ; p < 0.001 ) ( table 2 ) . a total of 230 vehicles with standard drls were involved in collisions with pedestrians , which is a crash rate of 2.9 per 10,000 vehicles ( 95% ci : 2.53.3 ) . in comparison , a total of 911 vehicles without standard drl were involved in collisions with pedestrians , which is a crash rate of 5.2 per 10,000 vehicles ( 95% ci : 4.85.5 ) ( table 2 ) . the rate ratio for vehicles involved in collisions with pedestrians was 1.77 ( 95% ci : 1.532.05 ; p < 0.001 ) . finally , for collisions with a bicycle , there were 358 vehicles with standard drls involved in such collisions for a crash rate of 4.5 per 10,000 vehicles . without standard drls , 1,379 vehicles were involved in collisions with bicycles for a crash rate of 7.8 per 10,000 vehicles . the rate ratio for vehicles involved in collisions with bicycles is 1.72 ( 95% ci : 1.541.94 ; p < 0.001 . based on our study results , drls had an association with vehicle crash reduction in motor vehicle collisions , consistent with two previous studies . farmer and williams showed that vehicles equipped with drls were involved in 3.2% fewer crashes . our crude crash rate reduction as reflected by the rate ratio was notably higher than in both of these previous studies . this may be due to the fact that our study was a retrospective study of all vehicle crashes in minnesota during the time period , whereas the preceding studies cited employed a case - control methodology to compare specific subsets of vehicles with and without drls . our study shows a statistically significant reduction in fatal crashes for vehicles with drls versus those without drls . this latter finding may be attributable to the relatively low numbers of vehicles involved in fatal crashes compared to all crashes reflected in the nhtsa study denominator . larger studies with greater numbers of fatal crashes would be helpful to further delineate the impact of drls in fatal crashes where causation is likely multifactorial . vehicles that collided with other vehicles showed lower crash rate in vehicles with standard drls compared to those without drls . this is a subtype of crashes that would expectedly be impacted by the drl feature , as increased visibility of other vehicles would likely decrease collisions . in addition , the rate of vehicles colliding with pedestrians may also be predictably lowered by the use of drls because these vehicles may be increasingly visible to pedestrians . our study does demonstrate a reduction in vehicle - pedestrian crashes not inconsistent with the 28% reduction rate reported by the nhtsa . to our knowledge , no traffic law revisions , such as lower speed limitations , or newer primary seat belt stop legislations , affected our crash rates . specifically , there were no traffic law changes in minnesota identified during the study period . first , unknown drl status excluded vehicles from analysis , and incremental value of layered standards or options in crash prevention is not quantified . second , snapshots of data streams may fail to demonstrate the whole picture in complex large volume relationships over time . we used a denominator from vehicles registered in 2004 and assumed a similar proportion of vehicles with drl standard to those without drl standard for all of the study years . we believe the proportion of vehicles remained reasonably constant over the study period , but there is no retrospective database to confirm this . third , use of best - case scenario assumptions to disprove the null hypothesis may limit capture of other significant differences between groups . lastly , confounders related to the driver or vehicle parameters such as age , experience , or safety record may significantly affect associations . driver and vehicle files containing private or privileged information ( insurance status , license qualifications , organ donor information , health outcomes , etc . ) first , unknown drl status excluded vehicles from analysis , and incremental value of layered standards or options in crash prevention is not quantified . second , snapshots of data streams may fail to demonstrate the whole picture in complex large volume relationships over time . we used a denominator from vehicles registered in 2004 and assumed a similar proportion of vehicles with drl standard to those without drl standard for all of the study years . we believe the proportion of vehicles remained reasonably constant over the study period , but there is no retrospective database to confirm this . third , use of best - case scenario assumptions to disprove the null hypothesis may limit capture of other significant differences between groups . lastly , confounders related to the driver or vehicle parameters such as age , experience , or safety record may significantly affect associations . driver and vehicle files containing private or privileged information ( insurance status , license qualifications , organ donor information , health outcomes , etc . ) minnesota vehicles equipped with drls were associated with a statistically significant decrease in crash rates compared to vehicles without drls , model year 1995 or newer , from 1995 to 2002 .

backgrounddaytime running lights ( drls ) are a safety feature intended to reduce crashes by increasing the contrast between vehicles and the background.aimsthe purpose of this study was to determine whether there is an association between vehicles in the usa being equipped with drls and crash rates.methodsthis was a retrospective study using the minnesota department of transportation ( mndot ) crash database from 1995 to 2002 . crash reports included in the analyses were limited to accidents involving vehicles 1995 or newer ( drls not available on prior models ) and limited to ideal conditions : ( 1 ) daylight , ( 2 ) optimal visibility , and ( 3 ) dry road surface . the vehicle identification number ( vin ) was used to determine the make , model , and year . this information was cross - referenced with a national highway traffic safety administration table of manufacturer listed drl conditions to determine vehicle drl status . crude crash rates for vehicles were calculated relative to the number of all registered vehicles in minnesota in 2004 , for models 19952002 . ninety - five percent confidence intervals ( ci ) for the rates were constructed assuming a poisson error distribution.resultsduring 19952002 , there were 184,637 vehicles ( 1995 or newer ) with identifiable vins involved in accidents which occurred under the specified test conditions . of these vehicles , 37,909 were determined to have standard drls and 146,728 were determined to be models without drls ( including those listed as drl optional ) . the crash rate among vehicles without standard drls was 1.73 ( 95% ci : 1.711.75 ) times higher than the rate for vehicles with standard drls . the rate ratio was also significant for fatal vehicle crash rates 1.48 ( 95% ci : 1.231.76).conclusionminnesota vehicles equipped with drls were associated with a statistically significant lower crash rate compared to vehicles without drls from 1995 to 2002 .

Introduction Methods Results Discussion Limitations Conclusion

PMC4064690

recent advances in computer hardware and software have greatly extended the time scales that can be covered by biomolecular simulations . these longer time scales ( beyond nanoseconds ) one important characteristic of these force fields is the ability to accurately model the formation of salt bridges , or pairs of amino acids whose oppositely charged side - chains are within hydrogen - bonding distance in proteins . however , it has long been suspected that the forces between oppositely charged amino acids are overly attractive in molecular dynamics ( md ) simulations with current biomolecular force fields , and there have been a number of efforts to reduce this artifact in the improvement of various force fields . previous theoretical studies have analyzed the contribution of salt bridges to protein or protein protein complex stability , using both implicit and explicit modeling of solvation . others have studied salt bridges using amino acid analogues , often employing biasing techniques in the simulations . more recently , a comprehensive comparison of force field / water model combinations was conducted for salt bridge interactions between the amino and carboxyl groups of zwitterionic amino acids , using extensive simulations in explicit solvent on the microsecond time scale . here , we evaluated six biomolecular force fields for their ability to accurately model the strengths of salt bridges between the side - chains of oppositely charged amino acids by unbiased , microsecond - scale md simulations in explicit solvent . in particular , we directly compared current amber , charmm , and opls force fields in simulations of association between the side - chain analogues of three different pairs of amino acids , arg / asp , lys / asp , and his(+)/asp . we further tested one of the pairs , arg / asp , by simulating association of blocked amino acid dipeptides . in addition , we evaluated the influence of the solvent model on the strengths of the salt bridges by simulating the side - chain analogue pairs using a selection of different force field / water model combinations . to our knowledge , our microsecond - scale simulations provide the most extensive sampling of salt bridge formation to date , yielding thousands of association / dissociation events , permitting quantitative comparisons , both between the force fields and with experiment . our results reveal considerable variability among the current force fields in terms of the resulting strengths of salt bridge interactions , as well as differences from experimental data . we modeled the formation of salt bridges between the following pairs of oppositely charged amino acids using side - chain analogues : arg / asp ( guanidinium cation / acetate anion ) , lys / asp ( butylammonium cation / acetate anion ) , and his(+)/asp ( imidazolium cation / acetate anion ) . our systems were constructed to be consistent with the experimental conditions under which the equilibrium association constants ( ka ) of guanidinium acetate and butylammonium acetate have been measured , i.e. , using the same concentrations ( 0.9 m guanidinium and 0.02 m acetate , which corresponds to 100 molecules of guanidinium and two molecules of acetate in the presence of 18 000 explicit water molecules ) . to ensure a net charge of zero , we included 98 chloride ions ( the same counterion that is present in the experiments ) . the same concentrations of the cation , anion , and chloride ions were also used for the model systems consisting of butylammonium / acetate and imidazolium / acetate . starting models for these simulations were constructed using the packmol software package , immersing the appropriate number of side - chain analogues in periodic , cubic boxes of explicit solvent . for the arg / asp salt bridge , we also used blocked amino acid dipeptides ( acetyl arginine only a single copy of each blocked dipeptide was included , corresponding to a concentration of 0.012 m for each salt - bridging partner , with a distance of 10 between the amino acids . all force field parameters of the side - chain analogues were based on those of the complete amino acids . for the chloride ions , parameters derived specifically for the water model were used when available ; otherwise , parameters derived for a similar water model were used . nonbonded parameters of the side - chain analogues , along with those used to model chloride ions and blocked amino acid dipeptides , are provided in table s1 of the supporting information . to alleviate any unfavorable interactions , each model was subjected to energy minimization followed by a two - stage equilibration with harmonic position restraints on all heavy atoms of the side - chain analogues ( force constant of 10 kcal mol ) using the desmond 3.0.1.0 software package . in the first stage , the energy - minimized system was equilibrated for 20 ps at constant temperature ( 25 c ) using a weak langevin thermostat ( frictional constant of 1 ps ) . during the second stage , the system was equilibrated for 1 ns at constant temperature ( 25 c ) and pressure ( 1 atm ) using the martyna klein thermostat and barostat ( coupling time constants of 1.0 and 2.0 ps , respectively ) . to enable a 2 fs time step , bonds to hydrogen a short - range nonbonded cutoff of 10.0 was used , and long - range electrostatics were calculated using the particle mesh ewald ( pme ) method . the frame from the second half of the npt equilibration with volume closest to the average was used to start the production simulation . to obtain extensive sampling of salt bridge association ( and dissociation ) events , 1-s md simulations were performed for each side - chain analogue system ; 10-s simulations were performed for the blocked arginine and aspartate dipeptide systems . all simulations were carried out in the nvt ensemble using a 64-node anton special - purpose supercomputer , which is able to run md simulations roughly 2 orders of magnitude faster than conventional hardware ( altogether , the simulations required a total of 40 machine - days ) . the temperature was maintained at 25 c using the nos hoover thermostat with a weak coupling constant of 0.5 ps . van der waals and short - range electrostatic interactions were truncated at 10.0 ; long - range electrostatic interactions were calculated using the gaussian split ewald method . to enable a 2.5 fs time step , bonds to hydrogen equilibrium association constants ( ka ) were calculated from the populations of the bound and unbound states of the oppositely charged side - chain analogues . for example , the ka for association between guanidinium and acetate was calculated using the following:1where pbound is the population of the bound state , punbound guanidinium and punbound acetate are the populations of unbound guanidinium and acetate , respectively , and c0 is the reference concentration of guanidinium ( i.e. , 0.9 m ) . in addition to species in which a single acetate molecule is bound to a single cation molecule , forming a 1:1 complex ( e.g. , the guanidinium / acetate complex ) , species in which acetate is bound to two cation molecules , forming a 1:2 complex ( e.g. , the diguanidinium / acetate complex ) were observed . ka values for the latter are included in table s2 of the supporting information ; the results discussed below focus on formation of the major complex , which is the 1:1 complex . standard errors in the ka values were calculated using a block averaging method . for each side - chain analogue system , the unbound and bound states were defined using the potential of mean force ( pmf ) as a function of the minimum distance between the nitrogen and oxygen atoms of the positively and negatively charged analogues , respectively ( minimum n o distance ; see figure 1 ) . in particular , the point of inflection between the bound state free energy minimum ( 2.53 ) and the desolvation barrier ( 33.5 ) was used as the bound state cutoff , while 4.5 was used as the unbound state cutoff . if the minimum n o distance between an analogue pair dropped below the bound state cutoff they were classified as bound until they crossed the unbound state cutoff , and vice versa . for simulations of the blocked arginine and aspartate dipeptides , the same definitions of the unbound and bound states were used as for the guanidinium / acetate system . potentials of mean force ( pmf ) between three different pairs of oppositely charged side - chain analogues using six biomolecular force fields with the tip4p - ew explicit water model . the dielectric constant of water in each simulation , water , was calculated using the following equation:2where mwater is the net dipole moment of water , vwater is the volume occupied by water , t is the temperature of the system , kb is the boltzmann constant , and 0 is the permittivity of free space . the net dipole moment of water was calculated using the following:3where q is the atomic charge of each water site , and r is its position vector . the dielectric constant of water , rather than that of the complete system , was used since it is impossible to calculate the contributions of molecules with a net charge to the system dipole moment from simulations with periodic boundary conditions . the appropriate volume was thus the volume of the water molecules present in the system . for each water model used , a pure water system of the same total volume as that of the side - chain analogue systems was equilibrated using the same protocol , and the molecular volume of water calculated . for each analogue system , the number of water molecules present was multiplied by the molecular volume to calculate the approximate volume of water present in the system . we compared the following six current biomolecular force fields in terms of their ability to model salt bridge interactions : amber ff99sb - ildn , amber ff03 , amber ff13 , charmm27 , charmm22 * , and opls_2005 . in particular , we simulated association ( and dissociation ) of salt bridges between the following three pairs of oppositely charged amino acids : arg / asp , lys / asp , and his(+)/asp . we focused primarily on simulating side - chain analogues ( i.e. , guanidinium , butylammonium , and imidazolium cations for arginine , lysine , and histidine , respectively , and acetate anion for aspartate ) since these analogues are the minimal systems for studying the formation of salt bridges ; in addition , equilibrium association constant ( ka ) values for such systems have been experimentally measured , providing an excellent opportunity to validate the simulations . while blocked amino acid dipeptides ( i.e. , acetyl amino acid n - methyl ) might be regarded as being more representative of the protein environment , no experimental ka values for the association of oppositely charged amino acid dipeptides are available . nonetheless , we evaluated the force fields in simulating such systems , focusing on just one of the three salt bridges , arg / asp . finally , in addition to the above simulations , in which each biomolecular force field was paired with the tip4p - ew explicit water model , which reproduces the liquid properties of water at the temperatures and pressures relevant to biology , we also evaluated the influence of the water model on the strength of the salt bridges by testing a selection of force field / water model combinations for all three pairs of side - chain analogues . for each force field a selection of water models drawn from tip3p , mtip3p , tip4p , tip4p/2005 , and spc / e were tested , including the water model with which each force field was originally derived . to validate our simulations of association between oppositely charged side - chain analogues , we computed ka values and compared these to those measured by experiments . experimental ka values have been measured for guanidinium / acetate and butylammonium / acetate association by monitoring changes in the pka of acetate in the presence or absence of either the guanidinium or butylammonium cation . our microsecond - long simulations yielded thousands of independent binding events , permitting the extraction of extremely precise ka values , with the mean lifetimes of the bound state ranging from 10300 ps and the mean lifetimes of the unbound state ranging from 20120 ps ( table s2 , supporting information ) . in general , the ka values computed from our side - chain analogue simulations are overestimated in comparison to experimentally measured values , with the amber ff03 force field overestimating the strengths of the salt bridges to the least extent and the opls_2005 force field to the greatest extent , when using the same water model ( table 1 ) . for example , when using the tip4p - ew water model , the computed ka values for the three types of salt bridges vary by as much as 4-fold , 3-fold , and 4-fold for the associations of guanidinium , butylammonium , and imidazolium with acetate , respectively , which amounts to 1.4-fold , 1.8-fold , and 1.9-fold differences in the probabilities of binding ( pbound ) ( see figure 2 ) . we note that our definition of the bound state is very conservative and that the use of less conservative definitions ( e.g. , use of the desolvation barrier as a cutoff ) yields even stronger association constants , without affecting our overall conclusions . one potential factor that could influence the degree of salt bridge formation in our simulations is the choice of force field parameters for the chloride ions . to verify that these parameters are not the cause for overestimating salt bridge strength , we carried out simulations of a single guanidinium / acetate pair ( corresponding to concentrations of 0.1 m ) with no chloride ions present . the resulting ka values are even higher than those measured in the presence of chloride ions , indicating that the chloride ion parameters do not cause disproportionate salt bridge stability ( table s3 , supporting information ) . results are from 1-s simulations and standard errors were calculated using a block averaging method . experimental ka values of guanidinium and butylammonium acetate permit only a qualitative estimate of the associated error . taking two experimentally measured ka values of guanidinium acetate using different protocols into account , we estimate an error of 0.05 , although the true uncertainty is not known . using this estimate , we have back - calculated the range of simulated pbound values that would be expected in our simulation , based on the experimental ka . probabilities of binding ( pbound ) between three different pairs of oppositely charged side - chain analogues using six biomolecular force fields with the tip4p - ew explicit water model . the pbound values that correspond to the experimentally determined ka values of guanidinium acetate and butylammonium acetate are depicted as horizontal gray bars ; no experimentally measured ka is available for the imidazolium acetate system . two obvious features of a force field that influence the strength of the salt bridges are the atomic charges and radii . as expected , the charmm22 * force field yields ka values that are closer to experiment than those from the parent charmm27 force field since the atomic charges for the arginine , aspartate , and glutamate residues were parametrized specifically to reproduce the experimental association of guanidinium acetate . however , the charmm22 * force field does not produce as close agreement with experiment as the amber ff03 force field , which shows good agreement for butylammonium acetate . given these results , it appears that the general strategy used to derive atomic charges for the amber ff03 force field is reasonably effective for modeling electrostatic interactions . this strategy involved the derivation of atomic charges in the presence of a continuum solvent model with a dielectric constant of 4 to mimic an organic solvent ( protein - like ) environment . the resulting atomic charges in the amber ff03 force field are notably less polarized than those in the amber ff9x family ( including the amber ff99sb - ildn force field tested here ) , which were derived in vacuum and share the same set of atomic radii . the amber ff03 atomic charges are also less polarized than those in the amber ff13 force field , with atomic charges possessing increased polarity relative to previous amber charge models . in the amber ff13 charge model , nonpolarizable point charges have been fit to implicitly account for solvent polarization , using iterative cycles of classical md simulations with explicit water ( i.e. , tip4p - ew ) to estimate the water charge density around the solute , followed by quantum mechanical calculations to determine updated solute charges . interestingly , although certain critical atomic radii ( e.g. , the nitrogen in butylammonium and the oxygen in acetate ) in the amber ff13 force field were adjusted from their original values in the amber ff99 force field to reproduce experimental hydration free energies of the relevant amino acid analogues , the resulting strengths of the salt bridges are more overestimated , relative to the other tested amber force fields . notably , the amber ff13 force field results in a free energy landscape for salt bridge formation that is significantly different from those of the other force fields . in particular , as shown by the pmfs as a function of the minimum n o distance between the oppositely charged analogues for the three types of salt bridges ( figure 1 ) , the free energy minima for the bound states are consistently shifted to the right in the amber ff13 force field relative to the other force fields . when we substituted the atomic radii in the amber ff13 force field with the original radii from the amber ff99 force field , the free energy minima for the bound states shifted back toward those of the other force fields and yielded significantly deeper minima as well as more pronounced desolvation barriers , particularly for the guanidinium / acetate and imidazolium / acetate systems ( figure s2 , supporting information ) . since the atomic charges of the opls_2005 force field are not significantly different from those of the other force fields , the most likely reason for the fact that this force field overestimates the ka values to the greatest extent is that the atomic radii of the nitrogen - attached hydrogen atoms are smaller than those used by the other force fields , potentially allowing the pairs to associate more closely and increasing their electrostatic attraction ( table s1 , supporting information ) . consistent with this notion , simulations using the opls - aa force field , which differs from the opls_2005 force field only in that it omits atomic radii for these hydrogen atoms , resulted in slightly more strongly associated salt bridges ( table s2 , supporting information ) . we note that the ranking of the strengths of the three types of side - chain salt bridges in our study by the amber ff99sb - ildn , charmm27 , and opls - aa force fields is consistent with that observed for their oppositely charged termini in a recent study by others . as mentioned above , we additionally tested salt bridge formation of the arg / asp pair by simulating association / dissociation of blocked amino acid dipeptides , testing six different force fields in conjunction with the tip4p - ew explicit water model . as shown in table 2 , the relative ranking of the force fields in terms of the ka is generally consistent with our results from the corresponding side - chain analogue system ( guanidinium / acetate ) . the only exception is the amber ff13 force field , which yields the weakest ka for the association of the amino acid dipeptides , as opposed to an intermediate ka value for the association of guanidinium / acetate . as indicated by the pmf between the arginine and aspartate dipeptides ( figure 3 ) , the bound state free energy minimum of the amber ff13 force field is the most shallow among the tested force fields , corresponding to the lowest frequency of salt bridge formation . the inclusion of the backbone groups , therefore , appears to alter its propensity for salt bridge formation , likely through the competition of side - chain / backbone interactions with the side - chain / side - chain interactions between the two amino acids . this result emphasizes the benefit of using unbiased simulations ; had the relative orientations of the amino acids been fixed as in previous studies , any effects of significant side - chain / backbone interactions on the frequency of salt bridge formation would not have been apparent . results are from 10-s simulations and standard errors were calculated using a block averaging method . potentials of mean force ( pmf ) between blocked arginine and aspartate dipeptides using six biomolecular force fields with the tip4p - ew water model . the larger noise level compared to the data presented in figure 1 is caused by simulating a single pair of binding partners , rather than a concentrated solution . to monitor side - chain / backbone association , we used the same minimum n o distance coordinate and bound and unbound state definitions as used for the side - chain / side - chain interactions . a comparison of the relative probabilities of side - chain / side - chain versus side - chain / backbone association ( figure 4 ) reveals that the force fields generally prefer side - chain / side - chain association by a factor of 2 or more over side - chain / backbone association . the exception is amber ff13 , which shows a lower preference for side - chain / side - chain association of 1.3 . this slight preference over side - chain / backbone association is likely due to the substantially more polarized backbone amide and carbonyl groups of the amber ff13 force field relative to previous amber force fields ( including the amber ff99sb - ildn and amber ff03 force fields ) . thus , as a result of the delicate balance of side - chain / side - chain and side - chain / backbone interactions , the strength of the arg / asp salt bridge appears to be most accurately modeled ( least overstabilized ) by the amber ff13 force field in a model system that is representative of a protein environment . the probability of side - chain / side - chain association ( pboundsc / sc ) over the probability of side - chain / backbone association ( pboundsc / bb ) for blocked arginine and aspartate dipeptides using six biomolecular force fields with the tip4p - ew water model . in addition to the force field , the choice of water model can affect the strength of salt bridges . to evaluate the influence of the water model , we tested the above three side - chain analogue systems with a selection of force field / water model combinations in addition to the force field / tip4p - ew combinations . regardless of the water model , the relative ranking of the force fields is unchanged in terms of the ka values , with pbound varying by 510% between the water models ( table 1 ) . we also evaluated the dependence of salt bridge interactions on the dielectric constant of the employed water model ( water ) . interestingly , despite the fact that the spc / e water model yields a computed dielectric constant ( water = 70 ; table s4 , supporting information ) that is closest to the experimental value ( water = 78.4 ) among all of the tested water models , the use of the spc / e water model results in stronger salt bridge interactions than seen with the tip4p - ew water model , in which the water value is underestimated ( water = 56 ; table s4 , supporting information ) . in fact , as shown in figure 5 , there appears to be no clear correlation between water of the water model and the strength of the salt bridges . as an aside , the charmm27 and charmm22 * force fields were tested with both the standard tip3p and the charmm - modified tip3p ( mtip3p ) water models with which they were developed . the mtip3p water model includes atomic radii on hydrogen as well as oxygen atoms , whereas standard tip3p includes only the oxygen atom . using the mtip3p water model consistently results in a lower ka values , in better agreement with experiment . this suggests that it may be advisible to use of the charmm - modified tip3p , rather than the standard tip3p water model with any charmm force field . relationship between the probabilities of binding ( pbound ) and the dielectric constant of the water model ( water ) for three different pairs of oppositely charged side - chain analogues . the displayed data are simulation results using six biomolecular force fields and six explicit water models . the pbound values that correspond to the experimental association constants ( ka ) for the guanidinium acetate and butylammonium acetate systems are depicted as horizontal gray bars ; no experimentally measured ka is available for the imidazolium acetate system . the water values were calculated from the first 100 ns of each simulation . for each model , the presence of the solutes lowers the water for each system by 1015 relative to that of pure water ( table s4 , supporting information ) . note that the error bars are not visible since 95% confidence intervals for both pbound and water lie within in the symbols area in the graph . we compared the modeling of salt bridge interactions using six current biomolecular force fields . three different salt bridges ( arg / asp , lys / asp , and his(+)/asp ) were simulated and considerable differences in their strengths were noted , both between the force fields and with experiment . given the availability of experimentally measured ka values for the association of oppositely charged side - chain analogues , we have focused primarily on modeling salt bridge formation using these systems . we also tested the applicability of our results to amino acids by simulating blocked amino acid dipeptides for one of the salt bridges , arg / asp . our side - chain analogue simulations reveal that the computed ka values are generally overestimated , relative to experimental values , with the amber ff03 force field overestimating the strengths of the salt bridges to the least extent and the opls_2005 force field to the greatest extent when using the same water model ( tip4p - ew ) . for the blocked arginine and aspartate dipeptides , we observed general agreement in the relative ranking of the force fields with that obtained from simulations with the corresponding side - chain analogues . the only exception is the amber ff13 force field , which resulted in the lowest probability of salt bridge formation , likely due to the presence of competing side - chain / backbone interactions . thus , while the amber ff03 force field overestimates the strength of salt bridges to the least extent for the side - chain analogue systems , the amber ff13 force field results in an even lower frequency of salt bridge formation than the amber ff03 force field for the complete amino acids . finally , we examined the influence of the water model on the strengths of the salt bridges . irrespective of the water model , the relative ranking of the force fields remained unchanged , with no clear correlation between the probability of binding ( salt bridge formation ) and the dielectric constant of the solvent ( water ) . in conclusion , when running md simulations in which salt bridge formation may be of interest , careful attention should be paid to the specific force field and water model in simulations of protein systems . several current force fields yield considerably higher ka values than those experimentally determined , a discrepancy that may lead to erroneous conclusions . our encouraging results with the amber ff13 force field suggest that charge derivation strategies that implicitly incorporate solvent polarization from explicit water may significantly extend the lifetime of fixed - charge force fields , which include all of the force fields tested in this study . for example , using polarizable force fields that permit varying the charge distribution within a molecule based on both its conformation and environment may alleviate such shortcomings . in the past , the solvation of ions and charged small molecules , have been modeled using polarizable force fields , resulting in improved agreement with experiment , compared to charmm27 and amber ff99 force fields ( equivalent to the amber ff99sb - ildn force field used here ) . future work will determine whether or not this also holds for protein salt bridges .

recent advances in computer hardware and software have made rigorous evaluation of current biomolecular force fields using microsecond - scale simulations possible . force fields differ in their treatment of electrostatic interactions , including the formation of salt bridges in proteins . here we conducted an extensive evaluation of salt bridge interactions in the latest amber , charmm , and opls force fields , using microsecond - scale molecular dynamics simulations of amino acid analogues in explicit solvent . we focused on salt bridges between three different pairs of oppositely charged amino acids : arg / asp , lys / asp , and his(+)/asp . our results reveal considerable variability in the predicted ka values of the salt bridges for these force fields , as well as differences from experimental data : almost all of the force fields overestimate the strengths of the salt bridges . when amino acids are represented by side - chain analogues , the amber ff03 force field overestimates the ka values the least , while for complete amino acids , the amber ff13 force field yields the lowest ka value , most likely caused by an altered balance of side - chain / side - chain and side - chain / backbone contacts . these findings confirm the notion that the implicit incorporation of solvent polarization improves the accuracy of modeling salt bridge interactions .

Introduction Methods Results and Discussion Conclusions

PMC3231754

additional -globin genes in sheep might produce extra -globin chains and , consequently , the subject carrying triplicated ( ) or quadruplicated ( ) haplotypes may exhibit different hematological phenotypes when compared to the normal duplicated ( ) homozygotes ( nn ) . both and heterozygous ( nd ) and and homozygous ( dd ) individuals were obtained by selection and inbreeding . chromatographic rp - hplc analyses of the globin chains of 65 subjects ( 15 dd , 20 nd and 30 nn ) were performed . a highly significant linear regression ( r2 = 0.967 ) of the / ratio on the number of -globin genes was found , and the / ratio ranged on average from 1.0 in nn individuals to 1.2 in the nd and 1.6 in the dd subjects . values for blood fell within the range of normality but were rather peculiar as a whole . when the erythrocytes of individuals carrying normal arrangements were compared with those of subjects with extra -genes , the latter had fewer erythrocytes that were bigger in size and had a higher hb content and a greater osmotic fragility . this hematological picture is consistent with the existence of an unbalanced / ratio .

None

PMC4496421

the surveyed races were flat races on turf and dirt tracks held by the jra from january 1st , 2002 , to december 31st , 2010 . the race lengths were 1,200 , 1,400 , 1,600 , 1,800 and 2,000 m on turf and 1,000 , 1,200 , 1,400 , 1,700 and 1,800 m on dirt . these were the respective top 5 races in terms of their number of starters . the base of a dirt track is a layer of mountain sand packed firmly and then covered with loose sand ( 9 cm ) to absorb the touchdown impact . only the final time data from firm or standard condition tracks were used in this study because track condition affects the final time and the firm and standard conditions had the most data . the final time is that officially recorded by the jra , which is measured on video in time increments of one - tenth of a second . the horses were divided by gender , with one group being females and the other being males & geldings ; male horses and geldings were grouped together because of the relatively low number of geldings belonging to the jra . the racing speed of each horse was calculated by dividing the race distance ( m ) by the horse s final time ( sec ) . average speeds per month for each age and distance condition were calculated for each gender group when there were 30 or more starters per month for each age and distance condition in each gender group . the average weight carried per month by each age group was calculated for each gender group when there were 30 or more starters per month for each age group in each gender group . in descending order , the greatest number of turf race starters were found in the 1,200 m , 1,800 m , 2,000 m , 1,600 m and 1,400 m races ( table 1table 1.distribution of gender and distance for starters on flat turf racesdistance ( m)gender1,2001,4001,6001,8002,000males & geldings22,0248,76116,00621,51422,013females24,2278,31011,00913,1008,962total46,25117,07127,01534,61430,975 ) . the greatest number of starters for dirt races , in descending order , were found in the 1,200 m , 1,800 m , 1,700 m , 1,400 m and 1,000 m events ( table 2table 2.distribution of gender and distance for starters on flat dirt racesdistance ( m)gender1,0001,2001,4001,7001,800males & geldings6,21718,01713,56020,91223,431females6,26413,8357,0369,8488,245total12,48131,85220,59630,76031,676 ) . the ratio of males & geldings to females was equal across shorter distances in both turf and dirt races but increased with distance ; the ratio was almost 2:1 for 2,000 m races on turf ( table 1 ) and 1,800 m races on dirt ( table 2 ) . therefore , the effect of geldings on the data of the males & geldings is believed to be very small . the common characteristic change observed across all distances on both turf and dirt was that the average speed increased slowly up until february of the age of 3 years old ( figs . 1.average speed for each race distance in males & geldings ( a ) and females ( b ) , the average weight carried ( c ) and the number of starters ( c ) on turf races.fig . 2.average speed for each race distance in males & geldings ( a ) and females ( b ) , the average weight carried ( c ) and the number of starters ( c ) on dirt races . ) . subsequently , from march to september of the age of 3 years old , speed showed a continual increase and then increased rapidly in october and november . thereafter , their average speed increased continuously until the first half of the age of 4 years old , after which it remained almost constant , with little variation . average speed for each race distance in males & geldings ( a ) and females ( b ) , the average weight carried ( c ) and the number of starters ( c ) on turf races . average speed for each race distance in males & geldings ( a ) and females ( b ) , the average weight carried ( c ) and the number of starters ( c ) on dirt races . the average weight carried increased in males & geldings at approximately 3.5 years of age ( figs . 1c and 2c ) . the average weight carried decreased in both males & geldings and females in june of the age of 3 years old . from december of the same year , the weight carried increased and then peaked in june to august of the following year ( at the age of 4 years old ) . the number of starters in each gender group , and each type of race , increased at 2 years old . in october of the age of 3 years old , this number decreased rapidly ( figs . the average speed of thoroughbreds surveyed on turf and dirt races increased slowly up until february of the age of 3 years old ( figs . one of the reasons why the average speed increase during this period was slower than in subsequent periods might be that increases in carried weight inhibited the average speed increases associated with growth . in the jra races , the weight carried increased with growth in the males & geldings group ( table 3table 3.weight carried and age of horse2 years old3 years oldgender / ageuntilseptemberoctober todecemberuntilseptemberoctober todecembermales & geldings54 kg55 kg56 kg57 kgfemales54 kg55 kg ) . as a result , the average weight carried increased in almost the same period as the relatively slow speed increases ( figs . however , this explanation is unlikely because the increase in average speed was also slow in females of the same age , whose carried weight had not increased ( table 3 , figs . another possible cause was the change in the group composition of starters during the study period because of many horses debuting in a newcomer or maiden race . because the starters that debuted during this period included more horses that were novices and therefore less trained than horses that had already debuted , which were well trained and faster , the average speed could be expected to increase slowly . subsequently , from march to september of the age of 3 years old , the average speed for all race lengths showed a continued increase ( figs . average weight carried decreased in both males & geldings and females from june of the same year because races between 3-year - old horses and those over 4 years old had started ; there was a weight allowance for 3-year - old horses in these races . however , the average speed for each track and length increased gradually , not rapidly . therefore , it appears that running performance increases related to growth have a more significant impact than decreases in weight carried . the average speed for most race lengths and track surfaces increased rapidly in october and november of the age of 3 years old ( figs . maiden races also ended at this time , at the age of 3 years old ; therefore , almost all maiden horses that showed less impressive running performance retired from racing in the jra . as a result , the total number of starters decreased significantly for both turf and dirt track races after october of the age of 3 years old ( figs . therefore , as only horses with higher running performance than maiden horses were left , the average speed seemed to increase rapidly . with regard to subsequent increases , the average speed thereafter increased continuously to the first half of the age of 4 years old . during this period , the weight carried increased , as the weight allowance for 3-year - old horses in races with those over 4 years old decreased in incremental steps ( figs . however , no inhibition of the increase in average speed by weight carried was observed ; it appears that growth is a stronger factor for average speed than inhibition by such weight . after this period , average speed remained at an almost constant level , with little variation ( figs . in a previous study on geldings , performance peaked at a real age of 4.25 to 4.75 years evaluated by the beyer speed figures in the united states . because a horse generally gives birth between march and april in the northern hemisphere , a real age of 4.25 to 4.75 years would occur between june and december , which is consistent with this result . the fastest average speed was recorded at age 4 in thoroughbreds in brazil , at 4 to 6 years old in trotters in germany and at 5 years old in thoroughbreds in japan . these results also agree with the result of the present study . under the rules of the jra , the weight allowance in a special weight race is removed between january and march of the age of 4 years old , as a function of race length . the average speed did not decrease with aging after the peak at the first half of the age of 4 years old ( figs . however , it has previously been reported that performance evaluated by beyer speed figures decreased gradually after the peak . in that study , only horses that had records spanning several years were selected . however , the present study targeted all horses that started in the intended period and races . the average speed might not have decreased in this study because horses whose performance decreased with aging were retired from racing and the remaining horses retained their performance . furthermore , the average weight carried by the horses in our study decreased gradually in both males & geldings and females from the latter half of the age of 4 years old , at which time their performance peaked . weight adjustments like this might inhibit the decrease in average speed with aging . the average speed after the latter half of the age of 4 years old for each race length on turf tracks was fastest in the shortest ( 1,200 m ) race and slowest in the longest ( 2,000 m ) race for both genders in most months . on the other hand , on dirt tracks , although the average speed after the latter half of the age of 4 years old in the 1,000 m race was fastest in most months , the average speeds in the 1,200 and 1,400 m races were sometime close to those in the 1,000 m race . the average speed in the 1,800 m race was also close to that in the 1,700 m race for most months . the difference in average speed was not remarkable on dirt tracks . therefore , while the average speed decreased on turf tracks as the race length was extended , the influence of this extension of race length was low on dirt tracks . the reason for this seems to be that the top speed on dirt tracks is limited by the nature of the race . the top speed for turf track races of 1,200 m was about 17.2 m / sec , while that for dirt track races of 1,000 m was about 16.5 m / sec . another reason might be that the dirt tracks used for the 1,200 and 1,400 m races included a short turf zone ( about 100 m ) just after the starting gate on a few racecourses , and horses can run faster on turf than dirt tracks , as demonstrated in this study . however , although both the 1,200 and 1,400 m tracks of a few racecourses have a short turf zone , the difference in average speed between these two types of dirt track was not remarkable . therefore , extending the length of a dirt track had little effect on average speed . although the 5 race lengths surveyed for the turf tracks were different , with equal intervals of 200 m , the difference in average speed between the 1,600 and 1,800 m distances was larger than the differences for the other tracks . the 1,600 m racetracks used at most jra racecourses include a single turn , while most of the 1,800 and 2,000 m racetracks include two turns . because racing speed decreases at a turn compared with straights , the difference in average speed between the 1,600 and 1,800 m races might be larger than the difference between other races without different numbers of turns . on the dirt tracks , races up to 1,400 m also include a single turn , while 1,700 and 1,800 m races include two turns at most jra racecourses . although the difference between 1,400 and 1,700 m races is 300 m , the decrease in average speed between these two races was larger than that between 1,000 and 1,400 m races , in which the difference was 400 m. therefore , turns induce a decrease in average speed regardless of the composition of the track . it was possible that the influence of track turns was greater on dirt tracks than on turf tracks . the average speeds on turf tracks after the age of 4.5 years old were about 17.1 m / sec and 16.6 m / sec in 1,400 and 1,800 m races , respectively , a difference of about 0.5 m / sec . on the other hand , the difference was about 0.7 m / sec on dirt tracks of the same length ( average speeds of about 16.4 m / s and 15.7 m / s , respectively ) . furthermore , the decrease in average speed with extension of the race length was less on dirt tracks than on turf tracks . therefore , larger decreases in average speed between the 1,400 and 1,800 m races on the dirt tracks seem likely to have been caused by the greater influence of turning on racing speed on the dirt tracks per se . one other potential reason is that the dirt tracks are generally located on the inside of turf tracks at all jra racecourses , and the turning radius of dirt tracks is smaller than that of turf tracks . another reason may be that under standard conditions , dirt tracks might have less grip than turf track ; as a result , horses might reduce their speed on dirt tracks during turning . the propulsive force on turf tracks was shown to be larger than on either synthetic or dirt tracks . in conclusion , the average speeds for all race distances surveyed on turf and dirt increased up until the first half of the age of 4 years old . the effect of the increase in weight carried on the increase in average speed with growth was only small . after the latter half of the age of 4 years old , the average speed remained at an almost constant level , with little variation . i speculate that the decrease in weight carried and retirement of less well performing horses kept the average speed high .

abstractthe running performance of thoroughbred racehorses has been reported to peak when they are between 4 and 5 years old . however , changes in their racing speed by month or season have not been reported . the purposes of this study were to reveal the average racing speed of thoroughbreds , and observe changes in their average speed with age . the surveyed races were flat races on turf and dirt tracks with firm or standard track conditions held by the japan racing association from january 1st , 2002 to december 31st , 2010 . the racing speed of each horse was calculated by dividing the race distance ( m ) by the horse s final time ( sec ) . average speeds per month for each age and distance condition were calculated for each gender group when there were 30 or more starters per month for each age and distance condition for each gender group . the common characteristic change for all conditions was an average speed increase up until the first half of the age of 4 years old . the effect of increased carry weight on average speed was small , and average speed increased with the growth of the horse . after the latter half of the age of 4 years old , the horses average speed remained almost constant , with little variation . it is speculated that decreases in the weight carried ; and the retirement of less well performing horses ; are responsible for the maintenance of average speed .

Materials and Methods Results Discussion

PMC3263024

when multiple hypotheses are tested in a single experiment , the risk of type i error is increased and with it the risk of promulgating spurious significant findings.[13 ] the likelihood of obtaining a false positive result increases proportional to the number of tests performed . for example , the probability of obtaining at least one false positive result when performing 10 tests is given by 1- p(a ) = 1 - 0.95=0.4013 ( 1 ) where p(a ) is the confidence level of the test . although the problems associated with multiple testing are well known , numerous studies still fail to correct their reported p - values . for instance , bennett et al . found that only between 60% and 74% of the neuroimaging articles published in several major journals corrected for multiple comparisons . similarly , a study performed by austin et al . also demonstrated that the failure to account for multiple testing resulted in statistically significant , yet implausible results . in both cases the lack of attention paid to this problem in the pathology literature stands in stark contrast to its recognition in other fields such as ecology where there has been intense interest for over two decades since the seminal publication by rice . that being said , even within the field of ecology this topic still engenders debate . a systematic exploration of this problem in the pathology literature has not been undertaken ; however we have previously reported on a convenience sample of 800 publications from the pathology literature in 2003 , of which 37 presented multiple comparisons . twenty one of these 37 did not attempt to control for increased type i error due to multiple comparisons . one means of reducing the type i error from multiple testing is the bonferroni correction , which controls the family - wise error rate ( fwer ) . the fwer is the probability of type i error among the entire set of hypotheses . the bonferroni correction is calculated as follows : pcorrected= poriginal .n ( 2 ) where n is the number of hypotheses tested . there is a lack of consensus as to what actually represents a family of statistical tests ; however it has been suggested that if it is appropriate to place multiple p - values in the same table , it may be appropriate to correct all values in that table for multiple comparisons . because the bonferroni correction is conservative with regard to statistical power the bonferroni - holm correction is calculated as follows : pcorrected= poriginal .(n - k+1 ) ( 3 ) where n is the number of hypotheses tested , and k is the ordered rank of the uncorrected p - values ( from smallest p - value to largest p - value ) . rather than controlling for the probability of one or more type i errors in the entire experiment , some of the more recent approaches to the multiple testing problem have focused on controlling the false discovery rate ( fdr ) in the experiment . by controlling the proportion of type i errors , this has the advantage of further increasing the statistical power of the algorithm , and is especially suitable when conducting numerous hypothesis tests . the benjamini - hochberg method is a commonly used way to control the fdr of an experiment . it is calculated as follows : where n is the number of hypotheses tested , and k is the rank of the uncorrected p value . several commercial statistical software packages are capable of performing one or more of these corrections as well as at least one open - source program ( gnu r ) ; however the cost of the commercial packages , and the learning curves involved , may discourage researchers from using these programs . online tools are also available ( e.g. , http://www.quantitativeskills.com/sisa/calculations/bonfer.htm ) but are limited in scope and available options and rely on continued access to the publisher 's website . using the open - source programming language python v 3.2 , we developed a program capable of performing bonferroni , bonferroni - holm , and benjamini - hochberg corrections for any number of p - values . the user is prompted for a set of p - values and the desired significance ( alpha ) level . from the main menu the user may choose to display the results of the desired correction to the screen , or to export the corrected p values to the hard disk ( text and csv file types ) . the source code is available free as a supplementary file to this article ( which may serve as a literature reference for the program ) . a copy of the source code the program requires the free programming language python 3.2 which is capable of running on microsoft windows , mac os , and linux / unix operating systems . it may be downloaded from http://www.python.org/getit/releases/3.2/ .. the program is available for free by emailing the senior author at christopher.naugler@cls.ab.ca . detailed instructions and a faq are available at https://sites.google.com / site / christophernaugler/. to use the bonferroni calculator software , place the files bonferroni calculator.py and lesack and naugler.txt in a folder on your hard drive . in windows , the program will run from the command line by double clicking on the bonferroni calculator.py icon ; however the preferred method is to right click on the icon and select edit with idle from the dropdown list . press f5 to run the software , and then maximize the size of the window . follow the instructions on the screen . if the option is selected to save the results to files , these will be found in the same folder as the bonferroni calculator.py icon . the program is also available from the authors as a stand - alone executable file . using the open - source programming language python v 3.2 , we developed a program capable of performing bonferroni , bonferroni - holm , and benjamini - hochberg corrections for any number of p - values . the user is prompted for a set of p - values and the desired significance ( alpha ) level . from the main menu the user may choose to display the results of the desired correction to the screen , or to export the corrected p values to the hard disk ( text and csv file types ) . the source code is available free as a supplementary file to this article ( which may serve as a literature reference for the program ) . a copy of the source code the program requires the free programming language python 3.2 which is capable of running on microsoft windows , mac os , and linux / unix operating systems . it may be downloaded from http://www.python.org/getit/releases/3.2/ .. the program is available for free by emailing the senior author at christopher.naugler@cls.ab.ca . detailed instructions and a faq are available at https://sites.google.com / site / christophernaugler/. to use the bonferroni calculator software , place the files bonferroni calculator.py and lesack and naugler.txt in a folder on your hard drive . in windows , the program will run from the command line by double clicking on the bonferroni calculator.py icon ; however the preferred method is to right click on the icon and select edit with idle from the dropdown list . press f5 to run the software , and then maximize the size of the window . follow the instructions on the screen . if the option is selected to save the results to files , these will be found in the same folder as the bonferroni calculator.py icon . the program is also available from the authors as a stand - alone executable file .

increased type i error resulting from multiple statistical comparisons remains a common problem in the scientific literature . this may result in the reporting and promulgation of spurious findings . one approach to this problem is to correct groups of p - values for family - wide significance using a bonferroni correction or the less conservative bonferroni - holm correction or to correct for the false discovery rate with a benjamini - hochberg correction . although several solutions are available for performing this correction through commercially available software there are no widely available easy to use open source programs to perform these calculations . in this paper we present an open source program written in python 3.2 that performs calculations for standard bonferroni , bonferroni - holm and benjamini - hochberg corrections .

BACKGROUND Bonferroni Calculator software

PMC4299885

our initial sample consisted of 87 hcv+ participants who were recruited from several hepatology clinics and infectious disease clinics located throughout the greater los angeles catchment area . controls ( n = 21 ) were recruited from the community through advertisements and flyers . all procedures received prior approval by the university of california , los angeles and va greater los angeles healthcare system institutional review board committees for studies involving human subjects . all hcv+ patients participating in this study met clinical criteria for initiating hcv therapy but had not yet begun treatment . all data reported in the current study were collected at participants ' baseline visit . as part of procedures outlined in the parent study , there were no statistically significant differences between hcv+ participants who underwent neuroimaging and those who did not on key demographic variables such as age , sex , ethnicity , past drug abuse , current drug use , model for end - stage liver disease ( meld ) score , and psychiatric functioning ( all p values > 0.05 ) . inclusion criteria were as follows : ( 1 ) 18 years of age , ( 2 ) able to read and write in english , ( 3 ) and completed at least 6th grade education . exclusion criteria were as follows : ( 1 ) decompensated cirrhosis / liver failure ( detected by blood tests or liver biopsy ) with meld scores > 12 ; ( 2 ) current or past psychotic spectrum disorder ; ( 3 ) significant depression as judged by the study psychiatrists / psychologists ( defined as current moderate or severe major depressive disorder ) or suicidal ideation ( factors that were controlled for initiating interferon treatment ) ; ( 4 ) history of learning disability , seizure disorder , closed - head injury with loss of consciousness in excess of 30 minutes , or any other neurologic disease ; ( 5 ) evidence of any other cns opportunistic infection or neoplasm ; ( 6 ) hepatitis b infection ; ( 7 ) diagnosis of hiv infection as evidenced by hiv antibody testing ; ( 8) recent illicit drug use ( confirmed by urine toxicology ) ; and ( 9 ) contraindications for mri ( for the nested cohort who underwent neuroimaging ) . after applying exclusion criteria , our final sample ( those eligible for analysis ) included 76 hcv+ participants and 20 controls . ten hcv+ participants were determined to be ineligible due to liver cirrhosis , and 1 control participant tested positive for stimulants at study visit . brain mri was performed using a 3-tesla trio mri scanner ( siemens medical system , erlangen , germany ) . high - resolution mri included t1-weighted images using a magnetization - prepared rapid acquisition gradient - echo sequence using the following parameters : repetition time ( tr)/echo time ( te ) = 2,220/2.2 msec , inversion time = 900 msec , average = 1 , matrix size = 256 256 , field of view ( fov ) = 240 240 mm , slice thickness = 1 mm , number of slices = 176 . dti was acquired using a single - shot echo planar dual spin echo sequence with ramp sampling . the b - factor was set to 1,000 s / mm , tr = 9,600 msec , te = 90 msec , flip angle = 90 , and averages = 1 . a total of 71 axial sections were acquired using an image matrix of 130 130 , a slice thickness of 2 mm with no interslice gap , and an fov of 256 256 mm . proton spectra were collected using the 2d proton magnetic resonance spectroscopy imaging ( mrsi ) technique with the volume of interest ( voi ) preselected by means of point - resolved spectroscopy ( press ) . volume - selective 2d mrsi was performed on a 20-mm slab superior to the ventricles with te of 30 msec and tr of 2,000 msec . the nominal voxel size was 2.82 cm ( 16 16 phase encode steps over an 18 20 cm fov ) . outer - volume saturation bands were applied to all 6 sides of the voi localized by the press sequence to suppress the lipid contamination . we assessed attention using the wechsler adult intelligence scale - third edition ( wais - iii ) letter - number sequencing subtest and the paced auditory serial addition test ( only the first 50 trials ) ; processing speed using the wais - iii digit symbol and symbol search subtests , trail making test part a , and stroop color naming and word reading ; learning and memory using the hopkins verbal learning test - revised and the brief visuospatial memory test - revised ; verbal / language fluency using the controlled oral word association test , executive functioning using trail making test part b and stroop color - word interference test ; and motor speed using the grooved pegboard test . raw scores were converted into demographically adjusted t scores , grouped by neurocognitive domain , and averaged to create domain t scores . the vas contains 18 visual analogue lines with bipolar anchors with descriptors relating to energy and fatigue . internal consistency of the vas in our sample was high ( cronbach = 0.86 ) . the total score from the vas - fatigue subscale was examined in the current study . we used roieditor for preprocessing procedures according to a modified version of the active contour method and diffeomap . white matter parcellation map was used to segment the brain into 130 regions based on anatomical labeling , which included both the gray and white matter . once these regions were extracted , the cortex and the surrounding white matter were segmented using an fa threshold of 0.25 . we focused our analyses on regions of the corona radiata , insula , internal capsule , external capsule , striatum , fronto - occiptial fasciculus , cingulum , thalamus , hippocampus , and amygdala based on prior studies . for mrsi , metabolites were quantitated in each voxel using the frequency - domain fitting routine called lc - model algorithm , which analyzes the in vivo brain spectrum as a linear combination of individual simulated metabolite spectra that constitute the basis set or prior knowledge . we were able to quantify total naa_naag ( naa + naa glutamate ) , total choline ( gpc_pch ) , mi , and glutamate / glutamine ( glu_gln ) for regions of frontal white and gray matter , parietal white and gray matter , and basal ganglia ( see figure 2 ) . the accuracy of the quantitation was characterized using cramer - rao lower bound ( crlb ) , and metabolite ratios with crlb < 30% were considered for further analysis . no statistically significant differences were found for potential confounding variables such as age , years of education , ethnicity , and estimated premorbid functioning ( as measured by the wechsler test of adult reading ) ( all p values > 0.05 ) . groups also did not differ in current drug use . between - groups analysis of variance ( anova ) was used to examine differences between hcv and control groups on our 31 outcome variables of interest : neuroimaging ( 23 variables ) , neurocognitive assessment ( 7 variables ) , and fatigue ( 1 variable ) . outcome variables were all analyzed on a continuous scale and assumptions for performing anova and pearson correlations were checked prior to running analyses . demographic and drug use characteristics of the sample we used the false discovery rate ( fdr ) to correct for multiple comparisons with our outcome measures . fdr is sometimes preferred over family - wise error rate methods ( e.g. , bonferroni ) because it provides greater statistical power and reduces the chance of making type ii errors . given our available sample size of 96 for the neurocognitive and fatigue data , adjusted level of 0.01 ( for multiple comparisons ) , and power of 0.80 , we had enough power to detect medium effects ( f = 0.31 ) . this is consistent with effect sizes reported in previous investigations that have examined neurocognitive differences between hcv+ patients and controls . for neuroimaging analyses , with a sample of 49 this is also consistent with the effect sizes reported by neuroimaging studies that have compared hcv+ patients to controls . all procedures received prior approval by the university of california , los angeles and va greater los angeles healthcare system institutional review board committees for studies involving human subjects . all hcv+ patients participating in this study met clinical criteria for initiating hcv therapy but had not yet begun treatment . all data reported in the current study were collected at participants ' baseline visit . as part of procedures outlined in the parent study , there were no statistically significant differences between hcv+ participants who underwent neuroimaging and those who did not on key demographic variables such as age , sex , ethnicity , past drug abuse , current drug use , model for end - stage liver disease ( meld ) score , and psychiatric functioning ( all p values > 0.05 ) . inclusion criteria were as follows : ( 1 ) 18 years of age , ( 2 ) able to read and write in english , ( 3 ) and completed at least 6th grade education . exclusion criteria were as follows : ( 1 ) decompensated cirrhosis / liver failure ( detected by blood tests or liver biopsy ) with meld scores > 12 ; ( 2 ) current or past psychotic spectrum disorder ; ( 3 ) significant depression as judged by the study psychiatrists / psychologists ( defined as current moderate or severe major depressive disorder ) or suicidal ideation ( factors that were controlled for initiating interferon treatment ) ; ( 4 ) history of learning disability , seizure disorder , closed - head injury with loss of consciousness in excess of 30 minutes , or any other neurologic disease ; ( 5 ) evidence of any other cns opportunistic infection or neoplasm ; ( 6 ) hepatitis b infection ; ( 7 ) diagnosis of hiv infection as evidenced by hiv antibody testing ; ( 8) recent illicit drug use ( confirmed by urine toxicology ) ; and ( 9 ) contraindications for mri ( for the nested cohort who underwent neuroimaging ) . after applying exclusion criteria , our final sample ( those eligible for analysis ) included 76 hcv+ participants and 20 controls . ten hcv+ participants were determined to be ineligible due to liver cirrhosis , and 1 control participant tested positive for stimulants at study visit . brain mri was performed using a 3-tesla trio mri scanner ( siemens medical system , erlangen , germany ) . high - resolution mri included t1-weighted images using a magnetization - prepared rapid acquisition gradient - echo sequence using the following parameters : repetition time ( tr)/echo time ( te ) = 2,220/2.2 msec , inversion time = 900 msec , average = 1 , matrix size = 256 256 , field of view ( fov ) = 240 240 mm , slice thickness = 1 mm , number of slices = 176 . dti was acquired using a single - shot echo planar dual spin echo sequence with ramp sampling . the b - factor was set to 1,000 s / mm , tr = 9,600 msec , te = 90 msec , flip angle = 90 , and averages = 1 . a total of 71 axial sections were acquired using an image matrix of 130 130 , a slice thickness of 2 mm with no interslice gap , and an fov of 256 256 mm . proton spectra were collected using the 2d proton magnetic resonance spectroscopy imaging ( mrsi ) technique with the volume of interest ( voi ) preselected by means of point - resolved spectroscopy ( press ) . volume - selective 2d mrsi was performed on a 20-mm slab superior to the ventricles with te of 30 msec and tr of 2,000 msec . the nominal voxel size was 2.82 cm ( 16 16 phase encode steps over an 18 20 cm fov ) . outer - volume saturation bands were applied to all 6 sides of the voi localized by the press sequence to suppress the lipid contamination . we assessed attention using the wechsler adult intelligence scale - third edition ( wais - iii ) letter - number sequencing subtest and the paced auditory serial addition test ( only the first 50 trials ) ; processing speed using the wais - iii digit symbol and symbol search subtests , trail making test part a , and stroop color naming and word reading ; learning and memory using the hopkins verbal learning test - revised and the brief visuospatial memory test - revised ; verbal / language fluency using the controlled oral word association test , executive functioning using trail making test part b and stroop color - word interference test ; and motor speed using the grooved pegboard test . raw scores were converted into demographically adjusted t scores , grouped by neurocognitive domain , and averaged to create domain t scores . the vas contains 18 visual analogue lines with bipolar anchors with descriptors relating to energy and fatigue . internal consistency of the vas in our sample was high ( cronbach = 0.86 ) . the total score from the vas - fatigue subscale was examined in the current study . we used roieditor for preprocessing procedures according to a modified version of the active contour method and diffeomap . white matter parcellation map was used to segment the brain into 130 regions based on anatomical labeling , which included both the gray and white matter . once these regions were extracted , the cortex and the surrounding white matter were segmented using an fa threshold of 0.25 . we focused our analyses on regions of the corona radiata , insula , internal capsule , external capsule , striatum , fronto - occiptial fasciculus , cingulum , thalamus , hippocampus , and amygdala based on prior studies . for mrsi , metabolites were quantitated in each voxel using the frequency - domain fitting routine called lc - model algorithm , which analyzes the in vivo brain spectrum as a linear combination of individual simulated metabolite spectra that constitute the basis set or prior knowledge . we were able to quantify total naa_naag ( naa + naa glutamate ) , total choline ( gpc_pch ) , mi , and glutamate / glutamine ( glu_gln ) for regions of frontal white and gray matter , parietal white and gray matter , and basal ganglia ( see figure 2 ) . the accuracy of the quantitation was characterized using cramer - rao lower bound ( crlb ) , and metabolite ratios with crlb < 30% were considered for further analysis . no statistically significant differences were found for potential confounding variables such as age , years of education , ethnicity , and estimated premorbid functioning ( as measured by the wechsler test of adult reading ) ( all p values > 0.05 ) . groups also did not differ in current drug use . between - groups analysis of variance ( anova ) was used to examine differences between hcv and control groups on our 31 outcome variables of interest : neuroimaging ( 23 variables ) , neurocognitive assessment ( 7 variables ) , and fatigue ( 1 variable ) . outcome variables were all analyzed on a continuous scale and assumptions for performing anova and pearson correlations were checked prior to running analyses . demographic and drug use characteristics of the sample we used the false discovery rate ( fdr ) to correct for multiple comparisons with our outcome measures . fdr is sometimes preferred over family - wise error rate methods ( e.g. , bonferroni ) because it provides greater statistical power and reduces the chance of making type ii errors . given our available sample size of 96 for the neurocognitive and fatigue data , adjusted level of 0.01 ( for multiple comparisons ) , and power of 0.80 , we had enough power to detect medium effects ( f = 0.31 ) . this is consistent with effect sizes reported in previous investigations that have examined neurocognitive differences between hcv+ patients and controls . for neuroimaging analyses , with a sample of 49 this is also consistent with the effect sizes reported by neuroimaging studies that have compared hcv+ patients to controls . there were statistically significant group differences in fa in the striatum ( f1,48 = 8.49 , p < 0.005 ) , with the hcv group demonstrating higher fa levels than controls . while group differences in fa in the thalamus ( f1,48 = 4.6 , p = 0.04 ) and insula ( f1,48 = 3.86 , p = 0.05 ) emerged , these did not survive multiple comparisons correction . there were no statistically significant group differences in fa values among our other brain regions of interest ( see figure 1a and table e-2 ) . proton magnetic resonance spectroscopy imaging ( mrsi ) regions of interest of frontal white , parietal white , and basal ganglia . dti = diffusion tensor imaging ; fa = fractional anisotropy ; fdr = false discovery rate . among the hcv+ group , higher levels of fa in the striatum were correlated with poorer overall neuropsychological performance ( r29 = 0.45 , p = 0.01 ; r29 = 0.53 , p = 0.003 ) and the domain of language fluency ( r29 = 0.41 , p = 0.02 ) . individuals with chronic hcv infection demonstrated greater md in the fronto - occiptal fasciculus ( f1,48 = 18.6 , p < 0.0001 ) and external capsule ( f1,48 = 10.8 , p = 0.002 ) than controls ( see figure 1b ) . there was a statistical trend toward group md differences in the insula ( f1,48 = 3.86 , p = 0.05 ) ; however , this did not survive correction for multiple comparisons . there were no statistically significant relationships between md in our regions of interest and neurocognitive performance . hcv+ participants demonstrated lower levels of naa in bilateral parietal white matter ( f1,48 = 5.37 , p = 0.02 ) and elevations of mi in bilateral frontal white matter ( f1,48 = 9.48 , p = 0.004 ) compared to controls ( see figure 3 and table e-3 ) . for the hcv+ group , greater levels of frontal white matter mi were significantly correlated with poorer cognitive performance in the domains of processing speed ( r29 = 0.43 , p = 0.02 ) and verbal / language fluency ( r29 = 0.41 , p = 0.007 ) . higher naa in bilateral parietal white matter was significantly correlated with lower diffusivity in the fronto - occipital fasciculus ( r29 = 0.36 , p = 0.02 ) , whereas greater levels of frontal white matter mi were significantly correlated with higher diffusivity in the fronto - occipital fasciculus ( r29 = 0.47 , p = 0.01 ) . glu_gln = glutamate + glutamine ; gpc_pch = gylcerophosphocholine + phosphocholine ( total choline ) ; mrsi = proton magnetic resonance spectroscopy imaging ; naa_naag = n - acetylaspertate + n - acetylaspartate glutamate . hcv - infected individuals demonstrated poorer overall neurocognitive performance than controls ( f1,94 = 5.6 , p = 0.02 ) . cognitive domains that contributed to global findings included reduced processing speed ( f1,94 = 3.91 , p = 0.04 ) and verbal fluency ( f1,94 = 8.52 , p = 0.005 ) ( see figure 4 ) . neurocognitive performance differences between the hepatitis c group and controls . * = statistically significant at fdr - adjusted p value . as expected , hcv+ participants reported greater fatigue ( f1,94 = 7.41 , p = 0.008 ) than controls . among the hcv+ group , fatigue was not correlated with any of the dti regions ( all p values > 0.05 ) . higher fatigue scores correlated with higher levels of mi in frontal white matter ( r29 = 0.53 , p < 0.01 ) . among hcv+ patients , higher fatigue scores correlated with poorer overall neurocognitive performance ( r75 = 0.36 , p < 0.001 ) and domains of attention ( r75 = 0.26 , p = 0.01 ) , processing speed ( r75 = 0.31 , p = 0.002 ) , and executive functioning ( r75 = 0.40 , p < 0.001 ) . there were statistically significant group differences in fa in the striatum ( f1,48 = 8.49 , p < 0.005 ) , with the hcv group demonstrating higher fa levels than controls . while group differences in fa in the thalamus ( f1,48 = 4.6 , p = 0.04 ) and insula ( f1,48 = 3.86 , p = 0.05 ) emerged , these did not survive multiple comparisons correction . there were no statistically significant group differences in fa values among our other brain regions of interest ( see figure 1a and table e-2 ) . proton magnetic resonance spectroscopy imaging ( mrsi ) regions of interest of frontal white , parietal white , and basal ganglia . dti = diffusion tensor imaging ; fa = fractional anisotropy ; fdr = false discovery rate . among the hcv+ group , higher levels of fa in the striatum were correlated with poorer overall neuropsychological performance ( r29 = 0.45 , p = 0.01 ; r29 = 0.53 , p = 0.003 ) and the domain of language fluency ( r29 = 0.41 , p = 0.02 ) . individuals with chronic hcv infection demonstrated greater md in the fronto - occiptal fasciculus ( f1,48 = 18.6 , p < 0.0001 ) and external capsule ( f1,48 = 10.8 , p = 0.002 ) than controls ( see figure 1b ) . there was a statistical trend toward group md differences in the insula ( f1,48 = 3.86 , p = 0.05 ) ; however , this did not survive correction for multiple comparisons . there were no statistically significant relationships between md in our regions of interest and neurocognitive performance . hcv+ participants demonstrated lower levels of naa in bilateral parietal white matter ( f1,48 = 5.37 , p = 0.02 ) and elevations of mi in bilateral frontal white matter ( f1,48 = 9.48 , p = 0.004 ) compared to controls ( see figure 3 and table e-3 ) . for the hcv+ group , greater levels of frontal white matter mi were significantly correlated with poorer cognitive performance in the domains of processing speed ( r29 = 0.43 , p = 0.02 ) and verbal / language fluency ( r29 = 0.41 , p = 0.007 ) . higher naa in bilateral parietal white matter was significantly correlated with lower diffusivity in the fronto - occipital fasciculus ( r29 = 0.36 , p = 0.02 ) , whereas greater levels of frontal white matter mi were significantly correlated with higher diffusivity in the fronto - occipital fasciculus ( r29 = 0.47 , p = 0.01 ) . glu_gln = glutamate + glutamine ; gpc_pch = gylcerophosphocholine + phosphocholine ( total choline ) ; mrsi = proton magnetic resonance spectroscopy imaging ; naa_naag = n - acetylaspertate + n - acetylaspartate glutamate . * = statistically significant at fdr - adjusted p value . hcv - infected individuals demonstrated poorer overall neurocognitive performance than controls ( f1,94 = 5.6 , p = 0.02 ) . cognitive domains that contributed to global findings included reduced processing speed ( f1,94 = 3.91 , p = 0.04 ) and verbal fluency ( f1,94 = 8.52 , p = 0.005 ) ( see figure 4 ) . neurocognitive performance differences between the hepatitis c group and controls . * = statistically significant at fdr - adjusted p value . as expected , hcv+ participants reported greater fatigue ( f1,94 = 7.41 , p = 0.008 ) than controls . among the hcv+ group , fatigue was not correlated with any of the dti regions ( all p values > 0.05 ) . higher fatigue scores correlated with higher levels of mi in frontal white matter ( r29 = 0.53 , p < 0.01 ) . among hcv+ patients , higher fatigue scores correlated with poorer overall neurocognitive performance ( r75 = 0.36 , p < 0.001 ) and domains of attention ( r75 = 0.26 , p = 0.01 ) , processing speed ( r75 = 0.31 , p = 0.002 ) , and executive functioning ( r75 = 0.40 , p < 0.001 ) . there was no statistically significant correlation between fatigue and motor functioning . the current study examined the effects of chronic hcv infection on microstructural brain abnormalities , cerebral metabolites , fatigue , and neurocognitive performance . major strengths of the current investigation include the use of dti and mrsi in combination with measures of neurocognitive functioning and fatigue , and the use of a control group for comparison . as hypothesized based on prior literature , we observed microstructural abnormalities in such areas as the striatum , external capsule , and fronto - occipital fasciculus , which is consistent with previous dti studies of hcv and findings among individuals with hiv infection . we observed greater fa in gray matter regions of the striatum in hcv+ patients compared to healthy volunteers . higher fa in the striatum has been found among patients with huntington disease and is thought to be due to degeneration of efferent pathways that increase the coherence of gray matter structures . in a study of patients with chronic subdural hematoma , increased fa was found in the striatum , which reduced following surgical intervention . therefore , our findings are consistent with other investigations of neuropathology in regions that are affected in hcv . increased diffusivity in the fronto - occipital tract and external capsule was also found in the hcv+ group compared to controls . the fronto - occipital tract has been suggested as modulating frontal lobe related inhibitory control and occipital lobe related sensory inputs . alterations of this tract may interfere with integrating sensory information and inhibiting control over impulses and emotion , which is problematic among drug abusers . the external capsule contains a variety of different nerve bundles and pathways connecting the cerebral cortex to subcortical nuclei as well as connecting different parts of the cortex to each other . therefore , disruption to fibers of the external capsule may result in dysfunction of frontal - subcortical circuitry . hcv+ participants demonstrated lower levels of naa in bilateral parietal white matter and elevations of mi in bilateral frontal white matter compared to controls , which was associated with poorer performance in the cognitive domains of processing speed and verbal / language fluency . specifically , higher naa in parietal white matter was significantly correlated with lower diffusivity in the fronto - occipital fasciculus , whereas greater frontal white matter mi was significantly correlated with higher diffusivity in the fronto - occipital fasciculus . that stated , our mrsi results were generally consistent with previous mrsi studies of hcv+ cohorts , although we did not observe abnormal cerebral metabolite levels in basal ganglia as was expected . however , in the current study we were careful to exclude participants with medical ( e.g. , cirrhosis ) and psychiatric conditions that potentially could have confounded interpretation of the neuroimaging findings . through this process we may have excluded hcv+ individuals with more severe neurologic impairments and neuropathologic changes in subcortical structures that are detectable by h - mrs . although hcv+ patients demonstrated poorer global neurocognitive performance than controls , examination of performance data suggests normal range of performance ( i.e. , t > 40 ) . again , because of the use of stringent inclusion / exclusion criteria , this group may not be fully representative of the general hcv+ population . despite the potential recruitment of higher - functioning hcv+ individuals , we still found the poorer performance in the cognitive domains of processing speed and verbal fluency ( relative to controls ) that has been reported across other studies , and this performance was independent of such factors as liver fibrosis and history of substance abuse . participants also reported greater fatigue than controls , which was associated with abnormalities in frontal white matter , whereas poorer cognitive performance was associated with abnormalities in both frontal white matter and subcortical structures . these results suggest that hcv - associated neurologic complications that are specific to changes in frontal - subcortical structures give rise to both reduced cognitive performance and fatigue . the specific cognitive deficits observed in verbal / language fluency and information processing speed are all regulated by frontal - striatal structures . in our sample , verbal fluency demonstrated the greatest degree of performance difference between hcv+ and control groups and the strongest correlation with elevated levels of mi in frontal white matter . first , while structural neuroimaging methods are helpful in identifying microstructural pathology that may not be detected on standard mri , they do not provide a clear understanding about the functions of these neural circuits . hence , existing disruptions in a neural circuit may make a patient more vulnerable to developing symptoms such as fatigue . second , although we attempted to control for a number of demographic variables between hcv patients and controls , we recognize that there are a myriad of psychosocial differences ( e.g. , stress , past drug use ) that may account for the reduced cognitive performance and structural brain differences that were observed in the current study . for instance , we were unable to examine past drug abuse differences between our hcv+ and control groups because information on past drug abuse was not collected from the controls . we recognize that in order to precisely rule out the effects of past drug abuse we would have needed to recruit a sample of past drug abusers who were hcv. however , considering that 61% of our hcv+ patients reported a lifetime history of cocaine or opiate use , we attempted to address this concern by examining the effects of past drug abuse within this subgroup . while we did not find significant differences in our neuroimaging or neurocognitive data as a function of past drug abuse ( all p values > 0.10 ) , we can not rule out the residual confounding effects of distant substance use on neurologic function . despite these limitations , the current study represents a significant extension of the extant literature on hcv 's effects on neurologic and neurobehavioral functioning by demonstrating how abnormalities in frontal / parietal and subcortical structures have independent and overlapping relationships with cognitive performance and fatigue . it has long been known that hcv is hepatotoxic ; increasingly there is reason to believe that it is neurotoxic as well . while the precise pathophysiologic mechanism remains unclear , findings from the current study as well as while advances in the pharmacologic treatment of hcv hold incredible promise , there remain millions of hcv - infected adults in the united states and approximately 100 million worldwide . thames was involved in the study design , was responsible for conducting statistical analyses of study hypotheses , assisted with processing of neuroimaging data , and drafted the initial version of the manuscript . dr . castellon was involved in the conceptualization of the parent study ; provided scientific input on study design , data collection , and interpretation ; provided consultation for statistical analyses in the current study ; provided critical revisions to the write - up of study findings ; and gave final approval of the manuscript for submission . dr . singer was involved in the conceptualization of the parent study design , provided scientific input on clinical and medical data collection and interpretation , provided critical revisions to the write - up of study findings , and gave final approval of the manuscript for submission . nagarajan was involved in the study design of the mr spectroscopy data acquisition and mr spectroscopy postprocessing , provided critical revisions to the write - up of mr spectroscopy findings and interpretation , and gave final approval of the manuscript for submission . sarma was involved in the study design of the diffusion tensor imaging data acquisition and processing , assisted with the analyses , provided critical revisions to the write - up of diffusion tensor imaging findings and interpretation , and gave final approval of the manuscript for submission . smith was involved in the interpretation of clinical data , provided critical revisions to the manuscript draft , and gave final approval of the manuscript for submission . thaler assisted with statistical analyses of the neuropsychological data , assisted with the write - up of results , provided critical revisions to manuscript drafts , and gave final approval of the manuscript for submission . truong provided scientific input on medical data acquisition , provided critical revisions to the manuscript draft , and gave final approval of the manuscript for submission . schonfeld was involved in data acquisition , formatted the tables and figures , assisted with the write - up of the methods for the manuscript , and gave final approval of the manuscript for submission . thomas was responsible for the conceptualization and study design of the neuroimaging data , provided scientific input on sequence parameters and data analysis , provided critical revisions to manuscript drafts , and gave final approval of the manuscript for submission . dr . hinkin is the pi of the parent project and was responsible for the conceptualization of the study design , provided scientific oversight for all study phases , provided critical portions of manuscript drafts , and gave final approval of the manuscript for submission . funding support for the current study was provided through the nih ( ro1mh083553 , pi : c.h . thames has received research support from the nimh and the american psychological association society for clinical neuropsychology . e. singer is on the nih study section advisory board , is a reviewer for orau grants , and receives funding from nimh , minds , and nida .

objective : this study examined neurologic abnormalities ( as measured by proton magnetic resonance spectroscopy imaging and diffusion tensor imaging ) , neurocognitive performance , and fatigue among a sample of adults with hepatitis c virus ( hcv ) . we hypothesized that hcv+ individuals would demonstrate structural brain abnormalities and neurocognitive compromise consistent with frontostriatal dysfunction as well as increased fatigue compared to controls.method:participants were 76 individuals diagnosed with hcv and 20 controls who underwent a comprehensive neurocognitive evaluation and clinical assessments . a subset of the hcv+ participants ( n = 29 ) and all controls underwent mri.results:individuals diagnosed with chronic hcv infection demonstrated greater fractional anisotropy in the striatum as well as greater mean diffusivity in the fronto - occiptal fasciculus and external capsule compared to hcv controls . hcv+ participants also demonstrated lower levels of n - acetylaspartate in bilateral parietal white matter and elevations in myo - inosital ( mi ) in bilateral frontal white matter compared to hcv controls ( all p values < 0.05 ) . hcv+ participants also demonstrated significantly poorer neuropsychological performance , particularly in processing speed and verbal fluency . hcv+ patients reported higher levels of fatigue than controls , and fatigue was significantly correlated with diffusivity in the superior fronto - occipital fasciculus , elevations in mi in frontal white matter , and overall cognitive performance.conclusions:our results suggest that hcv - associated neurologic complications disrupt frontostriatal structures , which may result in increased fatigue and poorer cognitive performance , particularly in those cognitive domains regulated by frontostriatal regions .

METHODS Standard protocol approvals, registrations, and patient consents. DTI and MRSI postprocessing. Preliminary analyses. RESULTS DTI. MRSI. Neurocognition. Fatigue. DISCUSSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

PMC4216325

despite its world - wide omnipresence in soil , a. fumigatus had been considered asexual until studies of an aspergillus - like anamorph isolated from brazilian soil revealed a heterothallic breeding system , and the teleomorph neosartorya fumigata was described . we describe the first reported case of acute respiratory distress syndrome ( ards ) attributed to neosartorya udagawae . the mold grew rapidly in culture of both sputum and bronchoalveolar lavage ( bal ) fluid from a previously healthy 43-year - old woman with ards , which developed as the consequence of an acute febrile illness that progressed over 5 days . but despite appearance of fluffy white , non - sporulating mold on a variety of culture media , the colonies did not produce conidiophores , and phenotypic identification was not possible . the conidia of n. udagawae require longer incubation in the laboratory to germinate , and misidentification of neosartorya sp . n. udagawae has been emerging as a cause of invasive infections in humans , but our case is the first report of ards associated with neosartorya infection and describes a new clinical entity . a 43-year - old woman presented to an outlying hospital with complaints of fever and progressive shortness of breath that developed over 5 days . the patient had a past medical history significant for depression managed with sertraline 50 mg per day , and tobacco use ( cigarettes ) . she was brought to her local hospital by the emergency medical service after developing profound weakness , with inability to get out of bed . she was intubated due to respiratory decline and initially required mechanical ventilation with 100% fio2 and positive end - inspiratory pressure ( peep ) of 12 mm / h2o . she was unable to achieve adequate oxygen saturations with mechanical ventilation and required extra corporeal membrane oxygenation ( ecmo ) at the time of transfer to our facility . upon arrival at our facility , the patient was hypothermic ( 35.9 c ) . she was intubated and sedated with a set respiratory rate on mechanical ventilation at 14 breaths / min . her pulse and blood pressure were 79 beats / min and 86/52 mmhg , respectively ; the fio2 was 99% ; her weight was 93 kg ( 204 lbs ) . endotracheal and nasogastric ( ng ) tube , foley catheter , and ecmo cannula were in place . neurologic exam was consistent with medically induced paralysis ; muscle tone , corneal and pupil responses were all normal . complete blood count was notable for leukocytosis of 17.810 wbc/l ( 78% neutrophils ) , and normocytic anemia ( hgb=10.7 g / dl ) . the complete metabolic panel was normal , outside of elevated chloride ; hypokalemia ( k=3.1 mg / dl ) and depressed protein and albumin ( 5.4 and 2.6 mg / dl , respectively ) . an arterial blood gas revealed acidosis and hypoxemia : ph=7.31 ; paco2=43 pao2=45 ; sao2=80% . semi - upright portable ap chest x - ray was notable for diffuse alveolar opacities . endotracheal aspirate and three sets of blood cultures and a urine sample were submitted to the microbiology lab for culture . she was empirically started on oseltamivir 75 mg q 24 h via the ng , along with intravenous azithromycin and ceftriaxone initiated at the outlying hospital . during the patients first 24 h in the icu , she progressed from hypothermia to a febrile state ( 39.1 c ) , resolving leukocytosis ( 10.910 wbc/l ) , and an unchanged physical exam . hiv elisa , mycoplasma serology , and fungal immunodiffusion antibody testing for aspergillosis , histoplasmosis , blastomycosis , and coccidiomycosis were performed , and all returned negative / non - reactive . a nasopharyngeal swab for multiplex pcr for respiratory viral and intracellular bacterial pathogens ( film array , biofire , inc . ) day two , the patient was noted to have a normal body temperature , but remained on paralytics in a medically induced coma . one additional blood culture was obtained , and ebv and cmv serologies were performed ; all returned negative . cultures of an endotracheal tube aspirate ( eta ) that were obtained on admission were notable for growth of a fluffy , sterile white mold with yellow center on standard blood agar media . on hospital day four , endoscopic bronchoscopy was performed . bal fluid was collected from the left and right lower lobes ( lll & rll ) of the lung and submitted for gram and gomori methenamine silver ( gms ) staining , which were negative . histoplasma and legionella antigen testing on the bal fluid was negative , but an aspergillus galactomannan antigen was positive ( > 3.75 ng / ml ) . pcr for influenza ( cepheid genxpert ) as well as multiplex pcr for respiratory pathogens ( film array , biofire , inc . ) were both negative . again , within 48 h , a similar appearing fluffy , white mold grew in cultures of both lll and rll bal fluid samples on all media ( blood agar , sabouraud agar , and mycosel agar ( becton dickinson ) ; a selective medium containing cycloheximide and chloramphenicol to inhibit bacterial growth ) . potato dextrose agar ( pda ) was inoculated with a sample of mold , but serial lactophenol cotton blue prep exams of the non - sporulating cultures were non - diagnostic , although clearly not consistent with mucormycosis . empiric treatment with lipid formulation of intravenous amphotericin b was initiated on hospital day 5 due to failure to improve on broad spectrum empiric antibiotic treatment . over the next 48 h respiratory status was unchanged and oxygenation parameters and radiographic imaging showed no improvement . on hospital day 7 , following two iv doses of liposomal amphotericin b , repeat bronchoscopy was performed . gram and gms stains , and histoplasma antigen testing were repeated , and returned negative for yeast , pneumocystis or fungal elements . methylprednisolone 125 mg iv every 8 h was added to the empiric iv amphotericin b for treatment of possible allergic bronchopulmonary aspergillosis ( apba ) . mold was not cultured from either bal fluid sample on pda or sabouraud agar after 45 days of incubation at 30 c , or blood agar at 37 c . our patient slowly improved and intravenous ( iv ) methylprednisolone was tapered from 125 mg every 8 h to 40 mg twice a day over a 7 day period . on hospital day 15 , methylprednisolone was discontinued and empiric antifungal treatment was changed from iv amphotericin b to voriconazole 40 mg iv every 12 h. on hospital day 18 , ecmo treatment was terminated , and our patient was weaned successfully from mechanical ventilation and extubated . she was continued on voriconazole , but developed transaminitis prompting cessation of the drug due to hepatotoxicity . further antifungal treatment or steroid treatments were not administered . on hospital day 27 , our microbiology laboratory and a state reference laboratory independently identified our mold as aspergillus fumigatus employing phenotypic criteria ( fig . partial calmodulin , -tubulin , and its gene sequences were obtained via pcr amplification of genomic dna extracted from our clinical isolate . based on dna sequencing , the mold was identified as the morphologically identical but phylogenetically distinct species neosartorya udagawae ( tables 13 ) . as a result of profound deconditioning , our patient was transferred on day 28 to an inpatient unit to complete intensive physical therapy . subsequently , she returned to work and had no evidence of recurrent infection on follow - up examination . neosartorya spp . are the complementary mating types of aspergillus and are required for sex to occur . although neosartorya infections are uncommon , cases of neosartorya infection misidentified as a. fumigatus are well represented in the literature , suggesting the incidence of neosartorya infections may be higher than suspected . in a retrospective evaluation of specimens banked from patients diagnosed with invasive pulmonary aspergillosis at nih between 2000 and 2008 , vinh et al . conducted multilocus dna sequencing to re - examine 36 cases of infection attributed to a. fumigatus based on phenotypic identification . of these , four cases were found to be caused by n. udagawae ; three were seen in patients with chronic granulomatous disease , and one was in a patient with myelodysplastic syndrome . the median duration of illness was approximately seven times longer than that typically observed for illness caused by a. fumigatus . the mics of various antifungals for the n. udagawae isolates were higher than those for a. fumigatus , and the disease caused by n. udagawae was refractory to standard therapy . to make a precise identification of the mold cultured from our patient , dna was extracted and partial its , -tubulin and calmodulin genes were amplified with touchdown pcr with the annealing temperature running from 55 to 45 c , and sequences were generated . an ncbi blast query of the 798 bp partial -tubulin gene sequence against the ncbi genbank nr / nt nucleotide database returned six n. udagawae isolates with 100% similarity , and returned 28 n. udagawae and seven aspergillus sp . sequences at the 99% similarity level ( table 1 ) . a query of the 373 bp partial its gene sequence returned seven n. udagawae with 100% similarity ( table 2 ) . a query of the 743 bp partial calmodulin gene sequence returned five aspergillus sp . with 100% similarity , and 10 n. udagawae and one aspergillus sp . hits at 99% similarity ( table 3 ) . sequences from type strains of species near n. udagawae , high similarity sequences found in the blast search , and the sequences from our clinical isolate were aligned and analyzed with mega 5.2 . 2 ) show large diversity among n. udagawae isolates and reveal that our isolate is in the n. udagawae clade . the aspergillus sp . sequences with identical or high similarity sequences to n. udagawae represent asexual colonies of this heterothallic species . because the species is heterothallic the morphology of the aspergillus phase is very similar to a. fumigatus and the definitive identification is through dna sequencing and phylogenetic analysis ( tables 13 ) . histopathology and culture , ideally in combination , are the current basis for diagnosis and identification of fungal infections . our patients isolate only produced diagnostic conidiophores after 12 days of incubation , and then it was misidentified as aspergillus fumigatus by two microbiology laboratories . although it took a week to grow the mold in culture and extract dna to definitively identify the isolate as n. udagawae using its , -tubulin and calmodulin sequence data , the identification could have been done in 23 days if genomic dna had been extracted directly from the original slant culture . prompt identification of invasive molds directly from clinical specimens is not feasible by conventional microbiologic techniques , but molecular methods for rapid detection of molds directly from bal fluid offer the potential opportunity to direct antifungal treatment and guide evaluation of underlying host susceptibility . pcr / esi - ms is capable of rapid identification of fungal pathogens directly from clinical specimens . institutional irb approval was obtained for submission of samples of our patients bal fluid for testing by pcr / esi - ms , as described by shin et al . . bal fluid from the first bronchoscopy was fixed for cytology testing and unavailable for pcr / esi - ms testing . pcr / esi - ms was performed on bal fluid from the second bronchoscopy , which was performed on hospital day 7 , 48 h after initiation of amphotericin b treatment . pcr / esi - ms detected a neosartorya sp . from both rml and left lingular bal fluid specimens . although airborne conidia of neosartorya sp . are conceivably capable of causing an apba - like syndrome that is seen with a. fumigatus in asthmatic patients , this has not been described . our patient did not have a history of asthma , nor did her laboratory testing exhibit peripheral eosinophilia or elevated ige titers suggestive of apba . her response to steroid treatment is consistent with what would be expected in patient with the fibroproliferative stage of ards . infection has not been previously described . due to uncertainty about the species of mold responsible for the pulmonary infection in our patient , she was unnecessarily exposed to toxic empiric antifungal treatment with amphotericin b. more timely identification of the pathogen would have resulted in earlier initiation of pathogen specific directed therapy . while colonization is always a concern with clinical cultures from intubated patients , identification of n. udagawae by gene sequencing of mold grown from multiple lobes in both lungs and by pcr / esi - ms directly from bal fluid samples taken after initiation of antifungal treatment strongly supports the biologic plausibility of n. udagawae as the etiology of ards in our patient . finally , given that the mics of most antifungals for n. udagawae isolates are higher than for a. fumigatus , and that disease caused by n. udagawae can be refractory to standard therapy , rapid recognition of n. udagawae as the cause of disease can be critical . presently , it is unclear how often neosartorya udagawae and other species within aspergillus section fumigati cause invasive pulmonary or sinus infections , or allergic bronchopulmonary or sinus manifestations . as molecular modalities such as -tubulin and calmodulin sequencing , and pcr / esi - ms become integrated into diagnostic clinical microbiology laboratories , detection of putatively less common invasive molds such as neosartorya sp . causing pulmonary disease may increase , thereby providing a more detailed picture of the spectrum of mycoses that infect humans and other animals . r. sampath and k.s . lowery are salaried employees of ibis biosciences , a division of abbott .

we describe the first reported case of acute respiratory distress syndrome ( ards ) attributed to neosartorya udagawae infection . this mold grew rapidly in cultures of multiple respiratory specimens from a previously healthy 43-year - old woman . neosartorya spp . are a recently recognized cause of invasive disease in immunocompromised patients that can be mistaken for their sexual teleomorph , aspergillus fumigatus . because the cultures were sterile , phenotypic identification was not possible . dna sequencing of its , calmodulin and -tubulin genes supported identification of neosartorya udagawae . our case is the first report of ards associated with neosartorya sp . infection and defines a new clinical entity .

Introduction Case Discussion Conflict of interest

PMC3894028

children brought for medical treatment are often found to be suffering from more than one morbid condition , making a single diagnosis impossible . the effective management of those conditions is more dependent on adopting a holistic approach using cheap , universally available and accessible strategies rather than sophisticated and expensive technology . according to the world bank report 1993 , for situations where laboratory support and clinical resources are limited , such an approach is more realistic and cost - effective , and therefore , has the potential to make the greatest impact on the global burden of disease . during the year 1992 , the world health organization ( who ) , who , in collaboration with united nations integrated children 's emergency fund ( unicef ) , and some other agencies , institutions and individuals , responded to the challenge by adopting a strategy known as integrated management of childhood illness ( imci ) . , priority has not been given to diagnosis of individual disease , rather the classification of the diseases and assessment of severity according to the common signs and symptoms were approached . along with curative care for common childhood illnesses like acute respiratory infection , diarrhea , measles , malaria and malnutrition , the strategy also addressed aspects of nutrition , immunization , and other important elements of disease prevention and health promotion . imci addressed the age group between 2 weeks and 59 months , but in india it was found that neonatal mortality constitutes 64% of under - five mortality , all neonates are included in the strategy , starting from the day of birth and it is adapted in the indian version as imnci . in the adapted version , the entire age group of 0 to 59 months ( as against 2 weeks to 59 months in imci ) was included to address the neonatal mortality challenge . in the imnci version , the age group was divided in two groups as 0 - 2 months and 2 months to 59 months . in both the age groups , through the combination of signs and symptoms classification of the condition is made . this classification indicates the severity of conditions and call for specific actions like red indicating urgent referral , yellow indicating management at existing health facility and green indicating management at home . the imci algorithm in both 7 days to 2 months and 2 months to 5 years age group was validated in india and other developing countries by several studies . as imnci is the adapted version of imci , followed only in india , there is paucity of published study testing the validity of imnci . however , two studies were done at department of pediatrics , kalawati saran children 's hospital and lady hardinge medical college testing the validity of imnci algorithm during 2002 - 2003 by goswami v , singh v , dutta ak and by kaur s , singh v , dutta ak , chandra j during the year 2005 - 2006 . in west bengal imnci algorithm was operational in purulia district since the year 2008 , but there is hardly any published study testing its validity . under this background , the present study has been done with the objective to assess the validity and reliability of the algorithm with provisional diagnosis of senior pediatricians for each imnci classifications . this observational , cross sectional study was done at department of pediatrics , in a tertiary care hospital of kolkata during january to march 2009 with the young infants between 0 - 2 months . all the young infants presented during this study period with a fresh episode of illness were included in the study with o an informed consent from their parents . altogether 117 mothers were interviewed with a pre tested , semi structured interview schedule and the young infants were assessed , classified and categorized for treatment using physician 's chart booklet for imnci along with the assessment form used for imnci . these cases were then sent to the pediatricians for further assessment without any mention of the classifications made by the researchers using the imnci algorithm so as to reduce observer bias . pediatricians examined the young infants as they did in any tertiary care hospital and recorded the presenting symptoms , clinical features and provisional diagnosis in the opd or emergency examination tickets . the imnci classifications were compared with those provisional diagnoses . the provisional diagnosis which could be compared with a particular imnci classification was determined after discussion with senior faculty members of department of pediatrics , medical college , kolkata . validity characteristics like sensitivity , specificity , positive predictive value , negative predictive value , and reliability characteristics like percent agreement , and kappa were assessed for individual imnci classifications against pediatrician 's provisional diagnosis . the kappa test is used to exclude the extent of percent agreement which was due to chance . out of the 117 young infants , 25.64% were within 7 days of age and 2.56% were presented within 24 hours . the young infants were presented with one or multiple presenting symptoms and among them cough and cold ( 33.33% ) was the main symptom . next were fever ( 12.82% ) , loose stool ( 11.11% ) and respiratory distress ( 11.11% ) . other important presenting symptoms were convulsion ( 7.69% ) , vomiting ( 7.69% ) , yellow discoloration of skin ( 5.98% ) , inability to suck ( 5.98% ) , unsatisfactory feeding ( 5.13% ) , and no cry after birth ( 2.56% ) . the common classifications were severe malnutrition ( 23.08% ) , feeding problem ( 22.22% ) , low weight for age ( 21.37% ) , possible serious bacterial infection ( 19.66% ) , possible serious bacterial infection , not able to feed ( 18.80% ) , severe jaundice ( 1.71% ) etc [ table 1 ] . distribution of study subjects according to the imnci classification ( multiple classification ) n = 117 when pediatricians assessed the cases , a single provisional diagnosis was made for each study subject based on initial clinical evaluation . the important provisional diagnoses as found in the opd or emergency tickets were common cold ( 22.20% ) , followed by septicemia ( 15.40% ) , jaundice ( 7.70% ) , breast fed loose stool ( 6.80% ) , birth asphyxia ( 6.80% ) , upper respiratory tract infection ( 6.80% ) , local infection ( 6% ) , lower respiratory tract infection ( 4.30% ) , low birth weight baby ( 3.50% ) , thrush ( 1.70% ) [ table 2 ] . distribution of study subjects according to provisional diagnosis by pediatricians n = 117 in eight cases ( 6.80% ) , the pediatricians found no abnormality and the initial diagnoses were healthy baby . for fourteen cases ( 12% ) , there were some other single provisional diagnoses like spina bifida occulta , anorectal malformation , cephalhematoma , congenital hypothyroidism , down 's phenotype , abdominal colic , congenital leukemia , stenosis of gut , hemorrhagic disease of newborn , meconium aspiration syndrome , excessive jitteriness , prune belly syndrome , congenital cyanotic heart disease [ table 2 ] . the classifications made by imnci algorithm were compared with the provisional diagnoses of the pediatricians . as per the opinions of senior faculty members of pediatrics department , possible serious bacterial infection was comparable with septicemia ; local bacterial infection with cellulites , boil ; whereas for jaundice , low body temperature , and diarrhea , the comparisons should be with similar conditions . there was no diagnosis as severe jaundice , severe dehydration , severe malnutrition , low weight for age , feeding problem and no feeding problem by the pediatricians and so comparison could not be made for those conditions . both imnci algorithm and pediatrician 's provisional diagnosis did not find any case of some dehydration , severe persistent diarrhea or severe dysentery and obviously no comparison could be done for those cases . in case of possible serious bacterial infection , in the present study , sensitivity was found as 88.89% , positive predictive value was 36.39% , negative predictive value was 97.26% and specificity was 71.72% [ table 3 ] . along with validity , the reliability of the classification was also assessed by percent agreement , by exclusion of chance by kappa test . but it was evident that most of the agreement was due to chance ; so when chance agreement was excluded by kappa test , the kappa value was only 0.38 [ table 3 ] , which indicates only minimal agreement . distribution of validity and reliability characteristics of imnci classification , against pediatrician s provisional diagnosis n = 117 in case of local bacterial infection , sensitivity was only 14.29% whereas the specificity was 99.09% [ table 3 ] . the percent agreement of local bacterial infection was 94% with the kappa value 0.20 , indicating only minimal agreement [ table 3 ] . in cases of jaundice , sensitivity was 66.67% whereas the specificity was 99.07% [ table 3 ] . the percent agreement was 97% with kappa value 0.73 , indicating good agreement [ table 3 ] . for no dehydration classification in algorithm , the sensitivity was 25% whereas the specificity was 94.50% [ table 3 ] . the percent agreement was 90% , the kappa value was only 0.19 , which indicates only negligible agreement [ table 3 ] . as per the algorithm , there were 44 cases of possible serious bacterial infection and among those , there were 21 cases , which had no ability to feed or suck . though in the algorithm , those cases were classified separately , pediatricians did not consider them as separate entity and diagnosed as septicemia as a whole . when chance agreement was excluded by kappa test , the kappa value was only 0.29 , indicating only minimal agreement [ table 3 ] . integrated management of neonatal and childhood illness ( imnci ) is already operational at the field level in india , but there is paucity of published study testing its validity and reliability . in the present study , it was found that young infants were presented with one or multiple symptoms and among them cough and cold ( 33.33% ) was the main symptom . the other important presenting symptoms were fever ( 12.82% ) , loose stool ( 11.11% ) and respiratory distress ( 11.11% ) . in the study done by sachdev et al . the most common presenting symptom was cough ( 65.3% ) , followed by fever ( 53.8% ) , running nose ( 40.3% ) , diarrhea ( 17.3% ) , respiratory distress ( 9.6% ) . the young infants were assessed and classified according to the imnci algorithm and it was found that majority ( 54.70% ) had single classification , 38.47% had two classifications , 5.98% had three and 0.85% had four classifications . the mean number of classifications was 1.53 with standard deviation 0.65 . in the study done by sachdev et al , the mean number of classification by imnci algorithm was 1.8 with standard deviation 0.8 . in the study done by kaur , singh , dutta , chandra , the mean number of morbidities was 1.75 . in the present study , the important provisional diagnoses as found in the opd or emergency tickets were common cold ( 22.20% ) , followed by septicemia ( 15.40% ) . in the study done by sachdev et al . the most common diagnosis of the pediatricians was low birth weight ( 75.20% ) , followed by diarrhea ( 27.9% ) , upper respiratory tract infection ( 26.3% ) and septicemia ( 21.7% ) . in case of possible serious bacterial infection , in the present study , sensitivity was found as 88.89% , positive predictive value was 36.39% and specificity was 71.72% , whereas in the study done by sachdev et al , the sensitivity was 96.5% and specificity was 51.8% . in the study done by kaur , singh , dutta , chandra , the sensitivity of algorithm to identify bacterial infection was 88.5% while the specificity was relatively low ( 57.4% ) . the reasons for this low specificity and positive predictive value might be that the predictors for possible serious bacterial infection as mentioned in the algorithm could predict other conditions also as convulsion or bulging fontanelle might occur in other cns disorders like hypoxic - ischemic encephalopathy , birth asphyxia , intracranial hemorrhage , meningitis , hypoglycemia , hypocalcaemia or any other causes of subdural effusion apart from septicemia . similarly increased respiratory rate , severe chest indrawing or nasal flaring could be the manifestation of hyaline membrane disease , transient tachypnea of newborn , meconium aspiration syndrome and other causes of respiratory insufficiency . dehydration fever , which is very much common particularly in an overheated nursery , might be an important cause of over diagnosis of possible serious bacterial infection . in the study by goswami , singh , dutta , algorithm tends to over diagnose serious bacterial infection by 8 - 20% ( in three age groups ) . in the study done by kaur , singh , dutta , chandra , also , it was found that out of 80 cases classified by the algorithm as possible serious bacterial infection with difference in diagnosis with the pediatricians , 31 ( 38.7% ) had birth asphyxia with hypoxic - ischaemic encephalopathy , 16 ( 20% ) had hypocalcemic seizures , 11 ( 13.7% ) had meconium aspiration syndrome , and 7 ( 8.8% ) had hemorrhagic disease of newborn . other conditions included respiratory distress syndrome ( 9 cases ) , transient tachypnea of newborn ( 4 cases ) , and neonatal seizures ( 2 cases ) . pediatricians diagnosed one big boil and more than 10 pustules as boil or cellulites , comparable with local bacterial infection whereas for the imnci algorithm , it was considered as possible serious bacterial infection . this difference in detection might be the cause of low sensitivity . in cases of jaundice , sensitivity was 66.67% whereas the specificity was 99.07 . pediatricians diagnosed all relevant cases as jaundice ; clinically they did not categorize any case as severe jaundice . however , in the algorithm when the jaundice appears in the 1 day of life or persists for more than 14 days and extends in the palms and soles , it was classified as severe jaundice . in the study done by kaur , singh , dutta , chandra , the algorithm under - diagnosed the severity of jaundice in few subjects ( 12/131 ) and over - diagnosed ( 8/131 ) the severity in few subjects . for no dehydration classification in algorithm , probability of low sensitivity might be due to some cases like lactose intolerance , breast - fed loose stool and not consideration of urination status by the algorithm . in the study done by kaur , singh , dutta , chandra , of the 76 cases identified as diarrhea by the algorithm , 22 ( 29% ) had breast - fed stools . in the algorithm , some conditions like birth asphyxia , down 's phenotype , abdominal colic , stenosis of gut , hemorrhagic disease of newborn , meconium aspiration syndrome , excessive jitteriness , prune belly syndrome , congenital cyanotic heart disease , anorectal malformation , cephalhematoma , congenital leukemia , breast - fed loose stool or lactose intolerance , spina bifida occulta , and congenital hypothyroidism were not covered . in the study done by kaur , singh , dutta , chandra , also it was seen that the algorithm under diagnosed some surgical conditions and congenital anomalies . therefore , in conclusion , it could be mentioned that imnci is a quite sensitive strategy and could identify the severe illnesses of the young infants requiring referral to higher facility . presence of other diagnosis with similar symptoms might result in false positive errors and low specificity . however , as this study was done in a tertiary care setting , further study particularly in primary health care setting is required .

background : integrated management of childhood illness ( imnci ) is already operational in many states of india , but there are only limited studies in indian scenario comparing its validity and reliability with the decisions of pediatricians . aims andobjectives : to assess the validity and reliability of the imnci algorithm with provisional diagnosis of senior pediatricians for each imnci classifications.materials and methods : the present study is done with all the young infants between 0 - 2 months presented during the study period with a fresh episode of illness to test the validity and reliability of the algorithm in comparison to provisional diagnoses of senior pediatricians . the study was done in a tertiary care hospital . validity characteristics such as sensitivity , specificity , positive predictive value , negative predictive value , and reliability characteristics such as percent agreement and kappa were assessed for individual imnci classifications.results:the sensitivity of possible serious bacterial infection , local bacterial infection , jaundice , no dehydration and possible serious bacterial infection , not able to feed were 88.89 , 14.29 , 66.67 , 25 and 44.44% respectively . the specificities for the same conditions were 71.72 , 99.09 , 99.07 , 94.50 and 86.87% . percent agreements for similar conditions were 74 , 94 , 97 , 90 and 80% respectively and the kappa ratios were 0.38 , 0.20 , 0.73 , 0.19 and 0.29 respectively.conclusion:it could be concluded that imnci is quite a sensitive strategy and could identify severe illnesses of young infants requiring referral to higher facility . further studies , particularly in primary health care setting , are required .

Introduction Materials and Methods Results Discussion

PMC4677765

in the age of health care reform , institutions are asking providers to continually improve the quality of care while reducing costs . patient satisfaction with pain management is a critically important measure of quality health care.1 while opioids are effective analgesics , their use is frequently associated with adverse effects such as dizziness , nausea , sedation , constipation , and in some cases , respiratory depression.2 guidelines suggest that opioid - sparing multimodal analgesia should be used whenever possible in order to optimize analgesic efficacy while minimizing the risk of opioid - related adverse events.2,3 regional anesthesia techniques are an effective mode of analgesia , considered safe with a low risk for complications , and are a frequently utilized component in multimodal analgesic regimens.35 transversus abdominis plane ( tap ) infiltration is a regional anesthesia technique that provides analgesia to the anterior abdominal wall . abdominal field blocks have been utilized for more than a century , but infiltration techniques used in tap were developed and refined within the last 15 years . tap infiltration has been demonstrated to be an effective regional anesthesia approach to controlling postsurgical pain , frequently allowing patients a faster recovery time after undergoing abdominal surgery.68 the tap is a potential anatomical space in the anterior wall that spans the entire abdomen between the internal oblique and transversus abdominis muscles ( figure 1).9 the anterior rami of the lower six thoracic nerves ( t7t12 ) and first lumbar nerve ( l1 ) pass through the plane and provide somatic innervation to the anterior and lateral abdominal walls.6,10,11 the nerves are bound to the transversus abdominis muscle by a layer of fascia , suggesting that local anesthetics should be deposited underneath the fascial layer to ensure optimal analgesia.11 detailed anatomic studies of the tap have revealed individual variability in the extent of spinal nerve branching . in a cadaveric dissection study of innervation of the anterior abdominal wall ( n=20 subjects),11 extensive branching was noted for t9l1 starting at the anterior axillary line to the midline . in addition , a large , longitudinal plexus of communicating branches was observed within the tap in the anterolateral abdominal wall lateral to the deep circumflex iliac artery . a second longitudinal plexus exists closer to the midline , running cephalocaudally with the inferior epigastric artery.11 another cadaveric study9 noted individual variation in the dimensions of the lumbar triangle of petit , usually situated just behind the highest point of the iliac crest ( figure 2),6,12,13 which serves as an anatomical landmark for tap infiltration . the study also noted that the t9l1 nerves held a relatively constant course in the tap prior to the midaxillary line , but that branching after this point was commonly observed.9 tap infiltration was originally characterized as a landmark - guided abdominal field block based on the lumbar triangle of petit in 2001.12 appropriate needle placement using the landmark technique resulted in a pop or sensation of giving way , which indicated that the needle had reached the fascial plane between the internal oblique and transversus abdominis muscles.12 the technique , initially described as a field block or a regional abdominal field infiltration , was eventually referred to as a tap block.7,14 this technique was evaluated and modified through a series of cadaveric and healthy volunteer studies.9,1518 an alternative method was described in which the needle is advanced perpendicularly until the first pop is felt in between the external and internal oblique muscles , and then advanced again until a second pop is felt , indicating entry into the transversus abdominis fascial plane.6,18 as spread of the local anesthetic is critical for analgesic efficacy , an ultrasound - guided ( usg ) technique was a logical progression for tap infiltration . the initial usg approach involved transverse application of the ultrasound probe to the anterolateral abdominal wall where the three muscle layers are most distinct ( figure 3).19,20 the needle , as well as the hypoechoic layer of local anesthetic in the fascia , was visualized in the plane.20 a series of clinical studies evaluated the extent of analgesia in multiple surgical models , including prostatectomy , abdominal surgeries ( bowel resections ) , appendectomy , cesarean section , and abdominal hysterectomy . initial clinical results deviated from the landmark - based technique , as the spread of analgesia did not seem to be as extensive.2125 although the reason for this is not well understood , it has been suggested that the key objective in ultrasound is visualization of the spread of the injectate between the internal oblique and transversus muscles , possibly causing the operator to ignore the tactile pop that indicates optimal needle placement in the anatomical space underneath the fascial layer.8 in response to this clinical variation , the technique evolved further . multiple and single injections as well as lateral , subcostal , and posterior approaches have all been described.17,19,24,26 a report describing four different approaches to tap infiltration found that local anesthetic spread resulting from the posterior approach mirrored most closely the original landmark - guided triangle of petit approach.17 spread of local anesthetic was measured radiologically , and was found to cover t5l1 levels using these techniques.17 a four - point single - shot technique that combines both the posterior and subcostal approaches has also been described and purportedly provides wider analgesic coverage.19 in sum , there are many factors that impact analgesic outcomes in tap : in addition to method of nerve localization and needle placement , population type ( pregnant vs nonpregnant , obese vs nonobese ) ; surgical procedure ; practitioner s level of skill ; type , dose , and volume of local anesthetic used ; timing of injection ; and clinical assessments used all have an impacting role.8 it was determined that extent of spread was variable and nondermatomal , indicating that the specific nerves that are blocked as a result of a tap infiltration likely vary from patient to patient based on their anatomy.27 the many nuances of the technique have led to a debate in the field : is there a need for standardization of techniques or technique nomenclature ? the tap block family and regional abdominal field infiltration have both been proposed to describe the group of techniques that have evolved from the initial triangle of petit landmark technique.7,8 however , the fundamentals of the technique remain consistent across approaches : no specific nerve within the tap is targeted ; localization of the plane and the spread of local anesthetic within the plane are the key determinants of analgesia.19 multiple surgeon - assisted approaches have been described , suggesting that techniques used to access the plane itself have also broadened . intra - abdominal laparoscopic cameras operated by surgeons , rather than ultrasound , have been used to visualize the plane . for example , one report describes a technique developed to enable identification of a peritoneal bulge in the area of injection that confirms delivery of local anesthetic into the tap.28 other reports describe a surgeon - assisted intraoperative transperitoneal approach in which the blunt - tipped block needle was advanced from inside the abdominal wall through the parietal peritoneum , into the transversus abdominis muscle , and then into the tap.29,30 direct visualization of local anesthetic spread in the tap using blunt dissection of the oblique muscles has also been described.31 the goal of the surgeon - assisted approach is to conduct the same field block as traditional surgical infiltrative techniques where local anesthetic is deposited near the incision . whether local anesthetics are utilized for a peripheral nerve block or for infiltration , the objective is the same to block the sensory response . however , key technical components diverge when comparing a peripheral nerve block to an infiltration technique . as described above , tap infiltration comprises multiple approaches used to identify an anatomical plane , instead of a specific nerve or nerve plexus . for example , in a peripheral nerve block of the upper or lower extremity , usg and/or nerve stimulation are frequently used to identify the nerve that is to be blocked along with surrounding anatomy.19 the optimal needle position is near or toward that specific nerve , and spread of local anesthetic around the nerve is observed.32,33 precision and ability to maintain appropriate needle placement are of the utmost importance in peripheral nerve blocks.19 while the objective of peripheral nerve block and tap infiltration are similar in that both approaches block sensory response in order to achieve analgesia , the technical components of these two approaches are different . unlike peripheral nerve block , which involves administration of a local anesthetic in close proximity to a specific nerve or nerve plexus , tap infiltration is a field block technique in which a local anesthetic is administered into an anatomical plane .

transversus abdominis plane ( tap ) infiltration is a regional anesthesia technique that has been demonstrated to be effective for management of postsurgical pain after abdominal surgery . there are several different clinical variations in the approaches used for achieving analgesia via tap infiltration , and methods for identification of the tap have evolved considerably since the landmark - guided technique was first described in 2001 . there are many factors that impact the analgesic outcomes following tap infiltration , and the various nuances of this technique have led to debate regarding procedural classification of tap infiltration . based on our current understanding of fascial and neuronal anatomy of the anterior abdominal wall , as well as available evidence from studies assessing local anesthetic spread and cutaneous sensory block following tap infiltration , it is clear that tap infiltration techniques are appropriately classified as field blocks . while the objective of peripheral nerve block and tap infiltration are similar in that both approaches block sensory response in order to achieve analgesia , the technical components of the two procedures are different . unlike peripheral nerve block , which involves identification or stimulation of a specific nerve or nerve plexus , followed by administration of a local anesthetic in close proximity , tap infiltration involves administration and spread of local anesthetic within an anatomical plane of the surgical site .

Introduction Anatomy of the TAP Evolution of TAP infiltration techniques Key differences between TAP infiltration and peripheral nerve blocks Conclusion

PMC3968616

smoking affects vascular and hormonal systems and is also involved in the development of atherosclerosis , thrombogenesis and vascular occlusion . chronic smoking adversely influences the prognosis of nephropathies . in primary hypertension , urinary albumin excretion as an index of renal damage the rate of progression to renal failure amongst smokers is twice in both type i and type ii diabetes . this study was intended for non - diabetic normotensive subjects to find out the effect of smoking on renal functions such as microalbuminuria , creatinine clearance and urinary albumin creatinine ratio ( acr ) . this was a community - based prospective cross - sectional cohort study conducted on 120 non - diabetic normotensive subjects as per the inclusion criteria . it was approved by the institutional ethics committee and conducted in accordance with the guidelines of the indian council of medical research , international conference on harmonization - good clinical practices instructions of drug controller general of india new delhi and institutional sop with a proper informed consent . inclusion criteria were age 30 - 70 years , normotensive ( 139/89 mmhg ) , non - diabetic ( fasting plasma glucose 125 mg / dl ) , non - obese ( body mass index [ bmi ] < 30 kg / m ) , no family history of premature vascular disease , normal total cholesterol level ( < 200 mg / dl ) , normal renal function ( urea 40 mg / dl and creatinine 1 mg / dl ) , clinically well and not on any regular cardiovascular medication and given informed consent . exclusion criteria were age less than 30 and above 70 years , diabetics or using insulin or oral hypoglycaemic agents , hypertensive or using antihypertensive medication , hyperlipidemic or using lipid lowering drugs , obese ( bmi 30 kg / m ) , abnormal renal function ( urea and creatinine ) , urinary tract infection , significant renal disease or using diuretics , angiotensin converting enzyme inhibitors , alcohol consumption or other significant drugs , fever , vigorous physical activity , menstruating / pregnant women and not willing to give consent . smoker was defined as anyone who had smoked at least 20 bidi / day for 5 years ( 5 pack - years ) or equivalent . smokers were divided into four sub - groups : very light smoker ( 5 - 9 pack - years ) , light smoker ( 10 - 14 pack - years ) , moderate smoker ( 15 - 19 pack - years ) and heavy smoker ( > 20 pack - years ) . out of 120 non - diabetic normotensive subjects , 80 ( 66.66% ) were smokers and 40 ( 33.33% ) were age matched non - smokers as control . the baseline physical characteristic and investigation were compared in these two groups using the statistical tests of significance ( p = ns ) [ table 1 ] . baseline subjects characteristics and their comparison in both groups overnight fasting blood sugar , blood urea , serum creatinine and lipid profile were measured and first morning void ( timed ) quantitative mid - stream urine was sample taken for screening of urinary albumin and urinary creatinine concentrations [ table 1 ] . laboratory methods used included ; clinitek micro albumin reagent strips ( urinary albumin and acr ) , jaffe colorimetric method ( serum creatinine concentration ) , glucose enzymatic colorimetric ( glucose oxidase / peroxidase ) method ( plasma glucose ) , enzymatic colorimetric cholesterol ( cholesterol oxidase / peroxidase aminophenazone ) method ( serum lipid profile ) , urine analyzer uriscan optima ii + combistix sg , korea ( urinary leukocyte and erythrocyte ) . all calculations were performed with the statistical package for the social sciences ( spss ) version 9.0 ( spss , chicago , usa ) software by chi - square analysis or analysis of variance . out of 120 non - diabetic normotensive subjects , 80 ( 66.66% ) were smoker and 40 ( 33.33% ) were non - smoker . 83 subjects ( 69.16% ) were male and 37 subjects ( 30.83% ) were female . the mean age in smoker group was 48.68 11.82 years and in non - smoker group was 46.10 10.77 years , ranged from 30 to 70 years ( p = 0.247 ) [ table 1 ] . the two groups were comparable in terms of all parameters except high - density lipoprotein ( hdl ) level ( p = 0.031 ) [ table 1 ] . smokers had higher mean urinary albumin level ( 52.84 mg / l ) than non - smokers ( 19.25 mg / l ) ( p < 0.0001 ) [ figure 1 ] . among smokers ( n = 80 ) , microalbuminuria was directly related to the amount of smoking ( pack - years ) [ figure 2 ] . seventy three smokers ( 91.25% ) and nine non - smokers ( 22.5% ) had urinary albumin level > 20 mg / l ( microalbuminuria ) . seven smokers ( 8.75% ) and 31 non - smokers ( 77.5% ) had urinary albumin level < 20 mg / l [ figure 3 ] . comparison of mean urinary albumin level between smoker ( n = 80 ) and non - smoker ( n = 40 ) relationship between quantity of smoking and mean urinary albumin in smoker comparison of smoker ( n = 80 ) and non - smoker ( n = 40 ) subjects for microalbuminuria smoker ( n = 80 ) had higher mean urinary acr ( 93.98 g / mg ) than non - smoker ( n = 40 , 18.99 g / mg ) ( p < 0.001 ) [ figure 4 ] . among smokers ( n = 80 ) , urinary acr level was directly related to the amount of smoking ( pack - years ) [ figure 5 ] . sixty four smokers ( 80% ) and two non - smokers ( 5% ) had urinary acr level > 30 g / mg . only 16 smokers ( 20% ) and 38 non - smokers ( 95% ) had urinary acr < 30 g / mg [ figure 6 ] . comparison of mean urinary albumin creatinine ratio level between smoker ( n = 80 ) and non - smoker ( n = 40 ) relationship between quantity of smoking and urinary albumin creatinine ratio in smoker comparison of smoker ( n = 80 ) and non - smoker ( n = 40 ) subjects for high urinary albumin creatinine ratio male and female smokers had no statistically significant distribution of urinary albumin , urinary creatinine and urinary acr ( p = ns ) . this study shows non - diabetic normotensive smoker had higher mean urinary albumin level , which is directly related to the amount of smoking ( pack - years ) among smokers . also , more smokers had microalbuminuria , which signify smokers have 4-fold higher prevalence of microalbuminuria than non - smokers . the heart outcome prevention evaluation study documented that smoking was an independent determinant of microalbuminuria in all participants , i.e. , non - diabetic and diabetic patients with a high cardiovascular risk profile . prevend study showed statistically significant difference in urinary albumin excretion in non - smokers and smokers . a recent study found that patients with hypertension and left ventricular hypertrophy smoking > 20 cigarettes / day had a 1.6-fold higher prevalence of microalbuminuria and a 3.7-fold higher prevalence of macroalbuminuria than never - smokers . several studies documented that smoking is an independent predictor of ( micro ) albuminuria in otherwise healthy hypertensive subjects . the prevalence of microalbuminuria is almost double in smoking than non - smoking lean patients with the primary hypertension . one of the underlying mechanisms by which smoking induces albuminuria and abnormalities in renal function is through advanced glycation end products ( ageps ) . ageps are cross - linking moieties formed from the reaction of reducing sugars and the amino groups of plasma proteins , lipids and nucleic acids . it is known that ageps are responsible for enhanced vascular permeability and that they accelerate vasculopathy of end - stage diabetic renal disease . , have shown that both aqueous extracts of tobacco and cigarette smoke contain glycotoxins , highly reactive glycation products that can rapidly induce agep formation on proteins in vitro and in vivo . it is reasonable to expect that the ageps formed by the reaction of glycotoxins from cigarette smoke with serum and tissue proteins will have the same effect on the systemic and renal vasculature as mentioned . another mechanism , based on the patho - physiological effect of smoking induced renal damage , is insulin resistance . several investigators have described smoking to be causally related to insulin resistance in non - diabetic subjects . insulin resistance has been known to be related to both albuminuria and abnormalities in renal function . both mechanisms act through endothelial dysfunction that is by inducing an imbalance between the contracting and relaxing substances produced by the endothelium . the plasma concentration of endothelin 14 has shown to be increased in smokers as compared to non - smokers , also indirect evidence available for a disturbance of endothelin , prostacyclin or nitric oxide release on stimulation in smokers . this study shows non - diabetic normotensive smoker had significantly higher mean urinary acr which is directly related to the amount of smoking ( pack - years ) among smokers . amongst non - smokers , females have low muscle mass compared with males ; therefore , this sex specific difference in acr is due to decreased urinary creatinine excretion in females . warram et al . , also proposed sex - specific acr cut points for microalbuminuria as 17 and 25 g / mg for men and women , respectively . a smoker had serum creatinine and creatinine clearance comparable with non - smoker , similar to prevend study . among smokers , both male and female had comparable urinary albumin , urinary creatinine and urinary acr ( p = ns ) , which signifies that the effect of smoking on renal function is not affected by gender . in this study , subjects having hypercholesterolemia ( total cholesterol > 200 mg / dl ) were excluded due to their known influence on renal function . smoker had significantly lower hdl than non - smokers , similar to several studies , which shows hdl is lower in smokers than in non - smokers . however , limitations of this study are a small number of subjects , single center data and screening with one timed urine sample , it is in comparison with one of the many ongoing prevention of renal and vascular end stage disease ( prevend ) group studies ( prevend ) , running in the city of groningen , the netherlands ( n = 7476 ) with the mean age of smoker ( 47 12 years who smoke < 20 cigarette / day , 46 10 years who smoke > 20 cigarette / day ) and non - smoker ( 47 13 years ) . this area of research needs further attention of physicians and nephrologists , looking to the highly prevalent smoking addiction in indian community as an independent risk factor and its impact on renal function , therefore ; more such studies can be planned at various centers . non - diabetic normotensive smokers have significantly higher level of urinary albumin and urinary acr than non - smoker , which is directly proportional to quantity of smoking . similarly , smokers have a higher prevalence of 4-fold for microalbuminuria and 16-fold for increased urinary acr than non - smokers . smoking significantly reduces the hdl level ; however , no significant effect on serum creatinine and creatinine clearance .

smoking is associated with an excessive morbidity and mortality from a variety of diseases . the aim of this study was to find out the effects of smoking on renal function study in non - diabetic , normotensive subjects . a community - based , prospective , cross - sectional cohort study was conducted on 120 subjects ; 80 ( 66.66% ) were smokers and 40 ( 33.33% ) age matched non - smokers ; with age range of 30 to 70 years . measurement of fasting sugar , urea , creatinine , lipids and one time screening of urinary albumin and urinary creatinine was done . smokers had significantly higher urinary albumin and albumin creatinine ratio ( acr ) ( 52.84 46.42 mg / l , 93.98 78.68 g / mg ) than non - smokers ( 19.25 7.77 mg / l , 18.99 6.65 g / mg ) , respectively ( p = < 0.001 , p = < 0.001 ) . microalbuminuria and urinary acr level were directly related to the amount of smoking ( pack - years ) . among smokers , 73 ( 91.25% ) had microalbuminuria ( > 20 mg / l ) and 64 ( 80% ) had increased urinary acr ( > 30 g / mg ) . smoker had significantly lower high - density lipoprotein level ( 36.66 10.28 mg / dl ) compared to non - smokers ( 41.22 11.72 mg / dl ) ( p = 0.031 ) . urea , creatinine , creatinine clearance , total cholesterol , low density lipoprotein , triglyceride levels were comparable ( p = ns ) . in conclusion , smokers have a 4-fold higher prevalence of microalbuminuria than non - smokers .

Introduction Materials and Methods Results Discussion Conclusion

PMC4525282

. introduced through the active ingredient , excipients , or the atmosphere , it can induce phase transitions , dissolve soluble components , and increase interactions between the drug and excipients , all of which can adversely affect the physical and chemical stability of the drug substance to the detriment of drug product performance . it must therefore be accounted for at all stages of drug substance and product manufacturing . not surprisingly , of the many possible critical quality attributes of a drug substance , hygroscopicity , a measure of the water vapor taken up by a solid with the potential to effect surface and bulk properties , is routinely evaluated , typically by moisture sorption analysis , at the earliest stages of drug product development . the measurement of water vapor sorption isotherms must , however , be tied more directly to the mechanisms by which water is taken up to truly understand the impact that water sorption has on the properties of the solid . on a molecular level , water has the ability to interact with a substance in many ways , including ( i ) physisorption or binding to the surface by hydrogen bonding , ( ii ) physical entrapment to form liquid inclusions , ( iii ) absorption in localized disordered regions , ( iv ) chemical addition , and ( v ) hydrate formation . hydrates often crystallize because water improves crystal packing efficiency and satisfies the hydrogen bonding sites of the drug better than the drug itself . hydrates may be stoichiometric or nonstoichiometric.(3,1013 ) stoichiometric hydrates show step - shaped sorption / desorption isotherms characterized by a fixed water content over a defined relative humidity ( rh ) range and generally convert upon dehydration to a distinct phase , crystalline or amorphous . the waters of crystallization usually play a crucial role in stabilizing the molecular network . nonstoichiometric hydrates , however , have a continuously variable composition within a certain rh range that is not associated with a significant change in the crystal lattice , except for possibly anisotropic expansion of the network to accommodate water . dehydrating a nonstoichiometric hydrate may result in an isomorphic dehydrate ( desolvate ) , a one - component phase that exhibits the main structural features of its parent phase . desolvated solvates are usually metastable and easily take up the original solvent or sometimes other solvents to minimize free volume ( void space ) in the crystal . in some cases , nonstoichiometric hydrates lose crystallinity and become amorphous when the very last water is forced out by extreme drying conditions . whether a crystalline hydrate is targeted as the delivery vehicle for a drug in the formulated product or designed around in favor of a neat form , the dehydration and rehydration processes of a hydrate forming system can be complex and difficult to control . hydrates are generally expected to be thermodynamically more stable , hence less soluble and slower to dissolve than anhydrate forms above the critical water activity for hydrate formation . however , as particle size / shape distribution , specific surface area , and other surface properties heavily affect dissolution , a hydrate may dissolve faster than an anhydrate . additionally , some hydrates are intrinsically more soluble ( less stable ) in water than neat forms . such exceptions to the general solubility trend include ly334370 hcl , n-{[(5s)-3-(4-{6-[(1r,5s)-6-cyano-3-oxabicyclo[3.1.0]hex-6-yl]pyridin-3-yl}phenyl)-2-oxo-1,3-oxazolidin-5-yl]methyl}acetamide , ly156735 ( a melatonin agonist ) , and norfloxacin . norfloxacin is considered unusual because its more soluble hydrates are zwitterionic and at least one of its anhydrates is charge neutral . in this case , the higher solubility of the hydrate can be readily attributed to the stronger hydration of the highly charged zwitterions relative to the uncharged molecule in the anhydrous form . another compound that shows water - induced proton transfer to form zwitterionic hydrates is the 5-ht2a and h1 inverse agonist , 3-(4-dibenzo[b , f]oxepin-11-yl - piperazin-1-yl)-2,2-dimethylpropanoic acid ( ly2624803 or db7 , figure 1 ) . we previously used db7 as a model compound to test the value of combining computational crystal structure prediction ( csp ) methods with an industrial solid form screening program . from the experimental screen for solid forms , which encompassed a broad range of crystallization conditions and totaled over 300 experiments , db7 was identified in nine solid forms , including three neat polymorphs ( forms i iii ) , a stoichiometric dihydrate ( hy2 ) , a nonstoichiometric hydrate ( hya ) , three unstable isostructural solvates ( from methanol , ethanol , and 2-propanol ) , and an amorphous form . for an amphoteric molecule with measured pka values of 2.95 ( oxepine - n ) , 4.03 ( carboxylic acid ) , and 7.81 ( piperidine - n ) , application of the pka rule of three would suggest that proton transfer from the carboxylic acid to the piperidine - n is essentially complete rendering db7 zwitterionic , at least in aqueous solutions at moderate ph . however , solid state n nmr spectra provided an initial indication that only hy2 and hya contain zwitterions ( db7 ) ; the neat forms contain the neutral db7 molecule . molecular diagrams of db7 in the charge neutral anhydrous forms i iii and amorphous form and in the zwitterionic hydrates . the atomic numbering given on the charge neutral form is also used for zwitterionic phases , which are indicated with superscript z . in this study , we explore through a combination of experiment and computation the structural and thermodynamic relationships between the hydrate / anhydrate phases of db7 and their interconversion pathways as a function of temperature , water activity , and ph . a range of experimental techniques , along with computed lattice energies and chemical shift prediction of experimental and hypothetical structures , are used to understand at an atomistic level two distinct ( de)hydration mechanisms of stoichiometric and nonstoichiometric hydrates complicated by both structural disorder and proton transfer . db7 ( form ii , purity 99.6% ) was obtained from lilly research laboratories . the dihydrate ( hy2 ) was prepared by dissolving amorphous db7 ( prepared by rotovapping a dichloromethane solution of the compound ) in stirred water ( ph 5.9 ) at rt . within 15 min a thick white slurry , hy2 , had formed . the solid product was isolated by vacuum filtration and dried in a 75% rh chamber . after stirring in water at rt for 10 days hy2 transformed to the second hydrate ( hya ) . the solid product was isolated by vacuum filtration , washed / transferred with water , air - dried , and equilibrated for several days at 43% rh to further dry the sample . experiments on hy2 and hya were performed with samples that had been stored at 75% rh over a saturated nacl solution . for solubility experiments the hydrate samples were equilibrated at 92% rh ( over a saturated kno3 solution ) , for ssnmr experiments at 75 , 58 , 43 , 33 , 22 , and 11% rh and for h2o / d2o exchange ( raman ) experiments at 98 and 11% rh . the three anhydrates , forms i iii , were prepared according to ref ( 47 ) . single crystal x - ray diffraction data were collected using a bruker d8-based 3-circle geometry diffractometer equipped with a cuk ( hy2 ) or mok ( hya ) radiation source and a smart apex ii 6000 ccd area detector . hy2 and hya1.95 crystals were taken freshly from the mother liquor and hya1.73 after storing the sample at ambient conditions ( 40% rh ) . the structures were solved by direct methods using the program package wingx ( sir2011 and shelxl2013 ) . the aromatic and aliphatic hydrogen atoms were generated by a riding model on idealized geometries with uiso(h ) = 1.2ueq(c ) for aromatic and nonterminal aliphatic hydrogens and uiso(h ) = 1.5ueq(c ) for ch3 . water hydrogen atoms and hy2 n h were located from the difference map and refined with constrained o / n h bond distances and uiso(h ) = 1.5ueq(o)/uiso(h ) = 1.2ueq(n ) . the hya n h proton was identified from the difference map and refined isotropically . proton disorder of the hy2 water molecules is addressed in section 3.2.1 and hya water position occupancies / disorder in section 3.2.2 . the structure models for both hydrates were supported with electronic structure calculations ( sections 1116 of the supporting information ) . xrpd patterns were obtained using an xpert pro diffractometer ( panalytical , almelo , nl ) equipped with a theta / theta coupled goniometer in transmission geometry , programmable xyz stage with well plate holder , cu k1,2 radiation source with a focusing mirror , a 0.5 divergence slit and a 0.02 soller slit collimator on the incident beam side , a 2 mm antiscattering slit and a 0.02 soller slit collimator on the diffracted beam side , and a solid state pixcel detector . the patterns were recorded at a tube voltage of 40 kv and tube current of 40 ma , applying a step size of 2 = 0.013 with 40s/80s per step in the 2 range between 2 and 40. for nonambient rh measurements a vgi stage ( vgi 2000 m , middlesex , u.k . ) was used . the diffraction patterns were indexed using the first 20 peaks with dicvol04 , and the space group , which was determined based on a statistical assessment of systematic absences as implemented in the dash structure solution package , agreed with the single crystal data ignoring temperature effects . hastings pseudo - voigt function was used for peak shape fitting . for the rietveld refinements the db7 molecule was refined with restraints ( distances , angles , and aromatic rings flattened ) and the water molecules as oxygens only . moisture sorption and desorption studies were performed with the automatic multisample gravimetric moisture sorption analyzer sps23 - 10 ( proumid , ulm , germany ) . the measurement cycles were started at 40% with an initial stepwise desorption ( decreasing humidity ) to 0% , followed by a sorption cycle ( increasing humidity ) up to 95% and back to 0% relative humidity ( rh ) . the equilibrium condition for each step was set to a mass constancy of 0.001% over 60 min and a maximum time limit of 48 h for each step . differential scanning calorimetry ( dsc ) was conducted using a ta q1000 dsc , operated with thermal advantage release 5.4.0 software ( ta instruments , usa ) or a diamond dsc equipped with a controlled cooling accessory ( intracooler 1p ) and operated with pyris7.0 software ( perkinelmer , norwalk , ct , usa ) . a few milligrams of accurately weighed ( mettler um3 ultramicrobalance ) sample was heated in perforated or sealed al - pans . heating rates ranging from 1 to 50 c min were applied with a nitrogen purge . the temperature and heat flow of the ta q1000 were calibrated against indium melting . the diamond dsc was calibrated for temperature with pure benzophenone ( mp 48.0 c ) and caffeine ( mp 236.2 c ) , and the energy calibration was performed with indium ( mp 156.6 c , heat of fusion 28.45 j g ) . the errors on the extrapolated transition onset temperatures and enthalpy values are 95% confidence intervals ( ci ) derived from at least three measurements . isothermal calorimetry was used to derive dehydration enthalpies ( see section 10 of the supporting information ) . thermogravimetric analysis ( tga ) was carried out with a tga7 system ( perkinelmer , usa ) using pyris 2.0 software . two - point calibration of the temperature was performed with ferromagnetic materials ( alumel and ni curie - point standards , perkinelmer ) . heating rates ranging from 2 to 20 c min were applied , and dry nitrogen was used as a purge gas ( sample purge , 20 ml min ; balance purge , 40 ml min ) . raman spectra were recorded with a bruker rfs 100 raman - spectrometer ( bruker analytische messtechnik gmbh , germany ) , equipped with a nd : yag laser ( 1064 nm ) as the excitation source and a liquid - nitrogen - cooled , high sensitivity ge - detector . the spectra ( 128 scans per spectrum ) were recorded in aluminum sample holders with a laser power of 300 mw and a resolution of 2 cm . temperature conditions were adjusted with a specac ( grasebury specac limited , orpington , u.k . ) variable temperature cell and a temperature control unit . for investigating h2o / d2o exchange , the number of scans and laser power were increased to 1064 and 400 mw , respectively . samples were stored and measured in hygrostats as detailed in ref ( 66 ) and section 9 of the supporting information . principle component analysis ( pca ) , a multivariate data treatment to reduce the number of variables and provide a representation of the spectra in low dimensional space , was used to interpret changes in the raman spectra during dehydration . max normalization ( opus version 5.5 , bruker optics , ettlingen , germany ) and first derivatives were calculated using simca - p ( version 11.0 , umetrics ab , umea , sweden ) . the spectral region of 1800 to 30 cm was used for constructing the pca models . infrared spectroscopy was used to characterize the ionization state of the crystalline and amorphous db7 forms ( see section 1 of the supporting information ) . cross - polarization / magic angle spinning nmr spectra were obtained on a bruker avance iii 400 wide - bore nmr spectrometer operating at h and c frequencies of 400.131 and 100.623 mhz , respectively , and using bruker 4 mm probes . the mas rate was set to 10 khz 2 hz using a bruker mas - ii controller . h decoupling was achieved using the spinal64 decoupling sequence at a proton nutation frequency of 100 khz . spinning sidebands were suppressed using a five - pulse total sideband suppression ( toss ) sequence . a 3.4 ms linear rf power ramp was used for cross - polarization from h to c. the acquisition time was set to 34 ms , and spectra were acquired over a spectral width of 30 khz with a recycle delay of 7 s. the c chemical shifts were externally referenced ( 0.05 ppm ) to the proton - decoupled c peak of neat ( liquid ) tetramethylsilane via the high - field resonance of adamantane ( = 29.5 ppm ) . the sample temperature was regulated to 25 c in order to minimize frictional heating caused by sample spinning . the crystal16 crystallization system ( avantium , nl ) was used to determine the kinetic solubilities of forms i , ii , hya , and hy2 in an acetonitrile / water ( 1:1 ) mixture . the temperature at the point the suspension becomes a clear solution upon heating or ( at 0.1 c per minute , with the exception of form i where a heating rate of 0.3 c per minute was used to avoid transformation to form ii ) was taken as the saturation temperature of the measured sample with known concentration . to make sure that solvent - mediated transformations had not occurred during the measurements , excess solid was stirred under the same conditions , and xrpd patterns of the residual solid were recorded after reaching the highest clear point temperature derived from the solubility experiments . periodic electronic structure calculations were carried out with the castep plane wave code using the perdew burke ernzerhof ( pbe ) generalized gradient approximation ( gga ) exchange - correlation density functional and ultrasoft pseudopotentials , with the addition of a semiempirical dispersion correction , either the tkatchenko and scheffler ( ts ) model or grimme06 ( g06 ) . for further details energy difference estimates between the zwitterionic and neutral forms are not given because the pbe functional has been shown to overstabilize the proton transfer phases of ammonia monohydrate ; this systematic error should largely cancel when comparing structures with the same ionization state , as should the known limitations in modeling water water interactions . nmr shielding calculations were performed on pbe - ts optimized structural models of db7 hya using the castep nmr code and on the fly pseudopotentials . the castep computed shielding constants , calc , were converted to chemical shifts , calc , according to calc = ref calc using a reference value , ref , taken from the zero intercepts of the fits of the calculated shielding vs. experimental chemical shift plot ( castep = xexp + ref ) for hya ( section 17 of the supporting information ) . our ability to meaningfully study the structures , stability relationships , and interconversion pathways of the db7 hydrates at a molecular level relied on securing highly crystalline , phase pure samples of each form through exquisite control over crystallization , filtration , and drying . since both hy2 and hya are metastable with respect to some or all of the neat polymorphs depending on the rh , they needed to be crystallized under carefully controlled conditions , then isolated and characterized before subsequent conversion to the more stable neat forms . selecting for the metastable hydrates in crystallization required that amorphous db7 be used as the starting material to ensure that the solutions were supersaturated with respect to the hydrates and free of crystalline seeds of the neat polymorphs . even then , the filtration and drying of these materials was surprisingly tricky , with under or over drying leading to form conversions , amorphization , particle agglomeration , and mostly brittle , chunky solids . eventually , conditions were identified to selectively crystallize and recover the hydrates in highly crystalline form , each with reasonably good material handling properties . the hy2 and hya materials used to fund our experimental effort were generally composed of well - formed crystals of similar size and shape commensurate with their high degree of crystallinity , phase purity , and homogeneity ( figure 2 ) . the molecular conformation in this zwitterionic form is closely related to one of the form iii conformers ( figure s2 of the supporting information ) , but with the carboxylic acid proton moved to the piperazine n3 . thus , the protonated piperazine is intramolecularly hydrogen bonded to the carboxylate ion ( n ho ) . the db7 molecules form centrosymmetric dimers ( figure 3a ) , which are arranged in layers parallel to the bc plane . each of the two water molecules ( w1 and w2 ) forms an o ho hydrogen bond to one of the db7 carboxylate oxygens ( figure 4 ) ; the other water protons are disordered over two positions each with a site occupancy of 0.5 . w1 and w2 form infinite corrugated chains , which are interlinked by the carboxylate group of db7 ( figure 4 ) , leading to zigzag sheets ( figure 3b ) . the zigzag sheets , containing all of the strong hydrogen bonds , are interleaved in a zip motif along a. packing diagram of hy2 viewed along the crystallographic b axis ( a ) and c axis ( b ) . hydrogen bonds are denoted as green dotted lines and the centrosymmetric db7 dimer and zigzag sheet are highlighted . the main difference in the molecular conformation of db7 in hya and hy2 is a 52 rotation of the c18c19c20o2 dihedral ( figure 5a ) moving the carboxylate group from an intra- to intermolecular hydrogen bonding conformation . the conformation of db7 in hya is closely related to that in anhydrate form ii ( figure s2 of the supporting information ) . owing to the shape complementarity of the db7 conformers in both hydrates , inversion related dimers similar to those formed in hy2 are also seen in hya ( figures 3a and 5c ) . however , the hya structure also features intermolecularly hydrogen bonded r22(12 ) dimers involving strong n the hya structures of the fresh and aged crystals differ in containing 1.95 ( hya1.95 ) and 1.73 ( hya1.73 ) mol of water per mol of db7 , respectively . the loss of 0.22 mol of water on aging resulted in a volume change of 3.5% , with the c - axis change of 2.5% being the most significant . three water sites ( w13 ) water site w1 is disordered over two positions , which are so close in proximity that only one of the two positions can be occupied at any time ( figure 6a ) . the w1 sites refined to an occupancy of 46% ( w1a ) and 52% ( w1b ) for hya1.95 , and 68% ( w1a ) and 30% ( w1b ) for hya1.73 . the site occupancy from the refinement of w2 was similar to that of w1b , i.e. , 52% for hya1.95 and 30% for hya1.73 . thus , if w2 is present , w1b is occupied , and if not , w1a is likely to be occupied ( figure 6b ) . water site w3 is also disordered over two positions , which are too close to be occupied at any one time . in hya , one of the w3 water protons is located on a special position , a 2-fold axis , so a maximum of 0.5 mol water per mol db7 would be present at full occupancy . refinement of the w3 waters revealed nearly full occupancy , 45% for both hya1.95 and hya1.73 , across the symmetry related sites . as in hy2 , the hya water molecules form strong hydrogen bonds to db7 and one another . w1b can form two ow1ho interactions , involving carboxylate o1 and the w2 oxygen ( figure 6 ) . w2 participates in four hydrogen bonds , as a donor to the carboxylate o2 and the oxepine n and an acceptor to w1b and w3 . together with the db7 carboxylate oxygens , w1b and w2 form an extended ring motif ( figure 6 ) the third water molecule , w3 , is hydrogen bonded to carboxylate o2 and w2 ( when present ) , linking the hydrogen bonded rings along the c axis , the axis most affected in length by the hya water content . the 1d hydrogen bonding network of hya is a marked contrast to the 2d sheets of water and db7 molecules found in hy2 . in hya the 1d hydrogen bonded chains ( figure 5c ) are close packed in the second and third dimensions through centrosymmetric db7 dimers . ( a ) overlay of db7 hy2 ( red ) and hya1.95 ( blue ) conformations ( rmsd1 = 0.38 ) . ( c ) packing diagram of hya1.95 viewed along c. centrosymmetric dimer , water position w2 , and 1d building block are highlighted ; w1 and w3 are obscured by the carboxylate groups in this view . the distinct water sites are coded in different colors : w1a , rose ; w1b , red ; w2 , blue ; w3 , green . note that only one of the two differently colored w3 orientations can be occupied as the two positions are related by a 2-fold axis at a proton position . ( a ) showing the time - averaged water occupancy and ( b ) one of many possible instantaneous snapshots of the water positions in hya in the range of occupancies seen in the two crystals . both hydrates are zwitterionic , but the n3h in each structure is close to one of the carboxylate oxygen atoms . computationally , we can confirm the zwitterionic assignment by a method used to distinguish between salts and cocrystals . hypothetical neutral hydrates are constructed from chy2 ( section 3.4.2 ) and chya2.0 ( section 3.4.3 ) by moving the acidic proton to the nearby carboxylate oxygen and adjusting the bond lengths to give a cis - carboxylic acid for hya and the less common trans - acid conformation for hy2 . full optimization of both charge neutral structures results in the acidic proton moving back to reform the observed zwitterionic hydrate structures , showing that both hydrates are more stable in zwitterionic than neutral form with no appreciable barrier to proton migration ( tables s5 and s7 of the supporting information ) . the converse was also found : full optimization of the corresponding hypothetical zwitterionic versions of forms i iii results in the proton moving to give the observed locally neutral structures . the hydration and dehydration behavior of the db7 crystal forms was investigated between 0% and 95% rh at 25 c ( figure 7 ) . two of the anhydrates , forms i ( figure 7a ) and ii ( figure 7b ) , show almost no water uptake ( form i < 0.32% and form ii < 0.04% ) up to 95% rh . in contrast , anhydrate form iii undergoes a phase transformation with a mass increase corresponding to 2 mol of water per mol of db7 at rh values > 70% ( figure 7c ) . the product hydrate phase , confirmed to be hy2 by xrpd , shows good rh stability with a marginal weight loss between 95 and 25% rh . hysteresis was observed in the form iii / hy2 isotherm despite the lengthy equilibration times ( up to 48 h ) at each rh . the profile of the moisture sorption / desorption isotherms for form iii hy2 with steps accompanied by phase changes , is typical of a stoichiometric hydrate . in contrast , highly crystalline hya shows the typical ( de)sorption isotherms of a nonstoichiometric hydrate ( figure 7d , see also figure s3 of the supporting information ) as the mass gradually changes depending on the humidity during sorption and desorption . the lowest water content measured by tga ( figure 7d ) for hya equilibrated at 0% rh corresponded to 1.29 mol of water per mol of db7 . the maximum water vapor uptake into the hya structure was estimated to be approximately 1.85 mol of water per mol of db7 at 95% rh . gravimetric moisture sorption and desorption curves of db7 solid forms at 25 c : ( a ) form i , ( b ) form ii , ( c ) form iii / hy2 , and ( d ) hya . the gray circles represent data points that fulfill the preset equilibrium conditions ( see experimental section ) , whereas the crosses mark measurement values that did not reach equilibrium within the allowed time limit ( 48 h ) . the automated gravimetric moisture sorption / desorption analysis of hy2 and hya was complemented with long - term drying experiments at 0% rh ( storage over p2o5 ) . dehydration of hy2 was observed immediately at this condition , and form iii was determined to be a kinetic dehydration product , with further transformation to the more stable forms i and ii occurring within 2 weeks . hya could be similarly dehydrated at 0% rh ; however , loss of the last strongly bound 1.3 waters of crystallization from this hydrate leads to a structural collapse to mainly amorphous db7 . the amorphous form has limited stability at 0% rh as there was evidence of forms i and ii nucleating in small amounts during the low rh drying . the gravimetric moisture sorption / desorption studies ( figure 7 ) were correlated with structural changes to hy2 and hya using variable - humidity xrpd at 30 c ( figures 8 and 9 ) . in agreement with the results obtained in figure 7c , distinct changes occur in the xrpd pattern of the hy2 sample at rh values < 30% ( figure 7a ) . the disappearance of high intensity hy2 peaks ( e.g. , at 6.82 , 10.62 , 16.37 , 16.71 , and 17.57 2 ) and appearance of form iii peaks ( e.g. , 7.33 , 12.68 , and 14.81 2 ) indicate a major change in the crystal structure ( figure 8c f ) on loss of the waters of crystallization . the unusual increase in volume / z upon dehydration ( figure 8b ) can be attributed to the less efficient packing of db7 with itself and ( a ) moisture - dependent xrpd measurements showing the dehydration of hy2 to form iii . peak positions marked with * correspond to hy2 impurities ( dehydration not complete due to the shorter time intervals of 12 h and 10% rh steps in contrast to 48 h and 5% steps in figure 7c ) . ( b ) moisture - dependent changes in the cell volume per asymmetric unit for the transformation hy2 to form iii . ( c , d ) molecular packing of db7 in hy2 and ( e , f ) db7 in form iii . disorder of the methyl group in form iii is indicated with different shades of gray . in contrast to hy2 , there is no significant change in the crystal lattice of hya with varying rh ( figure 9b ) apart from anisotropic expansion of the network to accommodate a variable amount of water . the anisotropy of the lattice expansion was quantified by indexation and rietveld refinement of the hya xrpd patterns recorded at different rh values ( figure 9a ) . using the hya1.95 structure as a starting model , rietveld refinement found the c axis to be affected the most by changes in the water content , in agreement with the scxrd experiments , but over a wider range of compositions ( table s2 of the supporting information ) . structural refinement also revealed that the water occupancy of w3 stays close to 50% ( i.e. , full ) independent of the rh , suggesting that the w3 position is integral to the hya structure . however , the occupancy parameters for w1 and w2 collectively decreased at progressively lower rh values . the w1w2 distance was also found to depend on the hydration state . at moderate rhs ( 2080% ) a plateau is reached with the modeled w1w2 distance being too short for two distinct sites , suggesting either water mobility or static disorder ( figure 9c ) . a further distinct change in the w1w2 distance is seen below 25% rh , the moisture range where in addition to continued diffraction peak shifting , selected reflections , in particular ( 002 ) , are broadened . the lowest hya water content observed in the rh - dependent xrpd experiments of hya was 1.32 mol of water per mol of db7 ( measured with tga ) . ( b , c ) changes in the hya cell volume , length of the c - axis , fractional occupancies of w1 + w2 and w3 oxygens , and distance between w1 and w2 oxygens on lowering the rh ( desorption ) . water position w1 was modeled as one site only . 100% rh data points given in ( b ) and ( c ) were derived from slurry experiments in water . ( d f ) packing diagrams of hya highlighting the water oxygen positions at different hya hydration states derived from single crystal x - ray data ( d , e ) and rietveld refinement of powder x - ray data ( f ) . red lines between water oxygens indicate the disordered sites for w3 and close proximity of the w1 and w2 positions in ( f ) . w1a and the moisture - dependent xrpd experiments were extended to water activity = 1 ( 100% rh ) by measuring a thick hya slurry in water . the powder pattern of the 100% rh hya sample refined to 1.96 mol equiv of water per db7 , comparable to hya1.95 observed ( by scxrd ) for a freshly crystallized single crystal . while a change in water content and unit cell volume was to be expected on increasing the rh from 90 to 100% , the sharp uptake of 0.10 mol water per mol db7 and unit cell volume increase of 0.5% ( figure 9b ) was unexpected and presumably reflects the slow hydration kinetics of hya in the solid state ( table s2 of the supporting information ) . an ordered hy2 structural model was derived from the experimental crystal structure by replacing the two disordered water molecules related by a 21 screw axis with one molecule in each orientation ( section 12 of the supporting information ) . this lower symmetry hy2 structure ( figure s10b ) was lattice energy minimized to give the computational model of the hy2 structure , denoted chy2 . the geometry optimizations of four hypothetical hydrate and framework structures derived from chy2 ( table s5 of the supporting information ) showed that proton transfer to a neutral db7 molecule occurred when there was no water hydrogen bonding to the carboxylate oxygen involved in the n although the layer structure ( figure 2 ) suggests that it might be possible to remove both w1 and w2 , doing this computationally results in proton transfer and an unstable , low density isomorphic desolvate with no strong hydrogen bonds between db7 molecules . partial removal of the w1 molecule gave hypothetical zwitterionic sesquihydrate ( chy21.5(0.5w1+w2 ) ) and monohydrate ( chy21.0(w2 ) ) structures , which differed in lattice energy from hy2 by 55 and 107 kj mol , respectively ( table s5 of the supporting information , averaged over the two dispersion models ) . estimating whether hy2 could undergo a phase transition to an isostructural sesqui- or monohydrate requires comparison of the lattice energies of the model hydrates with that of ice ( section 16 of the supporting information ) . this gives an unfavorable phase transition energy of more than 30 kj mol for each water removed . thus , the computational thermodynamic estimates show that a stepwise dehydration of the hy2 structure , preserving the hy2 db7 framework structure ( i.e. , a structurally related sesquihydrate or monohydrate as a transient , intermediate hydrate along the dehydration pathway ) , is highly unlikely . this is consistent with hy2 showing a single stepwise weight loss by moisture sorption analysis ( figure 7c ) and tga ( section 3.5.1 ) to water free db7 . the two hya structures show a similar nearly full occupancy of w1a + w1b ( 98% ) and w3 ( 45% ) , but differ in the w2 partial occupancy ( 52% or 30% ) . to form an ordered computational model of hya1.95 , the crystallographic 1.95 water molecules per db7 molecule had to be increased to 2 by modeling the 98% ( 46% w1a + 52% w1b ) , 52% and 45% occupancy ratios for w1 , w2 , and w3 as 100% , 50% , and 50% , respectively . all symmetry was removed from the c2/c , z = 1 structure of hya , resulting in a p1 , z = 4 cell in which the four w1 sites are independently occupied and half of the w2 and w3 sites are occupied ( section 13 of the supporting information ) . all possibilities leading to the described occupancy ratios were used as starting structures and the lowest energy structure after geometry optimization was used as chya2.0 hereafter . similarly , for the hya1.73 structure the occupancies were modeled as 100% , 25% , and 50% for w1 , w2 , and w3 giving a model chya1.75(2 ) ( table s7 of the supporting information ) , which agreed with the experimental structure . the confirmation that the ordered cell models chya2.0 and chya1.75 were reasonable representations of the disordered structures is given in section 15 of the supporting information . a set of hypothetical hya framework structures can be derived from the experimental structure , ranging from a hemipentahydrate ( chya2.5 , 2.5 mol of water per mol db7 at full w1 and w2 occupancy ) to the water free framework ( table s7 of the supporting information ) . the lattice energy of chya2.5 was higher ( less stable ) by about 2 kj mol than the sum of the lattice energies of chya2.0 and 0.5 mol of ice xi , implying that additional water to fully occupy the w2 site would destabilize the hya structure . computational dehydration of chya2.0 to chya1.75 , chya1.5 , and chya1.25 produces a range of possible structures , which are very close in energy ( table s7 of the supporting information ) . removal of water from hya was calculated to be energetically quite feasible and to require considerably less energy than removing comparable amounts of water from hy2 ( tables s5 and s7 ) . the calculated isomorphic hya desolvate ( chya0.0 ) also undergoes a proton transfer to the neutral molecule on optimization ( table s7 of the supporting information ) . in fact , proton transfer from db7 to db7 was observed when the carboxylate of the n hor22(12 ) dimer ( figure 5b ) was not also acting as an acceptor for an ow ho hydrogen bond . thus , the hydrogen bonded water molecules ( w1 , w2 , or w3 ) appear to be essential for zwitterion formation . since chya0.0 is estimated to be over 25 kj mol less stable than form ii , an isomorphic desolvate of hya containing zwitterionic or neutral db7 molecules is unlikely to form . this is consistent with a destructive dehydration mechanism on loss of all waters of crystallization . the suggestion of water mobility / disorder from the diffraction experiments and static lattice energy modeling led to the use of c cp / mas nmr spectroscopy to characterize hya as a function of water content . except for slight peak intensity variations , the ssnmr spectra of hya samples equilibrated between 22 and 90% rh were essentially identical ( figure 10a ; figure s5 of the supporting information ) . since partial occupancy of the hya water sites over this rh range would necessarily result in a distribution of hydrogen bonding arrangements ( figure 6 ) , the observation of a single c resonance for each carbon atom in db7 shows that the different hydrogen bonding environments are effectively averaged as the waters of crystallization move in and out of the water sites in the hya crystal structure . thus , ssnmr shows that the water of crystallization is highly mobile over a wide range of moderate and high rh . after equilibrating the sample at rh 11% ( figure 10b ) , peak splitting for carbon atoms c20 , c5 , and c17 and broadening of several c nmr resonances were seen across the ssnmr spectrum of hya . the additional peaks were not due to forms i iii or hy2 ( ref ( 47 ) and figure s6 of the supporting information ) phase impurities , as there is no match to their chemical shifts . instead , the emergence of low intensity peaks was ascribed to restricted water mobility in the contracted hya cell , which would also explain the diffraction peak broadening seen in figure 9a . support for this interpretation of the low rh ssnmr spectrum ( figure 10 ) comes from dft shielding calculations on 25 variably hydrated chya structures ( section 17 of the supporting information ) , which generated 80 c nmr spectra of db7 in different hya water environments . these showed that the chemical shifts of c20 , c5 , c18 , and c17 , because of their proximity to the water molecules , were particularly sensitive to hydration state and water positions , varying by 35 ppm ( table s11 of the supporting information ) . since the regions in the ssnmr spectrum shown computationally to be most sensitive to water in the hya structure coincide with those where peak splitting is observed at low rh ( figure 10 ) , we can account for the peak splitting as a manifestation of reduced water mobility on the ssnmr time scale . ( a , b ) representative experimental hya spectra differing in water content and ( c h ) six of 80 nmr - castep computed c cp / mas nmr spectra . ( c , d ) the two db7 environments observed in the ordered p1 chya2.0 model and ( e h ) the four environments observed in the ordered p1 chya1.25(13 ) model , which correspond to 0.75 w1 , 0.25 w2 , and 0.25 w3 . in contrast to hya , the ssnmr spectra of hy2 ( figure s6 of the supporting information ) show a distinct phase change to form iii in this case between 11 and 22% rh , in good agreement with the moisture sorption ( figure 7c ) and xrpd data ( figure 8a , b ) . hy2 and hya were exposed to deuterium oxide vapor ( 98% and 11% rh ) and characterized by raman spectroscopy at different time points ( figure 11 ) to investigate water dynamics in the two hydrates . the wavenumber region 34002800 cm is composed of peaks arising from (o h ) , (n h ) , and (c h ) stretching vibrations ; (o d ) stretching modes of d2o are seen between 2600 and 2300 cm . the emergence of o d peaks in the spectra of both hydrates on exposure to deuterium oxide vapor confirms that d2o displaces h2o in both crystal structures . thus , not only is the water of crystallization in hya mobile , but water exchange is also observed for the stoichiometric hydrate , hy2 . water diffusion in and out of the two crystal structures is rapid and quantitative , as derived from the fact that the raman spectra after exposure to d2o for 72 h were indistinguishable from fully deuterated hy2 and hya . db7 also has an ammonium proton that could exchange a hydrogen for a deuterium . to see whether the ammonium group had undergone exchange , the hydrate samples that had been exposed to d2o vapor were dehydrated and compared to the raman spectra of the neat forms . the comparison showed that within 72 h of d2o vapor exposure the ammonium group was not affected at all . raman spectra of db7 hy2 ( a ) and hya ( b , c ) as a function of time of exposure to d2o vapor , 98% rh ( a , b ) and 11% rh ( c ) . d stretching vibrations emerge over the course of 72 h. the tga curve of hy2 ( figure 12a ) shows a one - step mass loss of 8.67 0.05% , corresponding to exactly two mole equivalents of water . dehydration starts immediately under the dry conditions of a nitrogen purge . on removing the water of crystallization , the clear hy2 crystals turn opaque , but maintain their shape ( pseudomorphosis ) . slow heating dsc experiments of hy2 ( figure 12a ) using pinhole lids show four events : ( 1 ) dehydration of hy2 to form iii takes place below 45 c . ( 2 ) at 95 c there is an exothermic phase transformation of form iii mostly to form ii , with minor amounts of form i present . the measured enthalpy of transition is in agreement with the value derived using pure form iii as a starting material ( trshiii ii = 13.3 0.2 kj mol ) . ( 3 ) upon further heating , melting of the minor form i component is observed at an onset temperature of 176.0 0.4 c ( small peak ) . ( 4 ) at an onset temperature of 180.9 0.2 c , the form ii melts . by increasing the heating rate used in the hy2 dsc experiments to 5 c min or more ( figure 12a : 5 , 10 , and 50 c min ) , an additional thermal event is observed above 118 c corresponding to the inhomogeneous melting of form iii . melting is followed by the recrystallization of mainly form ii , then the melting of forms i and ii. by embedding the hy2 crystals in high viscosity silicone oil or using hermetically sealed dsc pans ( figure 12a ) , the incongruent melting of hy2 ( peritectic decomposition ) is observed at 89.8 1.3 c . compared to hy2 , higher temperatures are required to fully dehydrate hya . by tga , a hya sample pre - equilibrated at 75% rh ( stored over a saturated nacl solution ) showed a mass loss of 7.61 0.04% , corresponding to 1.74 mol equivalents of water ( figure 12b ) . toward the end of the dehydration process a loss of birefringence is observed in polarized light , indicating that the strongly bound water is released by a process that leads to a loss of crystallinity . the generation of mainly amorphous db7 with form i and ii impurities was independently confirmed by xrpd and raman spectroscopy . the loss of crystallinity upon dehydrating hya was also supported by dsc , based on the appearance of an exothermic event immediately following the dehydration endotherm . upon further heating , the few crystal nuclei produced at lower temperatures ( during dehydration ) grew into a mixture of forms i and ii , evidenced by the final two melting events . the lower the dehydration temperature , the less form ii was observed . dsc traces of hya collected at faster heating rates ( 10 c min ) show an overlap of dehydration and peritectic decomposition at 99 c . the peritectic decomposition of hya , measured in hermetically sealed dsc pans , is observed at 98.9 0.9 c . differential scanning calorimetry ( dsc ) and thermogravimetric analysis ( tga ) thermograms of ( a ) hy2 and ( b ) hya . tga curves were recorded in open pans at a heating rate of 5 c min . raman spectra were recorded for hy2 and hya every 2 to 5 c from 25 to 130 c , then subjected to pca to track the solid state transformations ( figure 13b , c ) by comparison to reference spectra of the db7 forms ( figure 13a ) . the raman spectra recorded throughout the hy2 dehydration appear as three distinct clusters , corresponding to hy2 , form iii , and form ii with form i impurities ; in between the clusters ( 50 , 100 , and 102 c , figure 13b ) are mixed phases . these results show that form iii is a distinct intermediate phase along the thermally induced dehydration pathway of hy2 . ( the close proximity of the dehydration product to phase pure form ii relative to phase pure form i in figure 13b shows that form ii is the main component , and the tightness of the final product cluster shows that the form i impurities did not transform to form ii . ) this dehydration pathway is in agreement with the dsc and tga results in figure 12a . the dehydration of hy2 does not go via an appreciably stable amorphous phase or phase pure form i because the triangles representing form i and amorphous db7 are not located on the dehydration pathway . the structural changes accompanying the dehydration of hya to a form i and ii mixture could be described with three pcs , but a continuous transformation pathway is evident from two of them , figure 13c . as the small structural variation within hya exhibiting variable water content between 25 and 62 c is apparent by pc2 , pc1 shows the transformation from the hya crystal lattice ( 70 c ) through the amorphous state ( 82 c ) to the form i / ii mixture ( 86130 c ) ( figure 13c ) . ( b , c ) raman principal component analysis ( pca ) plots for the first and second principal component ( pc1 and pc2 ) of db7 forms occurring during dehydration of hy2 ( b ) and hya ( c ) . each triangle corresponds to a raman spectrum : black , recorded during dehydration and used for constructing the pca models ; gray , reference spectra for forms i and ii and amorphous db7 . numbers correspond to the temperature at which each spectrum was recorded ; black ellipse corresponds to the 95% hotelling t2 , and areas of distinct db7 solid forms are encircled . the % -values on the axes indicate the data variance modeled by the respective pc . form iii was obtained for the first time following the salt disproportionation of db7 phosphate at ph 6 . only after we were unable to directly crystallize form iii from solution and systematically studied the dehydration mechanism of hy2 was it evident that hy2 was the kinetic phosphate salt disproportionation product and necessary intermediate phase for obtaining form iii . structural analysis has shown that the molecular conformation of db7 in hy2 with intramolecular hydrogen bonds seems to predispose it to dehydration to form iii , the only anhydrate , which also features intramolecular hydrogen bonding . to the best of our knowledge , following our observation of batch - to - batch variability in the disorder ratio of the dimethylpropionic acid side chain in form iii , we performed systematic drying studies of hy2 to estimate the effects of rh and temperature on the level of disorder present in the form iii dehydration product and to seek conditions that would select for the ordered structures . the disorder ratio was quantified from the intensities of the dimethylpropionic acid methyl c ssnmr peaks ( figure 14a , table s3 of the supporting information ) . unfortunately , despite the wide range of drying conditions surveyed , we were not successful in preparing either an ordered form iii sample or one having the minor conformation dominating . ( a ) c cp / mas nmr spectra of db7 form iii produced by dehydrating hy2 under a variety of conditions ( defined in the supporting information , table s3 ) . highlighted are the c18 and c21 peaks characterizing the structural disorder of the dimethyl propionic acid side chain . ( b ) dsc thermograms of db7 form iii lots n2/0/rt/24 and n2/0/5/48 measured at a heating rate of 1 c min . lot n2/0/rt/24 was prepared by drying hy2 under n2 purge at room temperature for 24 h and lot n2/0/5/48 under n2 purge at 5 c for 48 h. subsequent dsc investigations ( figure 14b ) were conducted to examine whether the disorder ratio in form iii influences its solid form transformation to forms ii and/or i. although the outcomes were somewhat variable , form iii samples obtained by drying hy2 under a variety of conditions ( table s3 of the supporting information ) ranged from having only small amounts of form i to as much as 90% form i after the solid state transformation . importantly , the heat induced phase transformation does not seem to be related to the disorder in form iii . form iii samples n2/0/5/48 and n2/0/rt/24 , for example , show a similar level of disorder in their c ssnmr spectra , but differ substantially in their thermal behavior ( figure 14b ) . thus , while the dehydration reaction of hy2 always produces the two different conformations in form iii , small differences in the phase purity and sample history also influence the outcome of the solid form transformation to forms i and/or ii. the db7 hydrates and db7 anhydrates showed appreciable kinetic stability in the solid state based on the gravimetric moisture sorption / desorption analysis . however , these forms were not sufficiently stable in suspensions to obtain equilibrium solubility data of interest for quantitatively establishing their thermodynamic stability relationships . the stability order of the two stable anhydrate polymorphs forms i and ii and the two hydrate forms was therefore qualitatively estimated as a function of temperature from kinetic solubility measurements in acetonitrile / water ( 1:1 ) . this solvent mixture was chosen because the db7 and db7 solid forms were sufficiently soluble to meaningfully estimate their relative solubility using the crystal16 parallel reactor system . unfortunately , the fast transformation kinetics of form iii to form ii in stirred solvent prevented any measurement of the kinetic solubility of form iii . the acetonitrile / water solubility data for forms i and ii ( figure 15a ) agree with the monotropic relationship previously derived from solubility measurements in methyl isobutyl ketone and dsc experiments , as the kinetic solubility data plotted in vant hoff form showed no signs of convergence or crossover below the db7 melting temperature ( figure 15b ) . hya is less soluble than hy2 in acetonitrile / water ( water activity , aw , of 1:1 mixture is 0.9 at 25 c , figure 15a ) , with the vant hoff plots further suggesting that hya is the thermodynamically most stable db7 hydrate at this water activity below the hydrates melting points . the zwitterionic hydrates are much more soluble than the charge neutral neat forms at all measured temperatures , but their relative solubility is clearly decreasing with decreasing temperature ( figure 15b ) . ( a ) solubility ( mole fraction ) of db7 forms i , ii , hy2 , and hya in acetonitrile / water ( 1:1 , aw 0.9 ) as a function of temperature and ( b ) vant hoff plot of the molar solubility as a function of temperature . the critical water activity defining the crossover in thermodynamic stability of the db7 hydrate db7 anhydrate pairs could not be determined from the solubility measurements at a single water activity ; however , these results did reveal that both forms i and ii are more stable than hy2 and hya at water activities up to at least 0.9 over the investigated temperature range . above aw = 0.9 , the rt thermodynamic stability order of the db7/db7 forms was investigated through slurry experiments in methanol / water mixtures ( section 8 of the supporting information ) . db7 hy2 and hya were separately added to methanol / water mixtures ( aw = 0.9 , 0.95 , and 1.0 ) and stirred for 21 days . slurries in mixtures of aw = 0.9 and 0.95 resulted in form ii , while the pure water slurry yielded hya . in showing that form ii is more stable at aw = 0.95 and hya is more stable in pure water ( aw = 1.0 , ph 5.9 ) , the critical water activity at which the solubility order of form ii/hya is reversed was more precisely bracketed between 0.95 and 1.0 . the hy2 to hya transformation in water did not allow us to estimate the hy2 aw dependency . therefore , we used gravimetric sorption / desorption experiments to estimate the influence of moisture . the desorption curve is similar to that of phase pure hy2 ( figure 7c ) . the water is released at rh < 25% , and the product contains forms i and ii from the spiking and form iii as the dehydration product . upon subsequently increasing the rh of the mixed anhydrate sample , hydration of form iii to hy2 is observed as well as a transformation to form ii ( figure 16 , 2 ) . the latter , which is indicated by the decrease in mass despite increasing the humidity at rh values > 85% , is consistent with the greater stability ( lower solubility ) of form ii than hy2 observed in 1:1 acetonitrile / water ( aw 0.9 ) , figure 15 . gravimetric moisture sorption and desorption curve of db7 hy2 spiked with forms i and ii at 25 c . the gray circles are data points that fulfill the preset equilibrium conditions ( see experimental section ) , whereas the crosses mark measurement values that did not reach the equilibrium within the allowed time limit ( 48 h ) . the forms noted in parentheses were identified as minor components by xrpd . to investigate the influence of ph on the db7/db7 form stability , slurry experiments were conducted in aqueous buffer solutions at 25 c . solvent mediated transformations ( smt ) starting from hy2/form iii showed that hya is obtained if ph 6 and anhydrous form ii if ph 5 . in contrast , the phosphate salt of db7 was shown to disproportionate in aqueous media , causing either hy2 ( ph 67.8 ) or form ii ( ph 2.85 ) to nucleate . the fact that hy2 converted to hya in the higher ph smt experiments confirms that hy2 was a kinetic disproportionation product at 25 c . thus , hya is the thermodynamically most stable db7 form in water , but this stability relationship has been established only at ph 5.9 ( from water slurry experiments ) . db7 has a rich solid form landscape composed of three neat polymorphs , two hydrates , three solvates , and an amorphous phase . with the goal of exploring the thermodynamic stability relationships between the hydrates and anhydrates , as well as their interconversion pathways , we have established through a combination of diverse experimental techniques and computational chemistry that db7 form appearance and stability depend on temperature , water activity , and ph . form ii is monotropically more stable than forms i , iii , and hy2 and usually more stable than hya . hya is the most stable db7 crystal form in saturated water solutions , but only at ph 5.9 . characterizing the structure stability relationships of the db7 crystal forms was complicated not only by the difficulty in isolating highly crystalline , phase pure samples of the metastable forms ( section 3.1 ) , but also the slow rate at which hya and hy2/form iii materials reached their equilibrium moisture content . equilibrium water contents were attainable for hya using extended equilibration times at most rhs , although complete rehydration to full crystallographic water occupancy was never achieved even in aqueous slurries . in contrast , the reversible dehydration of hy2 to form iii mediated by water vapor was readily apparent from a quick measurement of the moisture sorption desorption isotherms of either form , yet extended equilibration times did little to minimize the hysteresis ( figure 7c ) caused by slow transformation kinetics . the slow interconversion of hy2 and form iii in the solid state at moderate rhs frustrated attempts to establish the critical water activity relating these metastable forms . two distinctly different moisture sorption behaviors were established for hy2 and hya through detailed characterization of highly crystalline , phase pure samples as a function of water vapor pressure . curiously , our initial attempts to rationalize the different hygroscopicity of the db7 hydrates in terms of structural features ( solvent pockets , channels ) common to stoichiometric and nonstoichiometric hydrates yielded anything but expected results . figure 17 shows the void space ( default probe radius = 1.2 ) created by removing the waters of crystallization from the hya1.95 , hya1.73 , hya1.32 , and hy2 structures . counterintuitively , the water accessible voids in nonstoichiometric hya appear to be isolated from one another ( figure 17a c ) , while water channels are clearly seen running zigzag along the crystallographic b - axis in the stoichiometric hydrate , hy2 ( figure 17d ) . solvent accessible voids containing waters of crystallization in ( a ) hya1.95 , ( b ) hya1.73 , ( c ) hya1.32 , and ( d ) hy2 , calculated using a 1.2 probe radius . cursory inspection of the water channels in hy2 and the proximity of solvent pockets in hya suggests that the direction of water ingress / egress is likely to be along the b - axis in each structure . however , the 100 k structures individually do not reveal how either hydrate framework would respond to perturbations in rh , particularly at crystallization processing and storage relevant temperatures . water vapor sorption isotherms ( figure 7 ) , however , show that the db7/water stoichiometry is retained across a wide rh range for hy2 and that a fraction of the water in hya is adventitious . yet they reveal little as to how water is held in each crystal structure or the phase transformations that accompany dehydration and rehydration . indeed , only through a combination of experiment and computation were we able to paint a molecular level picture of two very different rh stability profiles and ( de)hydration mechanisms for hy2 and hya . nonstoichiometric hya retains between 1.29 and 1.95 waters of crystallization , all of which reside in the same solvent pockets in the crystal structure ( figure 17a c ) and are exchangeable ( figure 11b , c ) . the conspicuous absence of continuous water channels in hya suggests that for nonstoichiometric hydration to occur , cooperative movement of the carboxylate and dibenzoxepine groups ( figures 9d f and 17b c ) must temporarily open up diffusion pathways , similar to that seen in -cyclodextrin ( -cd ) hydrate and ciprofloxacin . water diffusion in and out of discrete crystallographic sites gives rise to varying disorder and occupancy in the diffraction time - averaged hya crystal structures , but is sufficiently rapid , at least under moderate to high rh conditions , that single , averaged db7c resonances are seen in the ssnmr spectra ( figure 10a ) . below 11% rh , water mobility is observably decreased with the resonances of c nuclei that are in close proximity to the partially occupied water sites showing splitting ( figure 10b ) . the decrease in water mobility witnessed on the ssnmr time scale at low rh is presumably caused by anisotropic contraction of the hya lattice as water is continuously lost from the crystal . in fact , rietveld refinement of the hya xrpd patterns collected across a wide rh range confirmed that the hya lattice essentially breathes to accommodate varying amounts of water with smooth changes in the cell volume and shifting of the water sites / occupancies ( figure 9 ) . void space analysis of the hya framework structures derived from hya1.95 , hya1.73 , and hya1.32 showed that as the unit cell contracts at lower hydration states ( figure 17a c ) , the solvent accessible voids decrease from 10.2% in hya1.95 to 4.2% in hya1.32 . isostructural dehydration modeling , in showing the small energy penalty that is incurred to attain full water site occupancy ( hemipentahydrate stoichiometry ) and the appreciably larger one for decreasing the water content below 1.25 mol equiv in the hya crystal structure , helped to rationalize the db7/water stoichiometries that were experimentally accessible . in the end , elevated temperatures or long - time storage over p2o5 ( 0% rh ) was required to overcome the activation energy for removing the final 1.3 equiv of water from the contracted hya structure . however , with no clear path to an appreciably stable neat polymorph , dehydration led to structural collapse as first observed by dsc ( figure 12b ) , then later confirmed by raman spectroscopy ( figure 13c ) . in contrast to hya , hy2 retains its full complement of water over a large rh range at 25 c ( figure 7c ) . stoichiometric hydration may not have been anticipated based on the location of water molecules in open channels in the hy2 crystal structure , but it was suggested by computational dehydration modeling ( section 3.4.2 ) . in revealing a significant energy penalty incurred by removing water molecules from this hydrate , lattice energy calculations for hy2 and hypothetical lower hydrate and framework structures derived from hy2 suggested that even partial dehydration to a lower isostructural hydrate would be highly unlikely . the inability to dehydrate hy2 ( without inducing a phase change ) can not be taken to mean that water exchange in this channel hydrate is not possible , however . in fact , water mobility was found to be similar in hy2 and hya by raman using h2o / d2o exchange ( figure 11a , for details see section 9 of the supporting information ) . that water is able to rapidly enter and leave the open channels in hy2 naturally raises the question : why is the stoichiometry of this hydrate conserved over such a wide rh range ? the answer lies in how the hy2 crystal structure responds to the loss of water . in this case , the hy2 framework , apparently incapable of making the type of adjustment that hya makes to accommodate appreciably lower amounts of water , experiences a significant increase in free volume on dehydration . the decreased packing efficiency , along with the loss of strong intermolecular waterdb7 hydrogen bonds , is considerably destabilizing . as a result , hy2 retains its full complement of water under most rh conditions at ambient temperature . with even modest increases in temperature ( figure 13 ) or decreases in rh ( figure 7c ) , however , hy2 gives way exclusively to a neat form ( form iii ) . the path to form iii , a neat polymorph that is more efficiently packed than the isomorphic dehydrate , is conceivably paved by the db7 conformations that allow intramolecular hydrogen bonding seen in the parent hy2 structure to be conserved ( after proton transfer ) . some of the propionic acid side chains in db7 undergo a significant conformational change on loss of water from hy2 , leading to a disordered form iii structure . this change probably accounts for the large rh range of coexistence , i.e. , hysteresis in the moisture sorption efficient and reversible nonstoichiometric hydration may be rationalized , and in favorable cases ( e.g. , cromolyn sodium , amg 222 tosylate , and pfizer s glycogen phosphorylase inhibitor ) , anticipated for open channel structures ; however , the hydrates of db7 highlight the dangers of inferring stoichiometric vs nonstoichiometric hydration from general structural features . the moisture sorption properties of hya may be unconventional , but they are not unprecedented . other carefully studied systems , such as topotecan hcl , paroxetine hcl form ii , gsk s apoa-1 up - regulator , ciprofloxacin , thiamine hcl , and -cd , show a similar ability to equilibrate with the humidity of their surroundings despite lacking continuous channels . however , hy2 is unusual in its ability to retain stoichiometric amounts of water in open water channels over such a large rh range . clearly , with neither hydrate behaving according to the conventional classifications based on either structure or absorption isotherms , jumping to conclusions about the hydration behavior from a few hasty experiments would have been very misleading . having a molecular level understanding of how water vapor is sorbed in hygroscopic materials and the risks that water uptake poses to the physical and chemical stability of a pharmaceutical product is invaluable , as some sources of water uptake are more manageable than others , careful evaluation of the stoichiometry , stability relationships , and transformation pathways of pharmaceutical hydrates should inform the solid form screening and selection process . nonstoichiometric hydration , for example , is an intrinsic and generally undesirable property of a solid form that , once established , might prompt further solid form screening and ultimately would have to be managed in the event that an alternate , nonhygroscopic form is not found . however , water uptake in the amorphous components of poorly crystalline materials is oftentimes correctable with improvements in crystallinity that are generally realized during crystallization process optimization . hygroscopicity is , of course , but one of many considerations taken in the selection of a crystal form for a commercial drug product . perhaps more important to delivering a drug safely and efficaciously are the solubility and stability , both thermodynamic and kinetic , of the solid - state form . as metastable forms of the 5-ht2a and h1 inverse agonist , hy2 and hya offer the advantage of generally higher solubility , which might be desirable for ensuring rapid oral delivery of an insomnia medication . however , from a manufacturability and control perspective , the added risk of form conversions established in our assessment of the structure stability relationships would have to be carefully weighed in the selection of either of these high energy forms for a commercial drug product . in this case , the difficulties we encountered with producing the db7 hydrates at small scale and avoiding conversions to the more stable neat forms prompted further evaluation of salt and cocrystal options to determine whether these were better suited for enabling a drug product . the computational modeling of static structures derived from the time and spatially averaged crystal structures supports the interpretation of the experimental results and accounts for the variability in disorder and water mobility shown by ssnmr and raman h2o / d2o exchange . however , current state - of - the - art calculations still struggle to obtain sufficient accuracy for the small energy differences involved and can not include the important effects of water activity or temperature , let alone proton - transfer energies . ideally , we would wish to simulate the nonstoichiometric hya at ambient temperature to confirm how the water moves and determine the correlated motions within the db7 framework ( cf . apoa-1 up - regulator ) . nonetheless , by combining such a range of experimental and appropriate computational work to get a coherent molecular understanding of this complex hydrate / anhydrate system , we show both the potential of computational chemistry for corroborating and assisting in an experimental program , as well as the need for further developments . a polymorphic system , 3-(4-dibenzo[b , f]oxepin-11-yl - piperazin-1-yl)-2,2-dimethylpropanoic acid , has two zwitterionic hydrate phases with disordered water , which differ remarkably in stability and hydration / dehydration mechanisms . the full multidisciplinary investigation of the behavior of the hydrate and anhydrate phases was complicated by the difficulty in isolating highly crystalline , phase pure samples , the sluggish equilibration of the hydrates at different rh , the metastability of the various forms at different conditions , and the variable hydration state of hya . however , structural based models for both hydrates and the metastable form iii rationalize this behavior . this system exemplifies the dangers in assuming behaviors based on cursory observation of stoichiometric and nonstoichiometric hydration . temperature , water activity , and ph determine the stability ranges of the db7 solid forms , considerably complicating the processing , storage , and handling of this unconventional pharmaceutical hydrate system .

elucidating the crystal structures , transformations , and thermodynamics of the two zwitterionic hydrates ( hy2 and hya ) of 3-(4-dibenzo[b , f][1,4]oxepin-11-yl - piperazin-1-yl)-2,2-dimethylpropanoic acid ( db7 ) rationalizes the complex interplay of temperature , water activity , and ph on the solid form stability and transformation pathways to three neutral anhydrate polymorphs ( forms i , ii , and iii ) . hya contains 1.29 to 1.95 molecules of water per db7 zwitterion ( db7z ) . removal of the essential water stabilizing hya causes it to collapse to an amorphous phase , frequently concomitantly nucleating the stable anhydrate forms i and ii. hy2 is a stoichiometric dihydrate and the only known precursor to form iii , a high energy disordered anhydrate , with the level of disorder depending on the drying conditions . x - ray crystallography , solid state nmr , and h / d exchange experiments on highly crystalline phase pure samples obtained by exquisite control over crystallization , filtration , and drying conditions , along with computational modeling , provided a molecular level understanding of this system . the slow rates of many transformations and sensitivity of equilibria to exact conditions , arising from its varying static and dynamic disorder and water mobility in different phases , meant that characterizing db7 hydration in terms of simplified hydrate classifications was inappropriate for developing this pharmaceutical .

Introduction Experimental Section Results Discussion Conclusions

PMC4410969

the major current use of genomic technology by the military is collecting dna from present and former members of the military in order to facilitate the identification of remains . the collection , called a biobank or biorepository , is the armed services repository of specimen samples for the identification of remains ( afrssir ) , which was established in 1993 . every member of the military is required to submit a blood sample to the repository upon enlistment , and as of 2012 , over 6.5 million blood samples have been collected and stored on bloodstain cards similar to those used for newborn screening . under current policy , afrssir can retain the samples for up to 50 years . aside from being used to identify remains , dna collected by the military can be decoded or sequenced , and the results linked to medical and personnel information obtained from service members or extracted from existing records . phenobank, which enables genetic and genomic variations to be correlated with medical , physical , and other characteristics and was one of the recommendations in the jason report . several military geno phenobanks are under construction . in 2009 , in collaboration with the national institute of mental health , the u.s . army began a $ 65 million , 6-year army study to assess risk and resilience in servicemembers ( starrs ) to identify risk factors for military suicides . one component of the research , the new soldier study which started in february , 2011 , asks new enlistees to volunteer to complete surveys and to undergo neurological and dna testing to identify genetic and other risk factors . as of november , 2012 air force office of the surgeon general established the patient - centered precision care research ( pc2-z ) program, which collects and sequences dna from saliva samples provided by volunteers from the air force medical service and matches the results with their medical information and family history . the purpose of the project is to create a personalized medicine profile that identifies genomic risks for clinically actionable , common complex diseases that are traditionally treated in the primary care setting, and to determine the impact of this information on the health of the volunteers . finally , in 2011 , the us department of veterans affairs ( va ) established the million veteran program , a geno phenobank that collects dna and health information from veterans and combines it with their va medical records for research purposes . the military may want to use the afrssir dna samples for research purposes rather than just for the identification of remains . the results of dna sequencing could be combined with medical and personnel records for present and former members of the military to form a very large geno , such an effort may be limited by the degree to which past records have been converted to an electronic format , which would be necessary in order to facilitate the research project . as noted earlier , the jason report calls for dod to create geno phenobanks to determine which phenotypes that might reasonably be expected to have a genetic component have special relevance to military performance and medical cost containment. once these genetic and genomic components are identified , genetic tests can be developed to identify which individuals possess genotypes that contain these components . currently , the u.s . military tests enlistees for sickle cell anemia and glucose 6-phosphate dehydrogenase deficiency . as the field of personalized genomic medicine expands , military physicians can be expected to use newly developed genomic tests in the same ways as their civilian medical colleagues , namely , to identify individual genomic characteristics associated with the prevention , diagnosis , and treatment of disease . for example , genetic testing can identify individuals with genetic disorders that will not become symptomatic until some point in the future , such as alzheimer 's or huntington 's disease . in some cases , there may be steps that can be taken to prevent or mitigate the symptoms . genomic testing also may be able to make or to help confirm a clinical diagnosis ; the air force , for example , is reported to be developing a genetic test for colorblindness that improves upon existing detection methods . as the jason report recognized , military physicians also increasingly will practice pharmacogenomics , in which genomic tests help determine the appropriate drugs to prescribe for specific patients based on their genomic profiles . like their civilian counterparts , moreover , military physicians might use genomic testing to identify individuals who were at risk for genetic disorders such as breast and colorectal cancers that can be prevented or mitigated by early detection and intervention . military interest in genomic testing will extend beyond the prevention , diagnosis , and treatment of disease . the jason report anticipates that tests will identify individuals who have genetic profiles that are of special relevance to military performance and medical cost containment. depending on whether test results revealed that individuals possessed desirable or undesirable genomes , the military could consider the results in deciding whether to incentivize or block enlistment , or to advance or curtail military careers , and the test results also could be useful in making duty , specialization , and geographic assignments . the national cancer institute , for instance , describes a young woman found to carry a mutation in a gene that increases the risk of breast cancer who may not be considered eligible for deployment for a 12- to 15-month period because access to recommended health care , such as mri screening , may not be easily accessible . in 2010 , a former military physician described an officer with a family history of huntington 's disease who was tested to see if he had inherited the disease gene before being promoted to flag rank , and a female pilot who was grounded after she developed a deep vein thrombosis and genetic testing revealed that she had a genetic mutation called factor v leiden polymorphism that increases the risk of such life - threatening blood clots . the military will be interested in identifying individuals with genomic variations of general medical interest , but also mutations that affect physical attributes that are of particular significance in military operations . the jason report , for example , mentions the value of genetic testing for susceptibility to traumatic bone fracture , prolonged bleeding , or slow wound healing . in 2009 , a group of researchers published an updated human gene map for performance and health - related fitness phenotypes containing over 214 entries that may be of interest to the military . the jason report also describes future genetic testing that would provide information about the ability to tolerate conditions of sleep deprivation , dehydration , or prolonged exposure to heat , cold , or high altitude. researchers already have developed and marketed a genetic test that identifies one variant of the actn3 gene in humans , called r577x , which codes for a protein called -actinin-3 . individuals with this genetic variation tend to have an abundance of slow - twitch muscles , which are associated with activities such as long - distance running that require endurance , while individuals who do not have this variant of the actn3 gene have more fast - twitch muscles , which are associated with activities requiring shorter bursts of energy such as sprinting and weightlifting . recruiters and training and assigning officers might be interested in knowing which of these variations their potential enlistees or trainees possessed . genomic testing for mental as well as physical traits will attract the military 's attention . a considerable amount of research on genetic variants associated with mental illness already is underway . for example , geneticists are searching for mutations that predispose warfighters to or protect them from post - traumatic stress disorder ( ptsd ) and have identified several genes , such as fkbp5 , pacap , tll1 , rs263232 in adcy8 , and rs71534169 in dpp6 , as susceptibility genes for ptsd . researchers also are seeking genomic mutations that are associated with superior mental abilities , and although claims of success are often met with skepticism , many geneticists are confident that genomic tests for superior mental capabilities eventually will be developed . scientists at the university of pennsylvania , for example , have created a strain of smart mice that produce more of a protein called nr2b , giving them superior memory and learning abilities . studies in humans have identified a gene called dysbindin on chromosome 6 and another called snap-25 on chromosome 20 that are associated with cognitive ability . genetic researchers have demonstrated in mice that a genetic mutation of the eif2 gene and genetic manipulation of the lynx1 gene could achieve enhancements in synaptic plasticity , learning , and memory . studies funded by the nih found that people with a certain variant of the gene catecholamine - o - methyltransferase can improve both memory and problem solving by taking a drug called tolcapone , which is prescribed for patients with parkinson 's disease , and exogenous medication may overcome a type of genotype - associated trait under which genetic variation can be associated with reduced dopamine neurotransmission to impair learning and cognitive performance . finally , a chinese research institute located in hong kong has purchased more than $ 90 million worth of advanced genetic sequencing machines and is using them , among other things , to sequence the dna of 2000 chinese school children to search for genes that correlate with educational testing scores . again , recruiters and trainers can be expected to want to use tests such as these in recruitment , training , and assignment . like their civilian colleagues , military physicians will develop and employ genomic technology not only to diagnose and predict but to prevent and treat both genomic and non - genomic diseases . a detailed description of the prospects for gene - based preventive measures and treatments is beyond the scope of this paper , but they include ( 1 ) the use of techniques such as recombinant dna to manufacture drugs ; ( 2 ) gene therapy , or the introduction or modification of endogenous or exogenous human dna to provide an increased ability to combat injury or illness ( for example , jonathan moreno cites the interest of the defense advanced research projects agency in using genetic manipulation to improve the immune system ) or to correct or compensate for genomic abnormalities ; and ( 3 ) altering the composition or characteristics of the microbiome , the non - human organisms found in the human digestive system and elsewhere that play a significant role in metabolism and human health . an especially controversial subject is germ line therapy , which entails making therapeutic alterations in an individual 's dna at such an early stage of development that the changes will be incorporated into eggs or sperm and therefore can be inherited by the individual 's descendants . the military may be interested in germ line therapy , for example , in order to reduce the frequency and costs of care for heritable genomic disorders in military families . the military will be intensely interested in exploiting the association between genomics and human capabilities and performance . in 2001 , the committee on opportunities in biotechnology for future army applications of the board on army science and technology at the national research council called on the army to lead the way in laying ground - work for the open , disciplined use of genomic data to enhance soldiers ' health and improve their performance on the battlefield. a 2002 report by dod information assurance and analysis center observed that because genomics [ sic ] information offers clues to improving human performance it could provide the army with means of increasing combat effectiveness. the jason report notes that both offensive and defensive military operations may be impacted by the applications of personal genomics technologies through enhancement of the readiness , and performance of military personnel. one way that genomic science can improve military effectiveness has been mentioned earlier : using genomic testing to sort individuals based on how they were expected to function in various military environments . in addition , the same genomic modification techniques described in the previous section on gene - based therapeutics could be used to actively improve warfighting ability . for example , until recently , one company , 23andme , offered direct - to - consumer genetic testing for a number of non - disease traits including avoidance of errors , eating behavior , food preference , measures of intelligence , memory , muscle performance , and pain sensitivity . the company acknowledged that the ability of the tests to actually measure these traits has not been established , but if the genomic variations could in fact be identified , it might be possible to manipulate them directly or by administering drugs that affected the proteins for which they coded . conceivably , then , warfighters could be made smarter and less error - prone , given better memories and muscles , or have their metabolisms altered to enable them to function with fewer calories or on unusual diets . air force office of scientific research reported that sleep deprivation disrupted the functioning of over 700 genes . in the future , these genes might be able to be reprogrammed so that they were not as affected by sleeplessness , enabling warfighters to carry out missions despite lack of sleep . a 2008 jason report predicted that , in contrast to elite sports , small improvements in warfighter performance would not have a dramatic effect on the outcome of military engagements ; a major breakthrough in the need for sleep , however , could seriously alter the balance of engagement. the uses of genomic technology by the military described in the previous section raise a host of elsi issues . how should the military , for example , conduct genomic research ? what types of genomic testing are appropriate in the military and how should the results be used ? what rules should govern how the military provides genomic therapeutics and enhancement technologies to its personnel ? similar questions arise in the civilian sector , and the elsi program at the nih has attempted to articulate solutions . however , the suitability of these civilian solutions in the military is limited by differences in core values . in his landmark work moral issues in military decision making , anthony hartle articulates freedom , equality , individualism , and democracy as the core american values . for the military on the other hand , hartle identifies country. in contrast to the civilian notion that the welfare of the individual is paramount , in the military it is subservient to the unit , mission , and country . in contrast to the civilian values of freedom , equality , and democracy , members of the military are obliged to obey lawful orders . the military also emphasizes taking responsibility for one 's decisions and actions , and an obligation to promote the welfare of subordinates to the extent consistent with the welfare of the unit , the accomplishment of the mission , and the safeguarding of the state . in a separate paper , i argue that these differences in core values require a different set of principles to govern bioethics in the military . the focus on the welfare of the individual patient in the clinical application of the civilian principle of beneficence , which stands in sharp contrast to the military value of selflessness , should be replaced in the military by the principle of proportionality . according to the principle of proportionality , a biomedical risk can be imposed on a warfighter only when there is no less risky alternative to accomplish a legitimate military objective , and the nature and degree of the risk are outweighed by the military advantage sought to be gained . in view of the lack of individual autonomy in the military , it also makes sense to downplay the civilian emphasis on voluntary choice reflected in the principle of respect for persons by substituting instead the principle of paternalism . combining the principles of paternalism and proportionality , military commanders have a duty to ensure that the biomedical risks that they impose on their subordinates are proportionate . these twin principles also guide privacy and confidentiality : commanders have a duty to protect their subordinates privacy and confidentiality unless and to the extent that it is outweighed by military necessity . commanders likewise have a duty to protect warfighters dignity by avoiding exposing them to biomedical risks that humiliate or demean them . finally , the principle of fairness should replace the civilian principle of justice and require members of the military to give their consent when commanders impose a biomedical risk only on a subgroup of subordinates or when the risk is especially great . fairness also precludes using biomedical risks as a punishment for bad behavior or imposing the risks in a discriminatory manner or on warfighters who are less able to bear them than others , such as physically weaker members of a unit . not only do basic principles of bioethics differ in civilian and military life , but several laws that play an important role in civilian genomics do not apply to service members , including the genetic information nondiscrimination act ( gina ) and , according to the so - called feres doctrine , the common law doctrine that provides damages for victims of medical malpractice . these legal differences must be taken into account in analyzing the elsi issues that arise in military genomics . the sections that follow identify the elsi challenges raised by military genomics explain how similar issues have been addressed by the civilian elsi program , and consider whether the civilian approaches are appropriate in the military due to their different bioethical and legal frameworks , and , if not , how these approaches should be adjusted to meet the needs of the military in an ethically , legally , and socially appropriate manner . a related set of issues involve the military operation of dna biobanks and geno phenobanks and the commercialization of research discoveries . a third series of issues relates to clinical and enhancement uses of genomic technologies by the military . the second is extramural research , where the military sponsors research that is conducted in civilian institutions such as hospitals and universities . the third setting is research conducted by the va on veterans who are receiving medical care from the va . each of these settings raises different eslp issues and therefore will be discussed separately . as discussed earlier , the military may wish to conduct genomic research to obtain general information relating to human health or to obtain information of particular relevance to the military . there are two types of genomic research involving human subjects : clinical research , in which experiments are conducted on live humans , and non - clinical research , which is conducted on human biological material such as dna samples . ( dod calls clinical research research involving a human being as an experimental subject to distinguish it from research involving human subjects, which includes both clinical and non - clinical research . ) the following discussion concerns clinical genomic research ; a discussion of non - clinical genomic research appears in balancing risks and benefits below . it might be thought that , under the principles of proportionality and paternalism , clinical research can take place without the informed consent of military subjects when the research is important enough to the unit , mission , or nation . an argument can be made that one major philosophical justification for informed consent , immanuel kant 's deontological categorical imperative that individuals should be treated as ends rather than as means , has little application in the military . military bioethicist michael gross asserts that , at least in wartime , human life is of but instrumental value , and observes that , there is very little compunction about using persons as means. no doubt based in part on this reasoning , the military gave soldiers lysergic acid diethylamide and other hallucinogens without telling them that they were being given the drugs but instead that they were participating in a study on the effectiveness of protective clothing and equipment against chemical warfare agents . similarly , voluntary consent was not always obtained from military personnel involved in radiation experiments that were conducted as part of atomic bomb testing . despite doubts about the applicability of a kantian perspective to the military , at least since the trials of nazi war criminals and the promulgation of the nuremberg code , which makes no distinction between civilian and military subjects , it is generally accepted that competent adults must give their informed consent to serve as subjects in biomedical research , and this consensus has long been reflected in official military policy . moreover , if the risks to subjects are more than minimal , it is required under the military bioethical principle of fairness since only a subset of the force is likely to be enrolled as participants . informed consent also has practical value in military research ; failure to obtain informed consent could discourage individuals from serving as research subjects , thereby reducing the amount of beneficial research that can be conducted , and even could discourage individuals from serving in the military in the first place . a 2000 congressional report on the dod 's mandatory anthrax vaccine program , for example , was especially concerned about the effect of the program on retention in reserve units , and noted that half of the air crew in one air national guard unit had resigned as a result . there is no reason to waive or weaken the requirement of informed consent for biomedical research employing military personnel as subjects when the research involves genomic science . informed consent is a central focus in the government 's efforts to protect human subjects in genetic and genomic research . indeed , it has been argued that genomic research requires even stronger protections for subjects than many other types of human experimentation because of the sensitive nature of personal genomic information that a study may uncover , such as predictive data about future illnesses for which the subject or the subject 's family members are at risk or misattributed paternity and maternity , that is , the discovery that a person 's presumed father or mother is not in fact biologically related . one reason to conduct genomic research on military personnel without informed consent might be to help preserve secrecy so that adversaries were not alerted to the production of information that might help them construct genomic weapons or defenses . for example , the military may feel the need to conceal the fact that it is obtaining genomic information about members of the same genomically differentiated subpopulation as enemy combatants . national security arguments have been put forth on behalf of military experiments conducted without consent in the past , such as atomic radiation experiments during the manhattan project and the cold war . although critics of those experiments argue that a measure of voluntary consent could have been sought from subjects , for example , by using the term dangerous radioactive substance in place of the classified word plutonium , it may not always be possible to use obfuscating terminology and still provide sufficient information to subjects to enable them to give informed consent . the solution would seem to be to enroll only those subjects who held security clearances high enough to permit them to be given the classified information . a requirement that members of the military give their informed consent to serve as subjects in genomic research does not mean that they actually can do so , however . in order for individuals to give informed consent , they first must have adequate information about the nature , potential benefits , and risks of the experiment , and they must be able to understand the information to a reasonable degree . serious doubts have been expressed about prospective subjects ability to comprehend and absorb the information they are given . there is little reason to expect members of the military to do better than other subjects ; on average , for example , they are less well educated than the general public . indeed , a strong argument can be made that warfighters should be regarded as a vulnerable study population , despite not being formally listed as vulnerable in the common rule . it was not until 1981 that army regulations were changed to prohibit penalties under the uniform code of military justice from being imposed on warfighters who refused to volunteer or withdrew from participating in military research . even without the prospect of formal punishment , warfighters may feel that they have no choice but to acquiesce when they are invited to serve as subjects . as victor sidel and barry levy explain in the textbook of military medicine , because they can not simply quit their jobs file a grievance with a union , government agency , or professional organization , military personnel may not believe that they can truly refuse to participate in these experiments . they may feel more like a captive audience than like volunteers . moreover , as the house committee on government reform pointed out in its critique of dod 's mandatory anthrax vaccination program , in a culture based on a chain of command and the power to compel , attempts at persuasion and education often devolve into intimidation. superiors may make subordinates willingness to serve as subjects a condition for allowing them to undertake certain attractive missions , for example , missions to test exciting new technologies such as genomic enhancements . unit cohesion and comradeship may lead warfighters to agree to be subjects if other members of their unit already have . warfighters may also feel that it is their patriotic duty to participate in research , a notion that some commentators are pushing in the civilian sector . dod is well aware of the risk that warfighters asked to give their informed consent to serve as research subjects will feel that they have no choice but to accede . dod rules prohibit superiors from influencing the decisions of their subordinates regarding participation as subjects and specifically forbid superiors from being present when informed consent is being sought . moreover , except for a payment of $ 50 in return for having blood withdrawn , military personnel on duty may not be compensated for serving as subjects . warfighter 's limited autonomy requires a shift in emphasis in military clinical research away from the reliance on informed consent as a primary protection for subjects in the civilian model and toward a reliance on paternalism . civilian research ethics contain a strong element of paternalism ; subjects are only asked to give their consent to participate in research after experts , in the form of institutional review boards , sponsors , government regulators , and the investigators themselves , are satisfied that the potential benefits of a study outweigh the risks . given the limits on warfighter autonomy , the need for paternalism in the military is more pronounced , however , and ensuring proportionality must be viewed as the primary responsibility of research directors , institutional review boards , and subjects commanders rather than the subjects themselves . to that end , dod rules require the appointment of an ombudsman and a research monitor ( who may be the same person ) to oversee the informed consent process when the research presents more than minimal risk ( and , in the case of an ombudsman , when the recruitment of subjects takes place in a group setting ) . individual services have added additional oversight in certain cases in the form of a requirement for a second level of protocol review in addition to review by an institutional review board ( irb ) . one approach that the military has taken to promote voluntary participation in research is to establish special units from which to draw subjects , beginning with operation whitecoat , in which 2300 seventh - day adventist church conscientious objectors between 1954 and 1973 participated in 137 protocols to develop vaccines and treatments for q fever , tuleremia , various viral encephalitides , rift valley fever virus , sand fly fever and plague , and continuing with the medical research volunteer subjects program using the us army medical research institute of infectious diseases ( usamriid ) troops recruited from although concerns have been voiced in connection with operation whitecoat that seventh - day adventist church officials endorsed the program , with one subject later pointing out that we grew up to trust the church, the head of usamriid emphasizes that participation in research among members of these units is voluntary , with only about 80 per cent of the troops consenting to serve as subjects in any one year . this has led some commentators to suggest that similar units should be the primary if not the exclusive source of military research subjects . but it is still unclear if the members of these units would feel sufficiently free to decline to participate in research , and specialized groups of subjects may not be representative of the troops who actually would be exposed to the instrumentalities being tested . an axiom of research ethics is that a study may proceed only if the risks are outweighed by the potential benefits . in balancing research risks and benefits , civilian and military principles of bioethics the risks to the subjects can be outweighed either by the benefits to the subjects themselves or by the benefits , in the form of the knowledge gained , to others . civilian research thus employs an approach similar to the military principle of proportionality in which the welfare of the individual can be subordinated to a greater good in appropriate circumstances . civilian research also embraces the military principle of paternalism ; as mentioned earlier , the balancing of risks and benefits is entrusted first to institutional review boards , study sponsors , government regulators , and the investigators themselves , and only after they have satisfied themselves that the benefits outweigh the risks are subjects asked to make their own assessment in giving their informed consent to participate . warfighters may be subjected to grave personal risks in order to achieve a military objective ; as bill rhodes , president of the international society for military ethics , states , a military member is expected to serve the state unto maiming , capture , or death. accordingly , a research risk that would be deemed excessive for civilian subjects , such as a significant likelihood of serious harm in a study intended to produce knowledge to benefit others but with no direct benefit to the subjects , might be deemed acceptable for military subjects , under the principle of proportionality if the military objectives were sufficiently important . in other words , while the risks might be greater than for civilians , so might be the corresponding benefits to the unit , mission , or country . along this line , military subjects ought to be allowed to participate in studies intended to produce information of special relevance to the military , such as tests of genomic interventions to treat ptsd , that posed greater risks than studies aimed at developing genomic interventions of no special value to the military . another question is how to weigh the risks and benefits of research on genomic enhancements , such as interventions to increase the ability to tolerate conditions of sleep deprivation , dehydration , or prolonged exposure to heat , cold , or high altitude mentioned in the jason report . although as discussed later , the distinction between enhancements and other types of biomedical interventions is not always easy to make , some commentators believe that enhancement benefits are less worthwhile than medical benefits and therefore that a risk that might be acceptable in a medical trial would be unethical in an enhancement experiment . one bioethicist states , for example , that : [ t]he cost / benefit analysis is different for enhancement . while those who are experimenting with treatments for serious diseases may only succeed in substituting one kind of misery for another , those experimenting on human enhancement are likely to substitute a miserable life for a happy one . [ t]he cost / benefit analysis is different for enhancement . while those who are experimenting with treatments for serious diseases may only succeed in substituting one kind of misery for another , those experimenting on human enhancement are likely to substitute a miserable life for a happy one . as my colleague jessica berg and i have argued , however , the value of a benefit clearly depends on the nature of the benefit ; an enhancement that potentially increased cognitive function substantially , for example , might be deemed more valuable than a treatment , say , for nail fungus . in both health - oriented and enhancement research , therefore , the potential benefit necessary to justify a set of risks will depend on the specifics of the study in question . moreover , it is difficult to distinguish between enhancement and other types of biomedical research in the military ; a study of a genomic intervention to provide combat troops with better - than - normal vision , hearing , or cognition , or a greater - than - normal ability to tolerate conditions of sleep deprivation , dehydration , or prolonged exposure to heat , cold , or high altitude , for example , could be regarded as the study of how to prevent injury as well as an enhancement study . in short , there is no reason to treat an enhancement experiment differently than a treatment study when it comes to balancing risks and benefits . a particularly controversial topic in genomic research is whether researchers have a duty to provide study findings to the subjects if the information could provide health benefits to the subjects . the issue arises especially when researchers are examining large amounts of genomic information on large numbers of subjects , such as searching a collection of dna samples for variations associated with particular disease risks or traits . as noted earlier , for example , military researchers are hunting for genomic variations that predispose warfighters to ptsd . the answer might seem to be of course , since individuals generally have a right to receive their personal biomedical information . but some geneticists worry that , since genomic information is often difficult to understand , subjects may misinterpret it and be harmed . genomic findings often are probabilistic , and the average person has difficulty understanding probabilities . the effect of a genomic variation may be small ; the genetic variation apoe4 , for example , is found in 40 per cent of persons with late - onset alzheimer 's disease but also in 2530 per cent of the general population . penetrance , so that individuals may never show symptoms or may only experience a mild form of the illness . since physicians may be uncertain about how to interpret genomic test results , one suggestion is to rely on specially trained experts called genetic counselors to convey results to subjects , but this requires more time and adds to the cost . another complication is that some diseases such as alzheimer 's can not now be prevented or treated , and some commentators question the value of informing subjects about risks for those kinds of disorders . furthermore , the clinical laboratories improvement act ( clia ) requires laboratories that test dna to be clia - certified if the results are reported to diagnose , prevent , or treat disease or to assess health , and obtaining and maintaining clia certification is burdensome and expensive . finally , commentators argue that researchers do not have the same obligations to subjects that physicians have to their patients , in particular , a duty to look after the subjects health needs . some of these objections do not apply in the military as much as they do in civilian research . the military is responsible for service members health , so military researchers , like other superiors , have an obligation to look out for the well - being of their subordinates under the principle of paternalism . moreover , military laboratories are not driven by the same profit motive as private civilian laboratories , and therefore may be less burdened by having to comply with clia requirements . but military researchers still need to carefully consider the pros and cons of returning results to subject . they should have the same compunctions about how well subjects understand study results as their civilian brethren , especially since they are held accountable for the welfare of subjects under the principle of paternalism . furthermore , there may be special considerations that justify withholding genomic information from military subjects . for example , if the military eventually discovers genomic variations closely associated with certain desirable or undesirable traits , it may decide to test prospective enlistees and use the results in making service assignments , but may not want to disclose the results to warfighters because of military exigencies . warfighters told that they were at increased risk for certain types of battlefield injuries , for example , they might lose effectiveness if military necessity required them to participate in combat missions . the need for security to keep sensitive genomic information out of the hands of adversaries also might justify withholding test results from warfighters in certain circumstances . another question is whether researchers who disclose results to patients to improve their health should also disclose the results to family members who may also be at risk for the same genomic conditions , and if so , should they do so only with the subjects permission . the question has been addressed mainly in the context of the relationship between patients and physicians rather than subjects and researchers , and there is a split of opinion . many prominent groups say that doctors may override the patient 's wishes if the physician has made a reasonable effort to obtain the patient 's consent , there is a high probability that imminent , serious harm will occur if the information is not disclosed , and only information necessary for the diagnosis or treatment of disease is disclosed . ( the institute of medicine goes further and says that the information must be disclosed . ) however , some physician groups maintain that notifying family members is the patient 's responsibility rather than the physician 's , and point out that laws in illinois , massachusetts , and new york prohibit any disclosure of genetic information without the patient 's consent . whatever the eventual consensus , the only reason for the rules governing disclosure of research to family members to differ between military and civilian life would be if the disclosure would pose a significant threat to national security or the successful completion of a military mission . a topic that is closely related to the return of research results is how researchers should handle information about subjects that is outside of the scope of the study , called ( a similar issue arises when a physician examining a patient 's genome for certain clinically relevant information discovers other medically relevant findings . ) researchers searching for genomic mutations that made subjects susceptible to ptsd , for example , inadvertently might discover mutations that placed certain subjects at risk for other diseases . again , the answer might seem clear that they should , since doing so could alert subjects to the need for future vigilance and beneficial medical care . is the medical risk reflected by the incidental information sufficiently important to outweigh the chance that subjects will misunderstand it ? how clear is it that the individual will show symptoms of the genomic disease or will experience a serious form of the illness ? what if little or nothing can be done to reduce or eliminate the health risk ? one particularly troublesome type of unintended finding , which can occur when both parents and offspring are studied in pedigree research , is that a parent is not biologically related to a child ; how should researchers handle this result , which can be extremely disruptive to the family unit ? for these reasons , some commentators maintain that researchers have no obligation to inform subjects about incidental findings . a growing consensus seems to be emerging , however , that researchers should disclose incidental findings that are sufficiently important for the subjects health , or at least that subjects should be asked during the informed consent process if they wish to receive this information . there is no reason to assume that military researchers have a lesser obligation regarding disclosure of incidental findings than civilian researchers . in fact , an argument can be made that military researchers have a greater obligation than their civilian counterparts to consider returning incidental findings to subjects because military personnel have less ability than civilians to obtain genomic testing outside of the military . another research controversy concerns whether researchers have a duty to contact subjects after a study is over to ascertain whether they are suffering any long - term or latent adverse effects , and to alert them to new information bearing on their health , such as the discovery of genomic risks that were not known at the time of the study or the availability of new treatments or preventive measures . the military has been criticized , for example , for not adequately following up with subjects in the man - break experiments on mustard gas during world war ii . civilian researchers object that they should not have a long - term obligation to follow up with subjects because of the difficulties of keeping track of their whereabouts , but military researchers should be better able to keep track of military subjects , who may still be in the military or receiving care from the va . in addition , the army maintains a volunteer registry for all subjects in more - than - minimal - risk studies , one of the purposes of which is to enable the military to exercise its obligation to ensure research volunteers are adequately warned of new risks and to provide new information as it becomes available, and the us army medical research and materiel command ( usamrmc ) stores the information for a minimum of 75 years . in sum , the military should endeavor to contact subjects about future developments of significance to their health bioethicists are concerned that offering people substantial amounts of money or other items of economic value in return for serving as research subjects could compromise the voluntariness of informed consent , but this is less of a concern in the military because dod rules forbid paying research subjects on active duty except for drawing a blood sample , for which they may receive $ 50 . ( off - duty personnel may receive compensation comparable to civilian subjects , which under health and human services ( hhs ) rules must be approved by an irb and be a reasonable amount in accordance with prevailing local rates . ) military irbs must be on guard however against offering troops non - monetary benefits for enrolling in research studies , such as desirable assignments or commendations . dod rules prohibit superiors from influencing the decisions of their subordinates regarding participation as subjects , but the rules could be more specific by defining what counts as influence. moreover , the prohibition against paying military subjects emphasizes the need to protect them from being exploited by being pressured to participate in disproportionately risky research . another financial issue that arises in civilian biomedical research is whether subjects are entitled to a portion of the financial benefits that accrue to sponsors of studies that lead to the commercialization of a lucrative new medical product . although withholding compensation has been criticized as unfair , there is a general consensus that sponsors can avoid compensating subjects if they disclose their policy to subjects during the informed consent process . the argument for rewarding military subjects is weakened by their obligation to serve the national interest , but the issue is complicated by the fact that a substantial amount of the fruits of military research often redounds to the benefit of private enterprises that commercialize the resulting discoveries , including prostheses and thermal imaging devices . a solution might be for dod to ensure that genomic discoveries with important medical value derived from research on military subjects were available to current and former members of the military through military medical services and the va . the common rule governing human subjects research does not require researchers to compensate civilian subjects for injuries sustained as a result of their participation , but subjects who have been injured by negligent investigators may recover monetary damages in a tort action . for example , the father of an 18-year - old subject who died in a university of pennsylvania experiment aimed at developing a gene therapy for a genetic disease called ornithine transcarbamylase sued the investigators for negligently enrolling his son without proper safety testing . no such remedy is available to military subjects , however , as a result of the so - called feres doctrine . dod rules require researchers to establish procedures to protect human subjects from medical expenses ( not otherwise reimbursed) that directly result from participation in more - than - minimal - risk military research , whereas civilian subjects may receive compensation for losses such as pain and suffering and reduced future earnings . it also runs counter to the principle of paternalism insofar as it shields the military from having to fully redress superiors mistakes . but congress has not seen fit to modify the doctrine to permit injured subjects to receive compensation , the courts have consistently given the doctrine a broad interpretation on the premise that immunity from suit is essential to maintain military discipline , and even some of the critics only want soldiers to be able to sue for intentional rather than merely negligent research misconduct . protecting the privacy and the confidentiality of subjects personal health information has long been a priority in civilian research , and its importance has been reinforced by the adoption of the privacy and security provisions of the health insurance portability and accountability act ( hipaa ) and its accompanying regulations . one of the requirements of informed consent under the common rule , for example , is giving potential subjects a statement describing the extent , if any , to which confidentiality of records identifying the subject will be maintained, and the government recently has proposed strengthening data security and information protection requirements for federally sponsored research . in addition , the gina specifies that genetic information is protected under hipaa . as noted earlier , dod has adopted the common rule / it also has subjected itself to hipaa . however , the privacy and confidentiality of members of the military is subordinate to military necessity . michael gross observes , for example , that among one 's own soldiers , the scope of the private sphere decreases and that of the public expands as collective welfare takes precedence over an individual 's private good. dod rules accordingly permit commanders to obtain health information to assure the proper execution of the military mission , and while service members have the right to an accounting of disclosures , there is no such right for disclosures for national security or intelligence purposes . one of the chief reasons for protecting privacy and confidentiality in regard to genomic information is to prevent stigma and discrimination . the legal protections against workplace discrimination for civilian federal employees in the rehabilitation act of 1973 and gina do not apply to uniformed members of the military . once warfighters leave the military , however , they come under gina 's protections against employment discrimination on the basis of genomic information . furthermore , dod policies implementing the national defense authorization act of 2008 changed a previous policy that had denied health and disability benefits to service members who experienced injuries or illnesses during their time of service if the condition was congenital or hereditary. now , service members are entitled to compensation and benefits so long as they do not have a disability that was noted at the time of enlistment , unless there is compelling evidence or medical judgment that the disability existed prior to enlistment . however , united states army special operations command ( usasoc ) found that the two strongest fears among service members with respect to behavioral health treatment , including ptsd , were confidentiality , fear of being labeled , and negative impact on career . as shown in part i.b . , the most recent research has shown a genetic contribution to ptsd and some scholars have raised concerns regarding the display of pertinent genetic information may also generate stigma and affect individual career outcomes. how should the status of subjects as members of the military affect the privacy and confidentiality of identifiable genomic information obtained in the course of military human subjects research ? one approach might be that , given the reduced privacy and confidentiality within the military , the military should be able to use identifiable genomic information obtained in the course of conducting human subjects research on military subjects without the subjects informed consent , so long as the information is used for a legitimate military purpose . but this approach conflicts with the spirit of the military 's policy of requiring military subjects to give their informed consent to serve as research subjects . another approach would be for irbs to give the potential loss of privacy and confidentiality less weight when balancing risks and benefits in research on military subjects than in research on civilians . but the federal government is contemplating removing privacy and confidentiality concerns altogether as matters for irb review and instead imposing mandatory data security rules on all human subjects research . the best approach therefore is to rely on the principles of proportionality and paternalism described earlier and place the responsibility on commanders , including those overseeing research programs and the researchers themselves , to protect subordinates against inappropriate disclosures of identifiable genomic information . in summary , using members of the military as subjects raises many of the same ethical issues as employing civilian subjects , but there are several notable differences . due to limitations on warfighter autonomy , members of the military need special protection from coercion and undue influence during the informed consent process , and the principles of proportionality and paternalism require commanders to assure themselves that the potential benefits of the study outweigh the risks rather than relying on individual subjects to make this judgement for themselves in uncertain cases . at the same time , risks that would be excessive for civilian subjects might be acceptable in the military if the military 's need for the research were sufficiently compelling . finally , the fact that the military is responsible for the health of its members and the relative ease with which it can keep track of warfighters , including after they are discharged , argue in favor of imposing a clearer obligation on military researchers than on civilian researchers to follow up with subjects and to return results and incidental and unintended findings to them in appropriate cases . dod spends approximately 75 per cent of its outlay for research and development on extramural research , that is , research conducted at civilian institutions . the allocation of such a large portion of the defense r&d budget to civilian researchers results from the need to fund industrial contractors that develop weapon systems and the desire to nourish a broad science base at u.s . academic institutions . some of this dod - sponsored research there are no published estimates of how much involves the use of live human subjects . some of these studies may involve genomic science , and the amount of genomic human subjects research can be expected to increase if dod follows the recommendations in the jason report described earlier . extramural studies of human subjects must be approved by irbs at the civilian institutions conducting the research as well as by internal military irbs . the army requires an additional layer of protocol review by the usamrmc commanding general 's human subjects research review board for studies involving gene transfer. although commentators have noted the potential for conflict between internal military and external civilian irbs , there is no information on how they interact , for example , how they resolve disagreements . nor is there an understanding of how the members of civilian irbs decide whether to approve protocols for military sponsored research . they may not be clear on how to weigh risks to subjects against military necessity and national security . on the one hand , they may feel that it is inappropriate for them to reject military studies that pose risks to subjects that would be unacceptable in purely civilian research . on the other hand , some irb members may be critical of military research for various reasons , leading them to block or unreasonably delay studies that should go forward . in addition , both dod and hhs regulations forbid irbs from considering possible long - range effects of applying knowledge gained in the research ( for example , the possible effects of the research on public policy ) as among those research risks that fall within the purview of its responsibility, and irbs may not know how this restriction should affect their consideration of the long - range effects of military research . bioethicist jonathan moreno notes the difficulties raised by conducting classified military research outside of the military , including requiring irb members and potential subjects to have security clearances . ( dod rules , for example , require irbs to determine if potential subjects need access to classified information to make a valid , informed consent decision. ) the advisory committee on human radiation experiments evaluated human subjects protections in classified government research and identified a number of shortcomings in written informed consent forms . academics describe resistance among academics and universities to conducting classified government research due to concerns about infringement of academic freedoms , including the freedom to publish research results . nineteen of 39 universities she studied had policies prohibiting either classified or non - publishable research . ( eisenberg notes , however , that in some cases faculty researchers at these institutions may be affiliated with off - campus laboratories that do not have similar restrictions . ) in addition , eisenberg reports that a number of other universities do not consider classified research for purposes of promotion , tenure , or academic credit . dod has adopted special protections for subjects in classified experiments regardless of whether the subjects are military personnel or civilians : the protocol must be approved by the secretary of defense ; the fact that the study is sponsored by dod and that the research is classified must be disclosed as part of the informed consent process , unless the secretary of defense makes an exception because providing this information could compromise intelligence sources or methods; irbs must do a full rather than expedited review ; and an irb member who disagrees with a majority decision approving the study may appeal to the secretary . the foregoing concerns about extramural military research raise the question of whether and in what circumstances it should take place at all . an argument can be made that all military human subjects research , including genomic research , should take place intramurally using members of the military as subjects . by being in the military , they have agreed to subordinate their interests to those of the military , and therefore seem more appropriate to bear research risks than civilians , especially in experiments that offer no prospect of direct benefit to subjects . furthermore , military subjects are protected by the principle of paternalism and therefore , at least in theory , commanders who expose them to disproportionate experimental risks are accountable and subject to punishment . in civilian bioethics , on the other hand , there is a dispute over how much of a duty civilian researchers owe to protect the interests of their subjects , with some commentators asserting that the investigators duty to subjects is superseded by their duty to the study sponsor . on the other hand , certain types of genomic studies for example , genome - wide association studies may need to use large civilian dna repositories in order to have the power to discover variations that account in small ways for phenotypes of military interest . science argues in favor of continuing to use military research funding to support civilian research institutions . finally , requiring military research to be conducted on military personnel could lead to public perceptions that they were being used as human guinea pigs , especially if the research was not on a topic primarily of military interest . the va has already staked a claim to genomic research by launching its million veteran program in may , 2011 , which collects voluntary donations of dna from veterans along with permission to correlate their genetic data with information in their medical and personnel records in order to establish a geno phenobank . ( the program is described further below in the section on biobanks . ) in addition , the va conducts genomic research on amyotrophic lateral sclerosis , bipolar disorder and schizophrenia , and ptsd . in 2006 , it established a genomic medicine program advisory committee to guide the start of a new genomic medicine program , and it also conducts pharmacogenomic research . the va , like dod , has adopted the common rule to govern live human subjects research . however , va patients asked to participate as subjects , like other civilian patients , may fear that they will be denied health care if they refuse . va researchers are required to make every reasonable effort to provide patients with an informational brochure called volunteering in research here are some things you need to know which states that they have a right to say no and that refusing to participate will not affect your va health care or benefits. beyond that , the agency has no special human subjects protections . for example , it does not recognize veterans as a vulnerable research population . while it is true that they are not among the groups formally designated as vulnerable subjects , they resemble those groups in their dependence on government - provided health care , and the fact that they served in the military may make them feel that they have a duty to participate in research whenever they are asked . these considerations require the va to employ a heightened degree of paternalism to protect veterans against disproportionate research risks . while the foregoing sections discuss the ethical , legal , and policy issues raised by clinical genomic research using human subjects , this section discusses another type of genomic research that uses dna samples rather than living human subjects . as noted earlier , the military currently possesses millions of dna samples from current and former service members , and the army , air force , and va are collecting additional samples for research purposes . the resulting biorepositories can be linked to the medical and personnel records of the individuals who contribute the dna to create geno phenobanks , enabling researchers to discover links between dna variants and physical and mental conditions and characteristics . phenobank research raises many of the same types of ethical , legal , and policy concerns as clinical research , including the need to obtain consent from dna contributors to use their dna for research , return of results and incidental findings , shared benefit , and maintaining the privacy and confidentiality of research results . in regard to consent , some commentators have suggested that , based on traditional research ethics , individuals whose dna is proposed to be used for research should be re - contacted and asked to give consent for each new research project . other commentators propose that , at the time that their dna is obtained , individuals should give multi - layered consent which would enable them to specify their wishes on a detailed form , such as giving consent to future research on specific diseases or being re - contacted and asked to consent to any future research . however , under increasing recognition that re - contacting participants to obtain their specific informed consent for every future research project may be impractical , expensive , and possibly impede socially valuable research , there has been a growing movement away from the model of specific informed consent . alternatives that have been put forward include ( 1 ) permitting human research ethics committees to waive individual consent under circumstances where obtaining consent is impracticable and there is a strong public interest in the research ; ( 2 ) broad consent that allows samples to be used for one or more general purposes , such as biomedical research in general , or research on one or more specific diseases ; and ( 3 ) to understand the issues involving consent , it is important to distinguish between research on existing dna samples and research on newly acquired samples . suppose dod wanted to combine existing dna samples in the afrssir with digitalized medical and personnel records of service members in order to conduct biomedical research . would dod have to obtain the consent of the dna contributors , and if so , what kind of consent ? on the one hand , it might be argued that consent is unnecessary . the dod policy that requires informed consent from military research subjects only applies to research involving a human being as an experimental subject, which dod defines as involving an intervention or interaction with a living individual, and moreover , providing dna to the afrssir is mandatory ; warfighters have been court - martialed for refusing , and their courts - martial have been upheld by the courts . furthermore , having to obtain consent could be burdensome in the case of individuals who are no longer in the service , who might be difficult to locate . the argument for proceeding without consent is especially strong in regard to research with distinct military applications , for example , on gene variants associated with coolness under fire , or genomic therapies that speed trauma recovery . on the other hand , the consensus in the civilian sector is that research on dna samples requires the consent of the contributors if their identity is revealed or can be determined from the data , which , as mentioned earlier , would be the case with a military geno phenobank . furthermore , the mandatory nature of the afrrssir program has been criticized , and in any event , dod currently permits afrrssir samples to be used for purposes other than identification of remains only with the consent of the donor or the surviving primary next - of - kin . finally , the whereabouts of personnel who have left the service may not be that difficult to determine since they may be obtaining health care from the veterans administration . if the consent of warfighters to use existing dna samples is necessary , what kind of consent must be obtained ? specifically , can warfighters be asked to consent to any research use of their dna ( referred to as broad or general consent ) , or must they give specific consent for each particular research project , including for different projects undertaken in the future ? this is the subject of controversy in the civilian sector , but there appears to be a growing consensus , reinforced by changes in consent rules proposed by dhhs , that broad consent is acceptable , and the reasoning supporting the consensus suggests that broad consent also should be sufficient for military research . if the military must obtain consent for research using existing dna , all the more reason that it should obtain consent for research on newly acquired specimens , since this can be done easily when the specimen is obtained . the common rule that governs civilian research does not apply to deceased persons , who are not deemed to be human subjects. however , some commentators have argued that researchers should honor preferences that a deceased person made known while alive , and that consent should be obtained from the next - of - kin . in the case of the military , when they submit samples to afrssir , enlistees could be asked to give their consent to posthumous research using their dna . as noted earlier , the va operates the million veteran program , a geno phenobank that collects dna and health information from veterans and combines it with their va medical records for research purposes . veterans at participating va medical centers are sent a letter asking if they would like to participate . if they opt in , they fill out a brief health survey and make an appointment at the medical center to accompany their next medical visit . during this visit , they are counseled about the program , given the opportunity to consent to be placed in the database and to make their medical records available for research , and asked to give a blood sample . the consent is broad , as the veteran is asked to consent to a general program of genetic research , and the sample is kept indefinitely . as of october , 2012 , as noted earlier , the va does not return any results to the donors , claiming that it is barred from doing so because the dna is not analyzed at a clia - certified laboratory . given the public nature of va funding and the relative ease in locating participating veterans , an argument can be made that the cost concerns raised by requiring samples to be analyzed at clia - certified laboratories are outweighed by the potential health benefits of returning results that are medically significant . testing in aid of medical diagnosis or treatment raises ethical and legal concerns . whether physicians can order hiv tests and drug screens without patient consent has been especially contentious , but objections also have been made to the widespread physician practice of ordering other routine medical tests without informing patients of the nature of the tests and obtaining their consent . when the testing in question is genomic , special concerns arise because the testing laboratory obtains access to the patient 's dna , which contains the patient 's entire genetic code rather than just information about the targeted illness . one issue that has been discussed earlier , for example , is whether the physician has a duty to inform the patient about when addressing ethical and legal concerns of genomic testing , it is common to distinguish between predictive genomic testing , which aims to ascertain if a person is at risk for a genomic illness in the future , and non - predictive testing , that is , testing to diagnose or aid in treating a manifested illness . as discussed earlier , military physicians can be expected to employ genomic testing for non - predictive purposes in the same manner as their civilian colleagues , that is , to help diagnose and treat illness in their military patients . non - predictive genomic testing generally has been less controversial , since it tends to be viewed as analogous to other types of medical testing . for example , a provision in massachusetts state law prohibiting genetic testing without informed consent excludes any test for the purpose of diagnosing or detecting an existing disease , illness , impairment or disorder from its definition of genetic test. but even when it is performed for non - predictive purposes , genomic testing can yield information about the patient 's risks for future illnesses , which can lead to social stigma if it becomes known to others and discrimination if it gets into the hands of employers or health insurers . from an ethical and legal standpoint , then , there is no meaningful difference between predictive and non - predictive genomic testing , and in the civilian sector , there is a clear consensus that no genomic testing should be performed without an individual 's informed consent . as discussed previously , however , the bioethical principles that govern military life are different from those that apply to civilians . the health status of warfighters not only affects their well - being , but the well - being of the unit , the success of the mission , and the security of the state . unlike civilians , therefore , warfighters are not free to refuse necessary medical care , including medical testing . should the fact that a test is genomic change this rule , that is , should warfighters be able to refuse a genomic test , and should the answer be the same when the genomic test is predictive rather than in aid of diagnosis or treatment ? finally , should warfighters be able to refuse genomic testing that pertains to non - disease characteristics , such as physical and mental abilities ? despite the special concerns raised by genomic testing , a strong argument can be made that it should be subject to the same rules as other types of military medical testing , meaning that warfighters can not refuse to submit to genomic testing , including when it is predictive rather than in aid of treatment and diagnosis . as noted earlier , providing a blood sample for dna testing under afrrssir is mandatory , and its mandatory nature has been upheld by the courts . furthermore , the military rules of evidence governing proceedings under the uniform military code of justice provide that , while communications between a patient and psychotherapist arguably the most sensitive type of medical information are privileged , the privilege does not apply when the information is necessary to ensure the safety and security of military personnel or the accomplishment of a military mission. moreover , although the military in a sense is an employer of its personnel , it is not subject to the prohibition in gina against requiring employees to undergo genomic testing . in any event , there is some sentiment that gina should be amended to allow employers to use genomic testing that could identify job applicants who might pose a threat to the safety of others as a result of their genomic endowment . at the same time , however , military genomic testing is subject to the principles of proportionality and paternalism that govern military bioethics generally , so it may only be undertaken if accountable superiors determine that the risks , including loss of privacy and potential breaches of confidentiality , are outweighed by military necessity . this is especially important in the case of genomic testing for non - disease characteristics . the military has a legitimate interest in obtaining information about warfighters physical and mental abilities , including genomic information , but only if the genomic test is a valid indicator of what it purports to show and the information is necessary in order to carry out the mission . furthermore , as noted earlier , the military has adopted the privacy rules of hipaa , including the limitation on disclosure of protected health information to the minimum necessary to accomplish the purpose of the disclosure , and the requirement that the information must be kept secure . finally , the military must be mindful of relying too heavily on the results of genomic tests that have not been adequately validated . the popular press is quick to tout purported links between genomic factors and traits or conditions that often turn out to be false or overstated . although military exigencies may create a compelling need for genomic data , the data must be viewed with caution until they are adequately confirmed . this raises the question of under what circumstances the military is entitled to rely on genomic technologies that have not yet been fully tested , discussed in the following section . as described earlier , the military has an interest in giving troops gene - based technologies that could help protect them , treat injury and illness , and improve their performance . earlier , this article discussed ethical and legal issues surrounding the testing of these interventions in military and civilian subjects . but the military might not want to pass up the opportunity to provide a promising genomic intervention to warfighters , especially those in harm 's way , even though the intervention had not undergone complete testing for safety and efficacy . a similar situation , although not involving genomic technologies , arose with the distribution of pyridostigmine bromide ( pb ) and botulinum toxoid ( bt ) vaccine to troops during the gulf war , and with the dod 's anthrax vaccine immunization program ( avip ) , which began in 1998 . these modalities were given to troops to protect against chemical and biological weapons rather than solely to determine whether or not they actually worked . in other words , they were deployment uses rather than formal research studies . at the time , the food and drug administration ( fda ) had approved pb for treatment of myasthenia gravis , but not for protection against nerve agents , while at the start of the avip program , the anthrax vaccine was approved by the fda to protect against cutaneous exposure but not against the airborne exposure that dod expected . ( the fda approved anthrax vaccine for all routes of exposure in 2004 . ) therefore , these were off - label or unapproved uses , a common medical practice . the fda had not approved bt vaccine for any use at the time of the gulf war , but the vaccine had an odd history ; it was the only vaccine available against botulinum toxin , had been used by doctors to protect people against it for years , and was manufactured for that purpose by the centers for disease control and prevention under an investigational new drug exemption , the fda 's mechanism for allowing unapproved products to travel legally across state lines . these deployment uses generated controversy over whether they should be voluntary , that is , whether troops should be allowed to refuse to take the drug and the vaccines . one concern was that making deployment use mandatory would encourage the military to use it to circumvent the informed consent requirements of formal research . ultimately , congress decided that drugs could be given to troops off - label only if a waiver of informed consent was issued by the president or under an emergency use authorization ( eua ) granted by the fda during a national emergency , and then not to all members of the armed forces but only to troops in a particular military operation. the same legal restrictions currently would apply to the deployment use of unapproved or off - label genomic technologies . in addition , the military should only seek a presidential waiver or an eua when the use of these genomic technologies would be ethical , that is , when it would comply with the principles of proportionality , paternalism , and fairness . commanders should carefully consider the available evidence concerning the risks and benefits of the technology in question and determine that there is no less risky alternative , so that imposing the risks on troops would be necessary in order to accomplish the mission . with the possible exception of deployment uses , warfighter access to genomic technologies will be through a physician intermediary , whether as a researcher , clinician , or operator of a genomic testing program . some commentators claim that physicians in the military should behave no differently than physicians in the civilian world and that the same ethical norms and legal rules should apply . the world medical association , for example , states that medical ethics in times of armed conflict is identical to medical ethics in times of peace, bioethicist peter clark asserts that the failure of medical professionals to recognize that military and civic duty can never trump medical ethical principles is clearly an injustice, and sidel and levy go so far as to claim that it is morally unacceptable for a physician to serve as both a physician and a soldier in the united states military forces , leaving open the question of who is going to provide medical services in the military . as noted earlier , the civilian bioethical principle that the welfare of the individual patient is the paramount is in conflict with the military principle that the welfare of the individual is subordinate to the welfare of the unit , the success of the mission , and the security of the state . as edmund howe emphasizes , the military physician , at least implicitly , promises to support the mission or greater good when and if this is necessary , even if this requires subordinating the medical well - being of the individual soldier. if military doctors acted according to civilian principles of bioethics when they provided services to their own troops , they might find themselves having to violate the fundamental principle of military bioethics . consequently , the rules governing military physicians can not be the same as those that govern civilians . military physicians are still medical professionals , however , and while this status can not relieve them of their obligations as members of the military , it imposes on them a special obligation to look out for the interests of warfighters for whom they are responsible . due to their medical expertise , they should be in a better position than non - physicians to assess biomedical risks and benefits , and therefore they have a duty to apply their medical expertise to determine if a genomic risk that commanders seek to impose on their subordinates is disproportionate . if physicians disagree with the commanders judgement , they must take the matter up the chain of command until they are satisfied that their professional concerns have received due consideration . as canadian brigadier general and former director of canadian military health services hilary jaeger states , the military physician must act as a counterweight , by being the voice of caution, and should not hesitate to challenge when they are not satisfied. a prime example of the special protective role of military physicians in fact involves a genomic technology , a recombinant - dna - manufactured clotting agent called factor vii . the army introduced the product into clinical practice in iraq in 2004 before full - scale testing , which later revealed both safety problems , such as an increased risk of blot clots that could cause strokes and a lack of efficacy . army physicians in baghdad became concerned in 2006 about the safety and efficacy of the clotting agent and urged their superiors in the medical chain of command to curtail its use . ethical and legal issues raised by military human subjects research on genomic enhancements were discussed earlier in balancing risks and benefits . this section addresses the issues raised by giving genomic enhancements to troops outside of research , such as during training or deployment . genomic enhancement technologies raise some of the same ethical and legal concerns that have been discussed in connection with other genomic technologies , for example , whether warfighters must be asked for informed consent . the question here is whether the fact that the genomic technology is an enhancement rather than medically related calls for different treatment . generally speaking the fact that a genomic test seeks information about a warfighter 's physical or mental abilities rather than their risk for genomic disease , for example , does not alter the ground rules for military genomic testing described in genomic testing , which mandate that any risks from the testing be proportionate to the military 's need for the information and that superiors protect the warfighter 's welfare to the greatest extent possible . the same is true for a genomic intervention such as a recombinant - dna - manufactured drug , an example of which would be erythropoietin , which provides greater endurance by increasing the oxygen supply to tissues , or even a direct genomic manipulation that aims to improve warfighter performance . in all these cases , the same considerations of safety , efficacy , proportionality , paternalism , and fairness obtain . one reason for not making special rules for genomic enhancement is that the distinction between an enhancement and other sorts of genomic technologies is difficult to make . consider a recombinant drug that improves mental ability or endurance . is this an enhancement or a preventive medical measure that helps prevent warfighters from becoming ill or injured ? some commentators argue that an enhancement is something that enables an individual to exceed population norms for the characteristic or ability . thus , a drug to improve cognitive function in persons with below - normal cognitive ability would not be considered an enhancement . , it refers to the frequency with which a trait or capability occurs within a population . in regard to height , the convention is to regard individuals who are more than two standard deviations below the mean height of the population as being of short stature . in other circumstances , what is considered normal may have no relationship to the distribution of a trait . for example , normal eyesight is deemed to be 20/20 , but only about 35 per cent of adults have 20/20 vision without some form of correction . standards of normality also may vary from place to place and time to time , and can be expected to change as the use of enhancements increases . body shapes that were considered healthy a 100 years ago , for instance , are now considered obese . furthermore , the concepts of disease and disorder themselves may be hard to pin down . before 1973 , the american psychiatric association regarded homosexuality as a mental disorder . moreover , there is a tendency to regard more and more health states as diseases and more and more interventions as treatments . instead of attempting to make a bright - line distinction between enhancements and other techniques , the emphasis should be on the effect of the intervention on the well - being of the warfighters who receive the intervention , the welfare of their units , and the completion of the mission . are there certain types of biomedical enhancements that should be out of bounds in the military ? arguably enhancements should not compromise warfighter dignity by producing socially stigmatizing or disfiguring physical characteristics . in the debate about the ethics and legality of human genomic engineering , one technology that causes particular concern , for example , critics object that even using this technology to combat disease , let alone for enhancement purposes , should be prohibited due to the risk of creating monsters and blurring the line between humans and other animals . on the other hand , an argument can be made that the benefits from giving warfighters an eagle 's daytime vision , an owl 's night vision , a dog 's sense of smell , a gorilla 's strength , or a cheetah 's speed might be so great that they should not be ruled out of the question . from a technical standpoint , however , it may well be necessary to produce such changes at a sufficiently early stage of embryonic development that the alterations showed up in all of the resulting individual 's cells , including their reproductive cells , and therefore would be passed on to their descendants . in short , germ line genomic engineering . this is another genomic approach that meets with strong objections , including that it could cause inadvertent harm to future generations . the question then would be whether the military ought to be allowed to produce modifications that made the offspring of their personnel especially valuable as warfighters if the same types of changes were forbidden in the general population . this scenario is sufficiently far in the future that it does n't have to be resolved at this time , but one conclusion that seems certain even now is that such modifications would be unethical without the warfighter 's informed consent . another ethical issue is what effect genomic enhancement use should have on promotions , commendations , and other rewards within the military . if warfighters are required to take the enhancements , then it does not seem justified to deprive them of resulting service benefits so long as access to the enhancements is fairly distributed . on the other hand , if warfighters use enhancements voluntarily , the situation might be thought to resemble enhancements in sport , where it is argued that abilities or accomplishments attributable in substantial part to enhancements do not deserve reward . as in the case of sports , however , one must inquire why from an ethical standpoint biomedical enhancements should be treated differently than permissible performance - improving practices such as dietary modification and extreme training . moreover , unlike in sport , where the use of enhancements may confer benefits on individual athletes and teams but produce little or no broader societal benefit , enhancements in the military could help protect warfighters and their comrades from harm , and aid them in accomplishing missions deemed to be in the national interest . in terms of the impact on military rewards , then , the willingness of warfighters to incur health risks from enhancements in order to benefit others as well as themselves might be deemed to be praiseworthy . an additional concern is the potential for genomic enhancement technology to migrate from the military to the civilian sector . the successful development of enhancements for the military is likely to become publicly known and create pressure to make them available to civilians , including from entities that have a proprietary interest in the technologies . warfighters also might illegally distribute enhancements that they were given for military use to family members and other civilians . just as the military legitimately prohibits public access to properly classified information and dangerous weaponry , it would be appropriate to prevent the public from gaining access to military enhancements that were overly dangerous or that were so effective that it would threaten national security if they became available to adversaries . a final question is whether warfighters should be allowed to benefit from being enhanced after they are discharged from the military or retire . the ability to do so , for example , might be used as a recruiting incentive , similar to being taught technical skills . a fairness issue would arise , however , if former warfighters continued to enjoy advantages from enhancements that were not readily available to civilians because they were illegal or too expensive . on the one hand , continued advantage might seem unfair to civilians who were not eligible for military service . on the other hand , enhancement advantages could be viewed as a legitimate part of warfighters compensation package , especially if they were in combat or had been given other especially risky assignments . similar considerations arise in connection with other veterans benefits such as government - subsidized college education , job preferences , and access to va care ; in all cases , the public must decide whether the benefits are justified based on factors such as the need for military volunteers and the value of the benefit compared to what is available to civilians . in the case of genomic enhancements , an additional issue is how difficult or dangerous it would be to remove or cancel the effect of the warfighter 's enhancement . although genomic technologies could produce significant benefits for the military and its individual members , they must be subject to appropriate ethical and legal constraints . the bioethical principles that underlie these constraints differ between military and civilian contexts , and therefore the rules that govern the military use of genomic technologies also must be different . given the exigencies of military operations and national security , the military in many respects deserves to have greater freedom to conduct research using military subjects , obtain genomic information from military personnel , create and establish military geno phenobanks , and provide genomic therapies and enhancements to warfighters than would be ethically acceptable in civilian biomedical contexts . nevertheless , warfighters deserve to be respected and to have their welfare maximized as much as possible under the circumstances . the essence of military bioethics is the duty of superiors to protect their subordinate 's welfare and impose risks on them only to the extent that the risk is appropriate in light of the military benefits to be gained . given the scientific uncertainty surrounding many aspects of genomics , public sensitivity to genomic misconduct , and the importance of maintaining an effective voluntary military force , adherence to these principles in the realm of genomics is especially critical .

advances in genomic science are attracting the interest of the u.s . military for their potential to improve medical care for members of the military and to aid in military recruitment , training , specialization , and mission accomplishment . while researchers have explored the ethical , legal , and social issues raised by the use of genomic science in a wide variety of contexts , there has been virtually no examination of these issues in connection with the use of genomics by the military . this article identifies potential uses of genomic science by the military , proposes an applicable ethical and legal framework , and applies the framework to provide ethical and legal guidance for military decision - makers .

USES OF GENOMIC TECHNOLOGY BY THE MILITARY LIMITATIONS ON THE USE OF CIVILIAN APPROACHES IN THE MILITARY RESEARCH ISSUES GENOMIC TESTING DEPLOYMENT USE OF UNPROVEN GENOMIC TECHNOLOGIES THE ROLE OF MILITARY PHYSICIANS GENOMIC ENHANCEMENT CONCLUSION

PMC2703923

the combination of structural genomics efforts and computational modeling has resulted in a large amount of 3d structure information for proteins . however , to a large degree , this structural information has not been translated into functional information . for example , understanding substrate specificity , catalysis or inhibition , is still largely dependent on biochemical and biophysical analysis of individual proteins . while protein structure in principle encodes this mechanistic information , reliable computational tools and approaches to establish a connection between structure and function are still lacking . the molecular function of proteins is largely determined by their interaction with other molecules at binding sites on the protein surface . thus , localization and characterization of a ligand binding site can contribute to functional annotation of a protein ; it can guide mutational experiments , and be useful in predicting or verifying interactions . the identification of ligand binding sites can also be an important part of the drug discovery process . knowing the location of binding sites facilitates virtual screening for hits , lead optimization and identification of features that influence the selectivity of binding . hence , several methods have been developed for the identification of binding sites from protein structures ( 16 ) and sequences ( 710 ) . the structure - based methods recognize geometrical features , such as clefts , or energetic features that describe the molecular interaction properties of the protein surface . in general , structure - based methods can be seen as complementary to sequence - based methods that exploit evolutionary information . here , we describe the sitehound - web server for identification of ligand binding sites in protein structures . it uses an energy - based approach to identify regions with high potential for interaction with ligands . a unique feature of sitehound - web is that it implements the use of different probes to characterize a protein structure , which enables not only the identification of different types of binding sites , but also a preliminary description of its interaction properties . the sitehound algorithm identifies potential ligand binding sites by recognizing regions characterized by favorable non - bonded interactions with a chemical probe ( 6 ) . depending on the nature of the probe , different types of binding sites currently , a carbon probe and a phosphate probe are available for the identification of binding sites for drug - like molecules , and ligands containing phosphate groups , respectively . affinity maps ( also called molecular interaction fields ) describing the interaction of the probe and the protein on a regular 3d lattice are calculated using either the autogrid program ( 11 ) for the carbon probe , or the easymifs program ( d. ghersi and r. sanchez , manuscript submitted for publication ) for the phosphate probe . the remaining points are clustered according to their spatial proximity using an agglomerative hierarchical clustering algorithm . the final output is a list of interaction energy clusters corresponding to putative binding sites , which are ranked by total interaction energy ( tie ) ( the sum of the energy values of all the points that belong to the same cluster ) . a test study carried out on 77 experimentally determined protein structures , corresponding to known protein ligand complexes , showed that the correct binding site was among the top three sitehound clusters in 95% of the cases ( 6 ) . sitehound - web ( http://sitehound.sanchezlab.org ) was implemented using a python - cgi and javascript based platform . wrappers integrate programs modeller ( 12 ) , autogrid ( 11 ) , easymifs ( d. ghersi and r. sanchez , manuscript submitted for publication ) , and sitehound ( 6 ) , resulting in a completely automated identification of ligand binding sites from a standard pdb file . the processed pdb file is then passed to either autogrid or easymifs , depending on the user - selected probe . the output is displayed using html pages including an interactive 3d representation of the protein structure and the putative binding sites using the jmol java applet ( http://www.jmol.org ) . sitehound - web requires a pdb file as input and the specification of a probe and clustering algorithm for the calculation . the input pdb file can either be uploaded or a pdb code can be specified . when specifying a pdb code the corresponding file is copied from the pdb database . the pdb file does not need to be preprocessed ( e.g. removal of ligands ) since the server does this automatically . two types of probes are currently available : a carbon probe for the identification of binding sites for molecules that interact mainly through van der waals contacts ; and a phosphate probe which is used to identify sites that bind to phosphorylated ligands . the carbon probe has been validated mainly with drug - like molecules ( 6 ) and the phosphate probe with phosphopeptides , phosphosugars , and atp ( d. ghersi and r. sanchez , manuscript in preparation ) . the clustering algorithm determines the way in which sitehound combines individual affinity map points into clusters corresponding to putative binding sites . the average - linkage clustering tends to result in relatively spherical clusters and is the default for both probes . while only the use of average - linkage clustering has been tested extensively in sitehound , the single - linkage clustering algorithm is provided as an alternative to be used with the carbon probe for the identification of larger elongated binding sites , like those of peptides . once a request has been submitted , the calculation proceeds unless a multiple chain pdb file has been uploaded or selected . in this case , the server will provide the option to select one or more chains from the pdb file to be included in the calculation . after chain selection the calculation proceeds . for a medium - sized protein ( 150 residues ) , a typical calculation takes 1 min . however , running time also depends on the shape of the protein , with elongated proteins taking longer than spherical ones . the output of sitehound - web has two components : an interactive web screen displaying a summary of results with a 3d representation of the putative binding sites on the protein structure ; and downloadable files for offline analysis . a cluster data table ( figure 1a ) displays the top 10 ranking interaction energy clusters ( i.e. putative binding sites ) . this table shows the rank , tie , coordinates , and volume for each cluster . the color of the rank corresponds to the color of the cluster in the 3d display . the tie , which is used to rank the clusters , is an indication of the strength of the clusters . significant clusters usually have ties that stand out against the background of weaker clusters ( see clusters 1 and 2 in figure 1a ; and cluster 1 in figure 2a ) . the cluster coordinates correspond to the x , y and z coordinates of the center of each cluster . this can be used , for example , to set up a docking box centered around a putative binding site ( 6 ) . finally , the volume of the cluster in is displayed in the last column . a 3d interactive view of the protein structure and the clusters ( figure 1b ) is provided using the jmol molecular viewer . this view interacts with the cluster selection panel ( figure 1c ) , which can be used to toggle the display of any of the top 10 clusters on and off . a cluster details panel ( figure 1d ) provides a list of protein residues in the vicinity of a selected cluster . clicking on its corresponding rank in the cluster data table changes the selected cluster . finally , the download data panel ( figure 1e ) provides links to various data files . the cluster data file provides the same information as the cluster data table , but for all identified clusters . the dx file stores cluster data in the dx format which is useful for display in programs such as pymol ( http://www.pymol.org ) and chimera ( 13 ) . the cluster pdb file contains the coordinates of the cluster points in pdb format ; it can be used to display the clusters in most molecular viewers ( figure 3 ) and is the file used internally by sitehound - web to display the clusters using jmol . it can be used with the offline version of sitehound ( d. ghersi and r. sanchez , manuscript submitted for publication ) to explore different parameters for cluster analysis . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown . ( a ) the cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy . the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows . two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest . the coordinates for the center of the cluster and the cluster volume are also displayed . ( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) . cluster selection panel allows toggling the display of individual clusters on or off . by default ( d ) the cluster details panel displays all residues in contact with the cluster selected in the cluster data window . ( e ) the download data panel provides links to various data files for offline analysis ( see text for a description of each file ) . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters . the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) . figure 3.combining sitehound - web outputs to describe the adenylate kinase binding site . ( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) . ( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) . the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions . the figure was prepared using the cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown . ( a ) the cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy . the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows . two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest . the coordinates for the center of the cluster and the cluster volume are also displayed . ( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) . cluster selection panel allows toggling the display of individual clusters on or off . by default ( d ) the cluster details panel displays all residues in contact with the cluster selected in the cluster data window . ( e ) the download data panel provides links to various data files for offline analysis ( see text for a description of each file ) . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and cluster data ( b ) panels are shown . cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters . the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) . ( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) . ( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) . the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions . the figure was prepared using the cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) . the sitehound algorithm identifies potential ligand binding sites by recognizing regions characterized by favorable non - bonded interactions with a chemical probe ( 6 ) . depending on the nature of the probe , different types of binding sites currently , a carbon probe and a phosphate probe are available for the identification of binding sites for drug - like molecules , and ligands containing phosphate groups , respectively . affinity maps ( also called molecular interaction fields ) describing the interaction of the probe and the protein on a regular 3d lattice are calculated using either the autogrid program ( 11 ) for the carbon probe , or the easymifs program ( d. ghersi and r. sanchez , manuscript submitted for publication ) for the phosphate probe . the remaining points are clustered according to their spatial proximity using an agglomerative hierarchical clustering algorithm . the final output is a list of interaction energy clusters corresponding to putative binding sites , which are ranked by total interaction energy ( tie ) ( the sum of the energy values of all the points that belong to the same cluster ) . a test study carried out on 77 experimentally determined protein structures , corresponding to known protein ligand complexes , showed that the correct binding site was among the top three sitehound clusters in 95% of the cases ( 6 ) . sitehound - web ( http://sitehound.sanchezlab.org ) was implemented using a python - cgi and javascript based platform . a series of python wrappers integrate programs modeller ( 12 ) , autogrid ( 11 ) , easymifs ( d. ghersi and r. sanchez , manuscript submitted for publication ) , and sitehound ( 6 ) , resulting in a completely automated identification of ligand binding sites from a standard pdb file . the processed pdb file is then passed to either autogrid or easymifs , depending on the user - selected probe . the resulting affinity map is then passed to sitehound . the output is displayed using html pages including an interactive 3d representation of the protein structure and the putative binding sites using the jmol java applet ( http://www.jmol.org ) . sitehound - web requires a pdb file as input and the specification of a probe and clustering algorithm for the calculation . the input pdb file can either be uploaded or a pdb code can be specified . when specifying a pdb code the corresponding file is copied from the pdb database . the pdb file does not need to be preprocessed ( e.g. removal of ligands ) since the server does this automatically . two types of probes are currently available : a carbon probe for the identification of binding sites for molecules that interact mainly through van der waals contacts ; and a phosphate probe which is used to identify sites that bind to phosphorylated ligands . the carbon probe has been validated mainly with drug - like molecules ( 6 ) and the phosphate probe with phosphopeptides , phosphosugars , and atp ( d. ghersi and r. sanchez , manuscript in preparation ) . the clustering algorithm determines the way in which sitehound combines individual affinity map points into clusters corresponding to putative binding sites . the average - linkage clustering tends to result in relatively spherical clusters and is the default for both probes . while only the use of average - linkage clustering has been tested extensively in sitehound , the single - linkage clustering algorithm is provided as an alternative to be used with the carbon probe for the identification of larger elongated binding sites , like those of peptides . once a request has been submitted , the calculation proceeds unless a multiple chain pdb file has been uploaded or selected . in this case , the server will provide the option to select one or more chains from the pdb file to be included in the calculation . after chain selection the calculation proceeds . for a medium - sized protein ( 150 residues ) , a typical calculation takes 1 min . however , running time also depends on the shape of the protein , with elongated proteins taking longer than spherical ones . the output of sitehound - web has two components : an interactive web screen displaying a summary of results with a 3d representation of the putative binding sites on the protein structure ; and downloadable files for offline analysis . a cluster data table ( figure 1a ) displays the top 10 ranking interaction energy clusters ( i.e. putative binding sites ) . this table shows the rank , tie , coordinates , and volume for each cluster . the color of the rank corresponds to the color of the cluster in the 3d display . the tie , which is used to rank the clusters , is an indication of the strength of the clusters . significant clusters usually have ties that stand out against the background of weaker clusters ( see clusters 1 and 2 in figure 1a ; and cluster 1 in figure 2a ) . the cluster coordinates correspond to the x , y and z coordinates of the center of each cluster . this can be used , for example , to set up a docking box centered around a putative binding site ( 6 ) . finally , the volume of the cluster in is displayed in the last column . a 3d interactive view of the protein structure and the clusters ( figure 1b ) is provided using the jmol molecular viewer . this view interacts with the cluster selection panel ( figure 1c ) , which can be used to toggle the display of any of the top 10 clusters on and off . the coloring of the clusters corresponds to their rank in the cluster data table . a cluster details panel ( figure 1d ) provides a list of protein residues in the vicinity of a selected cluster . clicking on its corresponding rank in the cluster data table changes the selected cluster . finally , the download data panel ( figure 1e ) provides links to various data files . cluster data file provides the same information as the cluster data table , but for all identified clusters . the dx file stores cluster data in the dx format which is useful for display in programs such as pymol ( http://www.pymol.org ) and chimera ( 13 ) . the cluster pdb file contains the coordinates of the cluster points in pdb format ; it can be used to display the clusters in most molecular viewers ( figure 3 ) and is the file used internally by sitehound - web to display the clusters using jmol . it can be used with the offline version of sitehound ( d. ghersi and r. sanchez , manuscript submitted for publication ) to explore different parameters for cluster analysis . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown . ( a ) the cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy . the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows . two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest . the coordinates for the center of the cluster and the cluster volume are also displayed . ( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) . cluster selection panel allows toggling the display of individual clusters on or off . by default ( d ) the cluster details panel displays all residues in contact with the cluster selected in the cluster data window . data panel provides links to various data files for offline analysis ( see text for a description of each file ) . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and cluster data ( b ) panels are shown . cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters . the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) . figure 3.combining sitehound - web outputs to describe the adenylate kinase binding site . ( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) . ( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) . the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions . the figure was prepared using the cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown . ( a ) the cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy . the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows . two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest . the coordinates for the center of the cluster and the cluster volume are also displayed . ( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) . cluster selection panel allows toggling the display of individual clusters on or off . by default ( d ) the cluster details panel displays all residues in contact with the cluster selected in the cluster data window . ( e ) the download data panel provides links to various data files for offline analysis ( see text for a description of each file ) . the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and cluster data ( b ) panels are shown . cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters . the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) . combining sitehound - web outputs to describe the adenylate kinase binding site . ( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) . ( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) . the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions . the figure was prepared using the cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) . ligand binding site identification is an important tool in structural biology because it can bridge the structure - function gap in a homology - independent way . sitehound - web is a ligand binding site identification server that can provide information about the location and binding preference of sites in protein structures . it has a simple interface that only requires the user to select a protein structure and two options ( probe and clustering algorithm ) . a unique feature of sitehound - web is its ability to identify different types of binding sites depending on the probe used for calculation . future development of sitehound will include the addition of more probes for characterization of a more diverse set of sites . because the method requires only the structure of the protein as input it can be used to complement sequence - based methods for identification of functionally important residues , which rely on evolutionary information . we expect sitehound - web to be especially useful in the context of structural annotation , and docking applications in which the binding site is unknown . while binding site identification methods can help in locating and characterizing the regions of the protein to which a ligand may bind , they can not guarantee that a given site will or will not bind a ligand . this is a problem that is better addressed by techniques such as virtual screening that can be carried out on the putative binding sites . national science foundation ( mcb 0517352 ) ; the national institutes of health ( gm081713 ) . funding for open access charge : national institutes of health ( gm081713 ) .

sitehound - web ( http://sitehound.sanchezlab.org ) is a binding - site identification server powered by the sitehound program . given a protein structure in pdb format sitehound - web will identify regions of the protein characterized by favorable interactions with a probe molecule . these regions correspond to putative ligand binding sites . depending on the probe used in the calculation , sites with preference for different ligands will be identified . currently , a carbon probe for identification of binding sites for drug - like molecules , and a phosphate probe for phosphorylated ligands ( atp , phoshopeptides , etc . ) have been implemented . sitehound - web will display the results in html pages including an interactive 3d representation of the protein structure and the putative sites using the jmol java applet . various downloadable data files are also provided for offline data analysis .

INTRODUCTION METHODS The SITEHOUND algorithm Server implementation SITEHOUND-web input SITEHOUND-web output CONCLUSIONS FUNDING

PMC4755455

it is where life begins , and it never ends . in this story , there once were two sisters who were happy and healthy and enjoying life together ( figure 1 ) . but then , all of a sudden , my sister was losing weight unexpectedly , and her weight was close to 90 lb . i remember my mother looking up her symptoms in her medical books and saying , i remember my sister saying , no , i do nt . the author ( left ) and her sister as children ( a ) , and the author ( right ) and her sister today ( b ) . there was no self - monitoring of blood glucose ( smbg ) at that time , and diets were strict . my sister was taught to weigh and measure her food and to follow the ada exchange diet the gold standard at the time . i remember being so impressed with how well she followed her diet in the beginning , and i felt so relieved . i thought to myself , she will be okay ; she knows what to do , and she can take care of herself . but i quickly learned that this was easier said than done . i noticed that , after the first 6 months of amazing diligence , my sister seemed weary of the process and was having challenges with high blood glucose . she kindly told me that it was her diabetes and that she just wanted me to be her sister and not get involved . for many years , i watched from the sidelines , hoping and looking for an opportunity to help . it is no surprise that i delved into the area of nutrition and diabetes and became very passionate about helping people with diabetes . as i counseled patients with diabetes , it was so very clear to me that the diet was the hardest part of the diabetes self - care regimen . one day , knowing that my sister still might not be open to discussing her own challenges with me , i asked her for help in my practice . i said that i wanted to know how i could help my patients more , and i thought that she could give me some guidance . i wanted to understand why it was that she did so well in the beginning with following the diabetes care plan , but then seemed to drop it all together . i was given a list of all the things i could nt eat and was told to weigh and measure all my foods . after 6 months of carefully weighing all my food portions , i decided that i could not live the rest of my life eating two - thirds of an apple for lunch . i decided i would just do the best i could to try to eat healthy and exercise regularly . as her sister , my heart sank . as a dietitian , i thought to myself , it was not doable , and she gave up ! clearly , she felt that , if having diabetes meant doing all of these things to perfection , she would have to risk living with the consequences of high blood glucose and hope for the best . this is my area , and i need to do whatever i can so that this does not happen to one more person with diabetes , at least not on my watch ! i became passionate about making sure that my messages about diet were realistic and doable and tailored for each patient . i wanted to be sure that i focused on realistic , patient - selected goals that instilled the hope that managing diabetes can be doable . , i had the amazing opportunity to get involved in research with the diabetes control and complications trial ( dcct ) , a study designed to compare the effects of conventional diabetes treatment to intensive therapy on the development of long - term complications in people with type 1 diabetes . the goals of intensive therapy were to maintain near - normal blood glucose levels and aim for an a1c 6.5% ( 1 ) . in 1983 , we had the advantages of smbg technology , multiple daily injection regimens , insulin pumps , and a1c testing to help tailor our treatment approaches , allowing us to focus on what really worked . here was my opportunity to contribute to making a difference in the lives of people with type 1 diabetes , particularly in the area of diet . we had an education checklist for dcct participants that outlined all of the diet - related topics we would review so that they could better manage their diabetes . i remember sitting at a meeting with my colleague , beverly halford , discussing how many different topics there were and how much information these patients needed to learn and process and how that could be overwhelming . at the same time , as a study group , we soon realized that the goal of intensifying insulin therapy to achieve a1c targets without hypoglycemia and weight gain could not be accomplished without attention to diet ( 1,2 ) . if only we knew which aspects of diet were most important in achieving better blood glucose control , then we could help patients prioritize which diet behaviors were most important to focus on . that is , maybe we could make the diet component more doable by focusing on the need to know versus the nice to know . we decided to conduct an ancillary study ( 3 ) to try to determine which diet behaviors were associated with better a1c outcomes . to do so , we asked 623 intensively treated patients to complete a survey about the methods they used in implementing dietary advice , adhering to meal plans , managing changes in food intake , treating hypoglycemia , limiting consumption of concentrated sweets , and timing insulin in relation to meals and snacking habits . we learned that , in the context of intensive therapy , there were six diet behaviors that were associated with lower a1c values ( table 1 ) ( 3 ) . the information learned through this study was used from that point forward by dcct dietitians to help study participants get closer to their a1c targets so that , as a research group , we could achieve the clinically meaningful separations in glycemic control that we needed to study the course of diabetes control and complications . dietary behaviors associated with lower a1c in the dcct ( 3 ) we now know , based on the dcct results , that every 10% reduction in a1c results in a 43% risk reduction in retinopathy and that the legacy of the dcct s intensive diabetes therapy regimen continues its imprint on diabetes - related health outcomes 30 years later ( 4,5 ) . i now know as a dietitian and diabetes educator how to help patients with type 1 diabetes separate the wheat from the chaff by focusing on the need to know instead of the nice to know . everybody wants to prevent diabetes . in 1996 , our research team became involved in the diabetes prevention program ( dpp ) . this randomized , controlled trial was exciting because it was designed to compare a lifestyle intervention aimed at 7% weight loss and increased activity to a diabetes medication ( metformin ) and to placebo with regard to effects on diabetes incidence in individuals with impaired glucose tolerance ( 6 ) . as a dpp co - investigator and lifestyle coach , i was involved nationally in helping design the lifestyle intervention . as we screened study participants , i remember wondering whether people who did not yet have a disease would be motivated to engage in a treatment to prevent it . we soon found out that , when people learned that they were at risk to develop diabetes , they were highly motivated to prevent it . the story of tim , one of my dpp participants , tells all . at 47 years of age tim s doctor told him he was highly likely to develop diabetes within 5 years of his twin brother because they had the same genes . he decided to join the dpp because he wanted to do everything he could to prevent diabetes . when tim joined the dpp , he was 510 tall and weighed 200 lb ; his bmi was 27 kg / m . he was excited to learn that he was randomly assigned to the lifestyle intervention program . he learned that his lifestyle intervention goals would be to lose 7% of his body weight to a target weight of 186 lb and to increase his activity level to 150 min / week . he rated his motivation to change his lifestyle at a 9 on a 10-point scale , and his self - confidence to do so as 5 out of 10 because he had never been on a weight loss program before . tim s wife attended almost all of his individual coaching sessions to learn how she could support his efforts . based on tim s initial weight , he was assigned a fat intake goal of 42 g / day based on his calorie goal of 1,500 kcal / day . at first , tim and his wife found keeping food records and learning about the sources of extra fat and calories in their diet to be time - consuming . however , with practice , feedback , and support , tim found that he could change his eating habits and lose weight while still enjoying his favorite foods . over time , he found that the process became easier , and the lifestyle changes became his new eating routine . after 6 months in the dpp , tim had lost 16 lb , to a weight of 184 lb , and he was exercising for 200400 min / week . through his participation in the dpp , tim now weighed 50 lb less than his twin brother ( figure 2 ) . for the remainder of the program , he continued to have individual follow - up visits every 46 weeks , and at the end of the dpp , he had maintained his weight of 184 lb . when he was debriefed on his results at the end of the dpp , he learned that , after 4 years of lifestyle intervention , he had reverted to normal glucose tolerance by both fasting and 2-hour postprandial blood glucose criteria at that time ( 7 ) . tim and the other dpp participants learned that , compared to placebo , metformin can reduce the risk of developing diabetes by 31% , and lifestyle intervention aimed at 7% weight loss and 150 min / week of activity can reduce that risk by 58% ( 6 ) . the effectiveness of a modest lifestyle change was approximately double that of the medication intervention . tim , at age 64 , had delayed diabetes for 17 years beyond his identical twin brother . his most recent weight was 192 lb ( 4% weight loss from his dpp baseline ) , and he is exercising 300 min / week , doubling the original dpp exercise goal of 150 min / week . tim s story tells us that modest lifestyle change seems to trump the expression of high - risk genes . lifestyle intervention participants who had the transcription factor gene tcf7l2 , which is associated with susceptibility to type 2 diabetes due to impaired -cell function , did not develop diabetes at the same rates as participants in the metformin or placebo groups with the same high - risk gene ( 8) . the same findings were replicated when those with the highest genetic risk score who received lifestyle intervention were found to have significantly lower diabetes incidence rates than their counterparts in the placebo group ( 9 ) . tim s story and these research results inspire me to continue to help patients with lifestyle change . i can provide a message of hope based on the evidence that preventing or delaying diabetes is doable with modest lifestyle change . i can encourage them by telling them that they are not doomed to develop diabetes if they have high - risk genes in their family history ; losing 810 lb could make a big difference , and every pound they lose matters . that is why one size does not fit all and no one diet is right for everyone . early in the dpp , i was excited about the potential of weight loss and increased activity to prevent diabetes . in my clinical practice , i had already witnessed the power of small weight losses and modest changes in activity to dramatically improve blood glucose control in my patients who already had type 2 diabetes . what if the lifestyle program works to prevent diabetes ? what will we need to know ? knowing that there are so many nutrition , activity , and behavioral topics to review with patients to help them lose weight , i asked myself , is there a way to make this more doable ? can we help patients prioritize their focus on the behaviors that are most important in predicting sustainable weight loss ? how can we discern the need to know from the nice to know? i decided to conduct another ancillary study to try to identify the most important modifiable predictors of achieving the activity and weight loss goals in both the short term and long term . at the end of the dpp , we learned that weight loss was the dominant predictor of diabetes prevention and that , for every kilogram of weight loss , there was a 16% reduction in risk for developing diabetes . we also know that lifestyle participants who did not meet the weight loss goal at year 1 , but who achieved the physical activity goal of 150 min / week of brisk activity , had a 44% lower incidence of diabetes ( 10 ) . although the lifestyle intervention was successful , not everyone was able to achieve the weight loss targets . it turned out that 49% of lifestyle participants achieved the 7% weight loss goal at 6 months , and 37% maintained the 7% weight loss at study end ( 11 ) . in our ancillary study , 274 lifestyle participants ( 25% of the cohort ) completed questionnaires to assess their weight loss history and a variety of psychological and behavioral factors . the questionnaires were administered at baseline and at 6 months after completion of the 16-session core lifestyle curriculum . we learned that the most important psychological predictor of weight loss was low - fat diet self - efficacy self - confidence in their ability to follow a low - fat diet . for each unit improvement in low - fat diet self - efficacy score during the core curriculum , there was an almost threefold greater likelihood of achieving the 7% weight loss goal at study end ( 12 ) . in the dpp , we promoted self - efficacy by teaching participants skills related to setting small achievable goals , self - monitoring and balancing fat intake , stimulus control , managing stress and high - risk situations , and problem solving , which could explain these self - efficacy improvements . we additionally learned that the most important behavioral predictor of weight outcomes was flexible dietary restraint . for each unit increase in dietary restraint skills during the core curriculum , there was a 4.3-fold greater likelihood of achieving the 7% weight loss goal at study end ( 12 ) . teaching patients flexible dietary restraint skills appears to be key . rather than trying to adhere to a specific diet plan , dpp participants had the flexibility to find their own way . participants self - monitored their weight and their eating and could compensate by eating less in response to any small increases in weight or any overeating episodes . they experienced this as a successful balancing of eating behavior rather than as a setback or failure to follow their diet and eat perfectly . as health care providers and diabetes educators , we can focus our energies on teaching dietary restraint skills and on other strategies that promote self - efficacy for both diet and activity behaviors . this evidence reminds me that people with diabetes need to be repeatedly reassured that they do not have to eat and exercise perfectly ; they need some structure , but they also need to learn to balance that structure with some flexibility with themselves by aiming to eat better on more days than not and by fitting activity into their lifestyle in ways that work for them . having the flexibility to find their own way to achieve clinical goals makes lifestyle change more doable and more sustainable . i was fortunate to be involved as a co - investigator and lead interventionist in the look ahead trial , helping to design the lifestyle intervention that was based on the dpp ( 13 ) . look ahead randomly assigned 5,145 patients with type 2 diabetes who were overweight to receive either a lifestyle intervention with goals of 7% weight loss and 175 min / week of activity or a program of diabetes support and education to see whether the lifestyle intervention would reduce cardiovascular morbidity and mortality over the long term ( 14 ) . after a median follow - up of 9.6 years , the lifestyle intervention did not reduce cardiovascular events defined as heart attack , stroke , hospitalization for angina , or death compared with a program of diabetes support and education ( 14 ) . for me as a diabetes educator , however , the clinical implications of the look ahead trial go way beyond the headlines . as we debriefed our lifestyle intervention group participants on the results of the trial , we heard a common refrain that was best articulated by bob , one of our trial participants . do nt lose sight of the impact that this lifestyle program has made on our quality of life . a multitude of published articles speak to the positive impact of the lifestyle intervention , not only on cardiovascular disease risk factors ( i.e. , improvements in a1c , blood pressure , and lipid levels with less medication use and associated costs ) , but also on numerous other conditions ( table 2 ) ( 15 ) . when we examine the list of benefits , it is so clear why the lifestyle participants felt so adamant about how much better they felt , both physically and psychologically . the initial weight losses with this lifestyle intervention are among the best achieved through lifestyle modification . benefits of lifestyle intervention versus diabetes support and education in the look ahead trial ( 15 ) diabetes remission rate body image dissatisfaction number of medications the look ahead lifestyle program , modeled on that of the dpp and including the proactive use of meal replacements , clearly needs to be translated into the clinical practice setting for patients with type 2 diabetes . this goal is particularly important in light of the findings from the shield study ( study to help improve early evaluation and management of risk factors leading to diabetes ) . the shield study , a 5-year , prospective survey of > 22,000 adults with or at risk for type 2 diabetes , had two key findings . first , people know what to do but do not know how to do it ; that is , they know they are supposed to lose weight and increase their activity , but they just do nt have the skills or know how to do it in a sustainable way . second , more effective interventions are needed to help patients improve health - related behaviors ( 16 ) . in 2012 , i received funding to conduct a pilot study adapting the look ahead lifestyle intervention for clinical practice in a program called improving diabetes outcomes through lifestyle change ( idolc ) . idolc compared an adapted look ahead lifestyle intervention to usual care . in this study , we randomly assigned 57 primary care patients with type 2 diabetes to receive either a 19-week group lifestyle program adapted from look ahead or usual care , defined as individual medical nutrition therapy sessions with a dietitian . compared to the look ahead participants , idolc participants were more likely to be male ( 60% in idolc vs. 40% in look ahead ) , slightly older ( 62 vs. 59 years of age ) , and had comparable ethnic representation . at baseline , the idolc intervention cohort was more medically complex and had a higher rate of insulin use ( 70% vs. 15% in look ahead ) and a higher mean a1c ( 8.2% vs. 7.3% in look ahead ) , with comparable initial bmis . our results show that , despite prevalent use of insulin and sulfonylureas , more than twice as many lifestyle group participants met the 5% weight loss target , with one - third losing 10% ( table 3 ) . this was achieved with simultaneous improvements in glycemic control and significant reductions in medication use ( 17 ) . patients reported greater improvements in dietary restraint skills , low - fat diet behaviors , and self - efficacy for following a low - fat diet . weight and diabetes outcomes at 6 months in the idolc trial ( 17 ) the idolc lifestyle intervention program has been well received , and kevin s experience in the program is just one of many positive , heart - warming stories that we have witnessed . he was 61 years old , weighed 276 lb , and participated in no regular physical activity . kevin was on 60 units of glargine insulin and metformin and had an a1c of 7.9% . his blood pressure and lipids were well controlled with medications . after participating in the 19-week program , he was able to discontinue insulin and lisinopril at the end of the intervention and had an a1c of 6.3% . eighteen months later1 year after completion of the intervention he had lost a total of 62 lb . kevin s story emphasizes an important message from the shield study : people know what to do ; they just need to learn how to do it . it s not about willpower , it s about lifestyle skill power , and we can teach skills to help people make sustainable lifestyle changes . twenty - two months after starting the program , kevin is 68 lb lighter , with the abcs of diabetes control ( a1c , blood pressure , and cholesterol ) all still in the target range . his a1c is 5.9% , and he is on a trial off of metformin treatment . based on the success of our pilot study , we have now received 5 years of national institutes of health funding to conduct a larger - scale translation study called real health diabetes ( figure 3 ) . in the real health diabetes study , we will randomly assign 210 community health center patients who have type 2 diabetes to receive one of three treatments : 1 ) dietitian referral for individual medical nutrition therapy , 2 ) a lifestyle intervention delivered via in - person group , or 3 ) a lifestyle intervention delivered via a new approach using a telephone conference call group to find out whether this could be a way to extend the reach of the program . our primary study outcome will be the mean percentage of weight loss at 6 months , 1 year , 2 years ( end of the intervention ) , and finally at 3 years ( after 1 year of no intervention ) to assess sustainability . we will also assess a variety of medical , psychological , and behavioral outcomes and quality of life . after 20 years of work designing and implementing these lifestyle programs , there is one thing i know for sure . lifestyle programs can provide patients with diabetes more of what they truly want and need : better health outcomes , less medication use , better quality of life , the continued ability to enjoy eating , and , most importantly , the know - how to achieve their weight loss and activity goals . so , what does it mean to make a meaningful difference ? for me , as a diabetes educator and dietitian , it is not just about contributing to landmark research studies in the area of diabetes and obesity . it is about creating a can - do feeling and improving the well - being of the patients who are struggling to manage these lifestyle - related conditions . we all know that the twin epidemics of diabetes and obesity are growing exponentially and that there is much more work to do . but each of us can commit to brightening the corner where we are . for me , it is not about what i do , but rather how i do it . patients come to me feeling discouraged about managing their diabetes or their weight , and we talk about the steps that they can take to improve their health . when i see their eyes brighten and their hopes rising , and i hear them say that s doable , my heart skips a beat because i know i have had a meaningful conversation , and that means it has been a successful day . for me , the final lesson and the moral of this story for diabetes educators is that it s not just about what you do , but also how you do it that can make living with diabetes more doable for your patients . our collective story on making a meaningful difference for people with diabetes is still being written .

editor s note : this article is adapted from the address ms . delahanty delivered as the recipient of the american diabetes association s ( ada ) outstanding educator in diabetes award for 2015 . she delivered the address in june 2015 at the association s 75th scientific sessions in boston , mass . a webcast of this speech is available for viewing at the ada website ( http://professional.diabetes.org/webcasts ) .

When Diabetes Hits Home: The Power of Love Diabetes Control and Complications Trial: Need to Know Versus Nice to Know Diabetes Prevention Program: The Power of Lifestyle Change Psychological and Behavioral Predictors of Weight Outcomes: The Importance of Balancing Structure and Flexibility Look AHEAD Trial: The Importance of Looking Beyond the Headlines

PMC4893435

recently , in addition to the high prevalence of infectious diseases , the incidence of noncommunicable disease is alarmingly increasing in the developing countries . although there are some more recent studies indicating the decline of cvd in africa , ncds are generally showing rapid rise in these countries . one of the explanations for the increase is the economic and social transformation taking place in these countries , and the lifestyle changes resulted from such transformation . hypertension is one of the noncommunicable diseases that is advancing at fast pace in african countries . the trend is unexpected given the socioeconomic status of people in these countries , as the disease is believed to be disease of the rich and developed nations . besides being a major cause of death , hypertension is a major risk factor for many chronic diseases like coronary heart disease , stroke , heart failure , kidney disease , and others . it is responsible for about 6% ( 9 million ) of deaths worldwide and affects about one billion people globally . its global prevalence among older adults aged 25 and over is around 40% in 2008 . the highest of this effect falls on the people in middle and low income countries . low income countries like sub - saharan africa are experiencing unexpected rise in the incidence of hypertension . the few studies conducted in ethiopia are also showing high prevalence of the disease in the country . according to one study another more recent study conducted in the capital shows that approximately 30% of adults in addis ababa have hypertension . added to the silent nature of the disease and the poor health seeking behavior of the people , as the country is going through a rapid economic development , and social and behavioral change , the figure is estimated to be higher . income , education , and occupation are the most commonly used indicators or measures of the socioeconomic status of an individual . although its measurement is difficult in the developing countries , household income has shown consistent association to the general measures of health . educational status is also widely used as a measure of socioeconomic status and is related to many health outcomes . educational attainment reflects a household 's ability to avoid risky behaviors and practice good health . it reflects health risks and protection factors related to the occupation and provision of source of income to practice good health behavior . there are significant evidences indicating an inverse association between socioeconomic status and hypertension in the developed countries [ 1214 ] . this is because in the developed countries individuals with high socioeconomic status may be the early adopters of healthy lifestyles that help to lower the risk of hypertension . but , in the developing countries , these groups are early adopters of harmful lifestyles characterized by smoking , diets with high energy and fats , and sedentary lifestyles . the inverse association of socioeconomic status with hypertension is not evident in the developing countries . the results from the few studies conducted in the developing countries have shown inconsistent results . for instance a study from nigeria among adults aged 3060 years found an inverse association between socioeconomic status and hypertension . in contrary to this finding , a study on civil servants in the same nation found that those at higher occupational levels had higher hypertension when compared with those at lower occupational levels . another cross sectional home to home study conducted in urban population in dar es salaam , tanzania , aged 25 to 64 came up with a result similar to that of the developed countries . however , the use of different measurement of socioeconomic status and the stages of their economic development in these countries have made comparisons across other literatures difficult . the results from many middle income countries are also showing positive association between socioeconomic status and hypertension . a study in rural indian adults shows the highest socioeconomic group had almost double the prevalence of hypertension as those from the lowest socioeconomic group . another study within the same country has also showed a significantly positive relationship between socioeconomic status and hypertension . in study undertaken among jamaican women , there are significant study results indicating the high prevalence of hypertension among the bank workers [ 19 , 20 ] . in contrast some studies undertaken in africa are showing the prevalence of hypertension being higher among teachers compared to the bankers . in ethiopia little therefore the current study aims to examine the association between socioeconomic status and hypertension among the bankers and teachers in the capital , addis ababa . this study is based on a cross sectional study conducted for the purpose of assessing the prevalence of noncommunicable diseases among working adults in addis ababa , ethiopia . the study was conducted by addis continental institute of public health in collaboration with university of washington multidisciplinary international research training program . study populations are permanent employees of the commercial bank of ethiopia and teachers in government schools in addis ababa . first two study sites , the commercial bank of ethiopia and public schools , were purposefully selected based on their work force stability . then probability proportional to size sampling procedures was used to select both commercial bank of ethiopia branch offices and government schools . from each of the selected locations , all employees were invited to participate . more details of the methodology are described elsewhere [ 21 , 22 ] . the study was conducted in accordance with the who 's step wise approach for noncommunicable disease surveillance in the developing countries [ 21 , 23 ] . it is often measured as a combination of education , income , and occupation [ 10 , 12 ] . the responses were grouped into two wider categories : diploma and below and degree and above . three income categories are identified based on the 50% median income classification and used for analysis [ 24 , 25 ] it was measured using a digital measuring device ( microlife bp a50 , microlife ag , switzerland ) with the participant sitting after resting for at least 5 minutes . blood pressure was measured 3 times , with at least 3 minutes between consecutive measurements . in accordance with the who recommendation the mean systolic and diastolic blood pressure from the second and third measurements were considered for analysis . for the purpose of this study people with systolic blood pressure greater than or equal to 140 mmhg , or diastolic pressure greater than or equal to 90 mmhg , or people with normal blood pressure who are taking antihypertensive drug therapy were classified as hypertensive . classification of hypertension was determined according to joint national committee on prevention , detection , evaluation and treatment of high blood pressure ( see table 1 ) . weight and height . participants were weighed using a solar - powered scale with an accuracy of 100 grams . their height was measured using an adjustable wooden measuring board , specifically designed to provide accurate measurements [ 22 , 23 ] . body mass index ( bmi ) was calculated as weight in kilograms divided by the square of height in meters . bmi < 18.5 represents underweight , 18.5 to < 25 stands for normal weight , 25 to < 30 refers to overweight , and obesity is decided when bmi 30 . data completeness and consistency were checked by running frequency on each variable . after excluding participants with missing bp measurement data and pregnant women ( n = 339 ) from the original study sample , frequencies and percentages were used for descriptive statistics to see the distribution of the different variables . bivariate and multivariate logistic regressions were conducted to see the association between the measures of socioeconomic status and hypertension . adjusted odds ratios ( aor ) with 95% confidence interval were reported . as the study has collected data on all the variables related to ncds , for this study participants with no bp measurement data ( n = 318 ) and pregnant women ( n = 21 ) were excluded from the original study sample . accordingly a total of 1866 study subjects ( 1124 men and 742 women ) with complete data on all the variables were included in the study . as table 2 indicates , the majority , 1,124 ( 60.2% ) , of the participants were male and 742 ( 39.8% ) were female with the male to female sex ratio of 1.5 : 1 . the mean age of the participants was 36.03 with a standard deviation of 11.91 years . majority of the respondents ( 54.1% ) were in the middle income category earning between 15,000 and 48,000 ethiopian birr per year . slightly more than half ( 51.9% ) of the participants had first degree and above and the other half ( 48.1% ) are diploma and below , making educational status normally distributed ( table 3 ) . as table 3 indicates , the majority ( 55.3% ) of the participants had a normal bmi , while 26.3% were overweight . 83 ( 4.4 ) of the participants are current smokers and 9.1% were previous smokers . the prevalence of hypertension in the study is 21% ( 95% ci = 19.15 , 22.85 ) . 127 ( 6.8% ) of the hypertensive participants are currently receiving bp treatment . only 278 ( 14.9% ) of the participants are aware of their hypertension status before the interview . the results from the bivariate logistic regression indicate that males tend to be more hypertensive than females ( or : 1.43 , 95% ci : 1.131.81 ) ( figure 1 ) . the odds of hypertension increases with increase in age : 3544 ( or : 3.11 , 95% ci : 1.944.99 ) , 4455 ( or : 7.786 , 95% ci : 5.02112.075 ) , and above 50 ( or : 14.23 , 95% ci : 8.7223.23 ) . a higher prevalence of hypertension was observed among middle income groups ( or : 2.26 , 95% ci : 1.663.07 ) ; a statistically significant prevalence is also observed among the higher income groups ( or : 1.75 , 95% ci : 1.212.52 ) . however , as table 4 shows , there was no statically significant difference in prevalence of hypertension among the educational categories ( p value = 0.536 ) and occupation of the participants ( p value = 0.197 ) . this study identified hypertension as a significant health problem among bankers and teachers in addis ababa . these figures are higher compared with the 10.5% prevalence among the general population of ethiopia and lower than the 30% prevalence among adults in addis ababa in previous studies . the prevalence of hypertension in this study is consistent with 19.3% reported in nigeria and 21.8% for uganda . all the significant factors which have proven to be related with the incidence of hypertension were entered into the multivariate regression model . in addition checks were conducted and no multicollinearity was found among the variables used for measuring socioeconomic status . from the socioeconomic status measures used , even after adjustment is made educational status and occupation have shown no significant association with the prevalence of hypertension . the association of income with hypertension in this study is consistent with the findings from many low and middle income countries [ 1 , 12 , 31 ] . this finding indicates that in the context of the developing country having a higher income is not necessarily protective of health as it is in the developed world . this is probably due to the lifestyle of this group of people in these countries . these groups may use this income to provide more resources that may be used mostly for purchasing calorie - dense foods and excessive drinking , and in some instances it is a cause of sedentary lifestyles which are the underlying risk factors of hypertension . among this study population no significant association between education and hypertension was found . the absence of the association in this study may be due to the nature of the study population . in this study however , studies done in the developing countries like tanzania , nigeria , and ghana found out that education is inversely associated with the odds of developing hypertension [ 16 , 31 ] . although there is a lack of studies that compared the two population groups , in studies conducted independently , the odds of developing hypertension is higher among the bank workers [ 19 , 20 ] . in those studies it is explained that bankers ' job is more stressful and sedentary ( table 5 ) . to the best of our knowledge this is the first study that assessed the association between socioeconomic status and hypertension in ethiopia . even though the study has come up with important findings , there are certain limitations worth mentioning here . first of all , the fact that our study participants are all working professionals who are urban residents and fairly well educated makes the generalizing of the result to the larger population difficult . secondly as the study is based on secondary data the problem of social desirability bias is mentioned in the original study . participants may withhold information regarding their life - style habits that may not be generally acceptable for working adults ( smoking , drinking , etc . ) which may result in an underestimation of these behaviors . this study highlights the high prevalence of hypertension in the study population . as in many other developing countries hypertension is becoming a serious public health concern among working adults like teachers and bankers in ethiopia . like many developing and middle income countries , in this study better income is positively associated with higher odds of having hypertension . however , no association between hypertension and measures of socioeconomic status like education and occupation was found in this study . as hypertension is becoming a serious public health concern in ethiopia , it has to be given due concern in the health agenda of the country as one of top priority . it rather influences the practice of risky behavioral factors that are responsible for hypertension . therefore promoting healthy lifestyles and interventions in lifestyle modifications related to the behavioral risk factors awareness creation and promotion of healthy behaviors have to be widely conducted , especially in the better income groups so that they will make rational decision in choosing their behaviors . particularly , the long term consequences that arise from these lifestyles have to be stressed to the society in general and these groups in particular . future studies are also highly recommended to confirm these findings in general population with varying measures of socioeconomic status .

background . the social and economic changes taking place in developing countries are influencing the pace at which hypertension and its risk factors are expanding . as opposed to the already established inverse association in developed nations , the association between socioeconomic status and hypertension in developing countries is poor and inconsistent . this study aims to determine the association between socioeconomic status and hypertension among teachers and bankers in addis ababa , ethiopia . methods . this study is based on a cross - sectional study conducted to assess the prevalence of ncds in addis ababa , ethiopia . the study was undertaken among workers of the commercial bank of ethiopia and teachers of public schools in 2010 . results . majority of participants were teachers ( 70.3% ) . most of the respondents ( 54.1% ) earn an annual income between 15,000 etb and 48,000 etb , and 51.9% of them have educational status of first degree and above . among the socioeconomic factors income was strongly associated with the odds of having hypertension ( aor : 2.17 with 95% ci : 1.582.98 ) . conclusions . higher burden of hypertension is observed among teachers and bankers in addis ababa , ethiopia . promotion of healthy behaviors and interventions that target higher income groups needs to be put in place .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions 6. Recommendation

PMC3249339

a 44-year - old man , who had a medical history of panic disorder , visited another hospital due to dyspnea on mild exertion . a coronary angiography showed a single coronary artery originating from the right coronary ostium . a single coronary artery bifurcated into the right coronary artery and left main coronary artery . the left main coronary artery coursed between the main pulmonary artery and aorta before bifurcating into the left descending artery and circumflex artery . we thought that panic symptoms or dyspnea on exertion might be a sign of myocardial ischemia due to compression of the left main coronary artery by the pulmonary artery and aorta . we decided that surgical treatment was the best option due to the high risk of sudden death associated with a coronary anomaly . under general anesthesia we dissected the left main coronary artery between the aorta and the main pulmonary artery on the beating heart . the proximal left main coronary artery was bifurcated from a single coronary artery that originated from the right coronary sinus . under cardiopulmonary bypass , only one coronary ostium was observed in the right coronary sinus , and a single coronary artery originated from the ostium . a 5-mm arteriotomy was made to the left main coronary artery at the site in which the left coronary ostium should have been located . neo - ostium formation was performed with a 5-mm puncher in the left coronary sinus . anastomosis between the neo - ostium and the left main coronary arteriotomy site was performed using a 7 - 0 prolene continuous running suture ( fig . the aortic cross clamping time was 88 minutes , and total cardiopulmonary bypass time was 117 minutes . follow - up computed tomographic angiography before discharge showed good patency of the neo - ostium in the left coronary sinus without stenosis at the anastomosis site ( fig . the patient remained asymptomatic without any complications or events for 15 months after the surgery . angelini reviewed 1,950 coronary angiographies and reported that the incidence of right coronary artery originating from the left coronary sinus was 0.92% and vice versa was 0.15% , with a total incidence of 1.07% . patients are usually asymptomatic . however , it may cause angina , syncope , and even life - threatening complications such as myocardial infarction or ventricular fibrillation . thus , we must consider surgical treatment or intervention if signs of myocardial ischemia are present . several surgical techniques can be utilized to treat coronary anomalies , such as coronary reimplantation to the original sinus , coronary artery bypass graft ( cabg ) , pulmonary artery translocation , unroofing , and neo - ostium formation . in this case , the left main coronary artery was bifurcated from a single coronary artery originating in the right coronary sinus . furthermore , the left main coronary artery passed between the pulmonary artery and aorta to reach the left heart . cabg , coronary reimplantation , unroofing or neo - ostium formation could therefore have been considered as viable surgical options . coronary reimplantation is one of the most physiologically beneficial repairs , but is technically difficult , and stenosis may occur at the site of anastomosis . cabg is technically feasible , but the arterial conduit has a competitive flow problem if no stenotic lesions are present on the natural coronary artery . also , a vein conduit may be problematic if the patient is young because of long - term patency . however , extended unroofing may cause valve insufficiency if the anomalous coronary artery is located under the valve commissure . unroofing was not proper for our case because a separated left main coronary artery originated from a single coronary artery , not the right coronary sinus . neo - ostium formation in the left coronary sinus without unroofing was considered to be a proper surgical treatment in this case . successful surgical treatment of anomalous of coronary anomaly depends on expertise in anatomic and hemodynamic pathophysiology , in addition to the selection of the appropriate surgical treatment option . we report that this case was successfully treated with neo - ostium formation in anomalous origin of the left coronary artery from the right coronary system .

anomalous origin of a coronary aortic artery is a rare cardiac anomaly . although it can cause angina , syncope , and palpitations , most patients are asymptomatic . this anomaly requires surgical treatment or intervention because it is associated with sudden death . several surgical techniques , such as coronary reimplantation , coronary artery bypass grafting ( cabg ) , unroofing , and neo - ostium formation , have been proposed as treatments . we report a case surgically treated with neo - ostium formation in anomalous origin of the left coronary artery from the right coronary sinus .

CASE REPORT DISCUSSION

PMC3855092

incidental vessel injury caused by some of these variations may require more extensive lung resection than would be necessary without repair during pulmonary resection . we herein describe a patient with a common trunk of the left pulmonary vein that was incidentally transected with a mechanical stapler during a left upper lobectomy . we also describe the reconstruction procedures that were successfully used for the concomitantly transected inferior pulmonary vein . a 62-year - old man complained of cough and bloody sputum and consulted our hospital . chest computed tomography ( ct ) showed a nodule with a diameter of 15 mm in the left upper lobe . positron emission tomography and brain magnetic resonance imaging revealed a clinical stage of t1n0m0 as categorized by the 7th edition of the union for international cancer control ( uicc ) classification . the common trunk of the left pulmonary vein was misidentified as a superior pulmonary vein until incidental transection with an endostapler during port access surgery . the left upper bronchus and pulmonary arteries to the left upper lobe were transected with endostaplers , and the common trunk of the pulmonary vein was left intact . the left main pulmonary artery was then clamped under intravenous heparin injection to achieve an activated coagulation time of 200 s. annuloplasty of the inferior pulmonary vein was completed during intermittent declamping of the left pulmonary artery . the orifice of the inferior pulmonary vein was augmented by a cuff technique using an orifice of the superior pulmonary vein , as shown in fig . 1 . the staples on the stump of the left common pulmonary vein on the atrial side were removed to adjust the augmented orifice of the left inferior pulmonary vein under partial clamping of the left atrium following pericardiectomy . end - to - end anastomosis was completed using running sutures with a 4 - 0 polypropylene thread . the postoperative course was uneventful , and the patient was discharged on postoperative day 14 . a pathologic study showed stage t1n2m0 small - cell carcinoma as categorized by the 7th edition of the uicc classification . postoperative enhanced ct demonstrated successful reconstruction of the left common trunk ( fig . 2 ) . 1.orifice of an inferior pulmonary vein was augmented with a cuff technique using the orifice of a superior pulmonary vein . the augmented orifice was anastomosed to the common pulmonary vein on the left atrial side after removal of staples as long as necessary under a partial clamp of the left atrium following pericardiotomy . fig . 2.postoperative enhanced ct showing ( frontal view ) successful reconstruction of an inferior pulmonary vein ( arrow ) . orifice of an inferior pulmonary vein was augmented with a cuff technique using the orifice of a superior pulmonary vein . the augmented orifice was anastomosed to the common pulmonary vein on the left atrial side after removal of staples as long as necessary under a partial clamp of the left atrium following pericardiotomy . postoperative enhanced ct showing ( frontal view ) successful reconstruction of an inferior pulmonary vein ( arrow ) . the branching pattern of the pulmonary vessels is variable . pulmonary vein variations such as upper lobe venous drainage posterior to the intermediate bronchus and a common trunk of the left pulmonary vein may cause lethal complications during and after anatomical pulmonary resection . based on ct findings , a common trunk of the left pulmonary vein reportedly occurred with a frequency of 14% among 201 cases . although three - dimensional ct and multidetector ct have been developed to detect pulmonary vessel variations preoperatively , incidental transection of the common trunk may be unavoidable when the pulmonary vein is anteriorly transected with no identification of the inferior vein during left upper lobectomy . furthermore , the use of video - assisted surgery and endoscopic devices has become more widespread , and skin incisions and exposure of anatomical structures have become more limited . a previous paper reported that the orifice of the inferior pulmonary vein was augmented with the use of a pericardial patch followed by anastomosis when the common trunk was incidentally transected . in this report , annuloplasty of the inferior pulmonary vein with a cuff technique using an orifice of the superior pulmonary vein allowed for much easier performance of end - to - end anastomosis , leading to successful reconstruction without a patch or prosthesis . this augmented technique may also be applicable to patients with lung cancer in the right upper lobe when the tumor is invading the right common superior trunk branching pulmonary veins to the upper and middle lobes .

a common trunk of the left pulmonary vein is an anatomical variation in the pulmonary vessels and may be incidentally transected during left upper lobectomy . difficulty in reconstruction of the left inferior vein often requires completion pneumonectomy . we herein describe a patient with lung cancer in the left upper lobe of the lung . his common trunk of the left pulmonary vein was incidentally transected with a mechanical stapler during a thoracoscopic left upper lobectomy . the concomitantly transected inferior pulmonary vein was augmented with a cuff technique using an orifice of the superior vein followed by end - to - end anastomosis . the postoperative course was uneventful . this technique should also be considered in patients with lung cancer when a right upper lobe tumor is invading the right superior trunk branching pulmonary veins to the upper and middle lobes .

INTRODUCTION CASE REPORT DISCUSSION

PMC3665034

in many developed and developing countries , there are many concerns about being overweight and obesity ; these concerns have become a universal and comprehensive problem progressing at different ages.[14 ] according to statistics by world health organization ( who ) , about 10% of 5 - 17-year - old school children are overweight and 3% of them are obese . in us , this number is roughly 33% ( 10% obese ) and , in europe , it is 20% ( 4% obese ) . nowadays , obesity in childhood is considered as one of the most important health problems in europe , especially in italy , greece , and spain . according to research on obesity by lobstin and ferlot , southern european countries have the most number of overweight children . in a study done at aragon ( spain ) , the body mass index ( bmi ) - old children were overweight , while it was comparatively less in 13 - 14 year - old children . moreover , in asian and middle - east countries , the matter of overweight and obese children turned out to be a universal problem so that , in the past decade , a significant and major increase in the number of overweight children has been reported.[813 ] also , in iran , as a developing country facing increasing phenomenon of industrialization and urban living , the amount of prevailing overweight children has been more than the expected percentage in recent years and , as people are aging , this percentage is increasing . as such , prevailing rate of 17% epidemic of overweight and obesity at the beginning of primary school has made iran correspondent with the big cities in europe . among the reasons of rapid overweight growth is the nutrient transition that iran experienced from 1990s onward and then it confronted an increased amount of received energy and decreased physical activity , leading to overweight during recent years . in a study by tilaki et al . , in 2011 , the amount of overweight and obesity epidemic among 7 - 12-year - old children was reported , respectively , as 12.3% and 5.8% and , in another survey done at female primary school in tehran , this amounts were reported , respectively , as 13.2% and 7.7% . in addition to the annually increasing costs imposed on world health organizations by overweight and obesity , their relationship with mortality is fairly proved and is an issue that has been evaluated and revisited many times , which in long term can affect children 's health,[1921 ] and it is more probable that the adolescents afflicted by weight issues and obesity may be obese adults in future ; however , most of these children may experience negative consequences of obesity and being overweight in next decades . nevertheless , obesity affects musculoskeletal system including lower limbs ' misalignment , fracture risk , and mobility weakness , leading to appearance of some health problems for infants and adolescents . in comparison to other children of same age , obese children are less willing to do activities.[2628 ] also , the results of a previous reports shows that obese children have problems in gait , running , and flexibility , leading to appearance of some changes in their foot structure . overweight children may suffer a flat foot probably due to increase of fat layer amount in their mid - foot . moreover , by aging , stretching power of plantar increases and flat foot problem is diminished automatically . on the other hand , if being overweight lasts for a medium or long term , the weight gaining process overcomes the stretching power of plantar and foot flatness forms at mid - foot . executed studies show physical changes in children due to obesity as it could reduce their balancing ability . berrigan et al . , in 2006 demonstrated that body fat mass increase in adults causes decrease of their postural stability . in 2003 utilized romberg 's test in order to control posture in 13 - 17-year - old adolescents and concluded that control posture of these children is weaker than in other same age adolescents . according to the related literature , being overweight has mal - effects on children 's physical features , so it seems that also balance , as one of the most important factors of physical fitness , is affected by these features . however , since musculoskeletal system is a consolidated and interrelated collection , disorder in one of its parts affects the other parts too . hence , the researchers in this study attempted to determine if being overweight affects balance and footprint parameters . we selected 22 participants randomly and divided them equally in two groups of normal and overweight . , ( 2000 ) table of bmi , which is based on age and sex . physical details of participants in normal and overweight groups at the beginning , we asked parents of the participants to fill in the personal consent questionnaire . they also filled in self - report questionnaires of health that showed no disorder in visual and vestibular balance systems , proprioceptive sensory system , and every kind of injury , especially in the lower limb . in order to record the footprint of participants , we used roll papers of 200 70 cm and gouache color , which were not harmful to their health . after explaining the protocol , in order to record static footprint , we asked participants to stand on a stamp painted with gouache and while they were gazing at the horizon , they should step comfortably by their right foot on the paper and then with their left foot . participants exercised this process several times before the final record of their footprint was done . then , we selected the best footprint after 3 repetitions and scanned it using canon 550 scanner with dpi of 200 . then , we used imagej software ( for 32 bit windows ) to calculate the anterior area , mid area , posterior area , total area , arch index ( ai ) . to find these information to calculate the variables of area , we considered footprint without toes [ figure 1 ] . different sections of footprint for determining area variables instructions for determining the variables using software are given in analogous studies . the footprint was divided in to three sections : anterior , mid , and posterior areas . the total area was determined through sum of the area of anterior , mid , and posterior sections . the ai was determined through dividing the mid area by total area . to test the balance of participants previous studies have reported a good reliability of this test ( icc = 0.85 - 0.91 ) . when we explained the protocol to all participants , we asked them to try the test in three trials . during the test , all participants stayed on the center of the grid with a single leg while they were wearing sport shoes . we asked them to maintain this position and make their contralateral lower limb to touch as far as possible along three lines of posteriolateral , posteriomedial , and anterior . after completion of all three trials in three lines , we determined the average value of each line . to determine the total score of each participant , we divided the average value by the length of participants ' legs , which were from the anterior superior iliac spine to medial malleolus . then , we multiplied the product by 100 in order to convert it into a percent of leg length . smirnov test to make sure about the normal distribution of scores , and one - way analysis of variance ( p 0.05 ) was used to compare the characteristics of footprint and balance of participants with overweight and normal children . the mean and standard deviation of the data of footprint and balance of participants were determined , and the results from descriptive analysis showed that the area of anterior , medial and posterior sections in overweight children was higher than in normal children ( m = 4519.20 , m = 2691.18 , m = 3055.7 , respectively ) . thus , the total area in this group was also higher than in the normal group ( m = 10266.09 ) . the ai was the same in both groups ( normal group : m = 0.2590 ; overweight group : m = 0.2573 ) . however , participants in the normal group had a better status in balance indices of y test in posteriolateral , posteriomedial , and anterior lines ( m = 110.23 , m = 106.7 , m = 99.86 ) and , therefore , their total balance indices were higher too ( m = 105.6 ) . we used one - way analysis of variance to compare footprint characteristics and balance of normal participants and overweight participants . one - way analysis of variance to compare variables in normal and overweight groups ( significant difference p.0.05 ) results from one - way analysis of variance between normal and overweight groups show that there is a significant difference among anterior , posterior , and total areas between overweight and normal participants ( p < 0.05 ) . however , there was no difference in terms of mid area between the two groups ( p > 0.05 ) . in addition , there was significant difference between the total areas of these two groups ( p < 0.05 ) . also , there was no significant difference in ai between them ( p > 0.05 ) . we found a significant difference between the two groups when comparing parameters of y test in anterior line ( p < 0.05 ) . we also found no significant association between weight and balance of the participants [ table 3 ] . the aim of this study was to investigate the effect of being overweight on the balance and footprint parameters of children . based on the findings of this study , there is a significant difference between parameters of footprint , anterior , posterior , and total area of the subjects , while there was no significant difference in the mid area . , who carried out their study with participation of two groups of male and female students aged 11.6 0.5 years with normal and overweight characters ; the findings of this study , taking into consideration the mid area of the subjects ' feet , showed that there was no significant difference between subjects who were in normal and overweight condition , which again approves the findings of nieto et al . , also , there is significant statistical difference in the parameter of total foot area of both groups of subjects ; this finding corresponds to that in , riddiford- harland et al . and nieto et al.[2937 ] in this study , the method of footprint was used to record the trace of foot on the paper but , in previous studies , riddiford- harland et al . and nieto et al . , the methods that were applied to record the trace of foot were pedograph and photopodogram , respectively . results indicated that these methods are more reliable than the method used in the current study . after calculating different segments of the foot with method of footprint , another factor , ai , was calculated by dividing mid area of the foot on total area of the foot , which showed no significant difference between the two groups of ( overweight and normal ) subjects ; this does not agree with results reported by the abovementioned researchers . furthermore , another study by mickal et al . , stated that subjects who are overweight have more noticeable ai than those with normal weight , this difference in result may be because the factor of total area between these two groups showed no significance difference based on the literature of the current study . on the other hand , concerning the mid area of the foot based on the results of balance testing that was carried out in this study , there was no significant difference between both groups of subjects , which is in contrast with the findings of goulding et al . , which included teenage boys aged 10 - 21 years and which reported significant difference between body weight , bmi , and percent of total fat mass with balance examination . teenagers who suffer from obesity have less balance than others with a normal weight . according to these findings , bernard et al . , reported the same result on examining obese subjects aged 13 - 17 years , stating that the existing differences between these two result could have been due to the differences between the age of subjects . , was single - leg stance test on the pedograph that is a static test and can be considered as a laboratory method of balance testing . biodex was used by bernard et al . , to assess and calculate the balance factors , the main reason of difference between the findings of this study with the current one could be due to the difference in the methods applied in these researches as the current study is based on field test method that evaluates dynamic balance of subjects . therefore , study of dynamic balance on the special devices such as pedograph may lead to different results , this attitude is relevant to specialist claim that recording of foot area in dynamic status represents more precisely the features of foot structure . in order to evaluate and define the pathology of foot structure , most of specialist use ai . according to the findings of this study , in static condition , being overweight has no significant effect on ai . further studies need to be done to survey the effect of being overweight on dynamic balance .

background : the present study was done in order to compare balance and footprint parameters in two groups of normal and overweight children.methods:this semi - experimental study included randomly selected 22 male children ( 11 normal and 11 overweight boys ) . to measure the footprint parameters , an ink paper system was used , i.e. , after putting their feet in the ink , the subjects were asked to stand comfortably on paper and their footprints were recorded . then , with the use of imagej software , the areas of anterior , middle , and posterior parts , the total area , and the arch index parameter were calculated . for measuring balance in three posterolateral , posteromedial , and anterior directions as well as the total balance , y - balance test was done . finally , to analyze the data , mean and standard deviation were calculated and anova test was used to compare the parameters.results:data analysis showed a significant difference between normal and overweight subjects in the anterior and posterior areas , whereas , in balance test , only the anterior areas showed significant difference ( p < 0.05).conclusions : it seems that area parameters in these two groups do not have significant difference ; hence , it can not be used as the criteria for analyzing the effects of being overweight on these parameters . in addition , it is probable that , in a dynamic situation , recorded footprints are more valid parameters for analyzing foot structure .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC4594086

since the cases of tertiary syphilis can be seen very rarely in western society today , it is very difficult for us to diagnose as a syphilitic aneurysm preoperatively when we encounter the thoracic aortic aneurysm whose etiology is unknown . major cardiovascular manifestations of tertiary syphilis are asymptomatic aortitis , aortic regurgitation , coronary ostial stenosis and aortic aneurysm . here , we report the patient who had suffered from giant ascending aortic aneurysm with positive syphilitic test , moderate aortic regurgitation and fistula between aorta and superior vena cava ( svc ) . a 52-year - old man had suffered from severe edema of his face and upper extremity . he was diagnosed with svc syndrome and referred to our institution for the treatment of svc syndrome . computed tomography scan revealed giant ascending aortic aneurysm with the dimension of 79 mm in size and svc was compressed severely by ascending aorta ( fig . 1 ) . although we had checked serodiagnostic tests for syphilis , treponema pallidum hemagglutination reaction ( tpha ) and fluorescence test assay absorption ( fta - abs ) , the results of their examination were not revealed preoperatively . we performed surgical repair of the aortic aneurysm via median sternotomy in a usual manner . cardiopulmonary bypass ( cpb ) was established between bilateral vena cava drainage and right femoral artery perfusion . the patient was cooled to the core temperature of 25 c , the aneurysm was opened under deep hypothermic circulatory arrest and selective cerebral perfusion was established . the marked adhesion was detected around distal aortic arch suggesting the inflammatory process around the aorta . the distal anastomosis was performed at 6 cm distal site from left subclavian artery . after the distal anastomosis under open distal technique , three neck vessels were reconstructed individually . when we started systemic rewarming , we detected the bleeding from the hole of the opened aortic wall . the hole was slit shape and 8 1 mm in size . we diagnosed the fistula between ascending aorta and svc , and closed the fistula with 4 - 0 polypropylene in a running fashion . postoperatively , we suspected syphilitic aneurysm because we could find that the preoperative serodiagnostic tests were all positive . although postoperative histological examination of the aneurysmal wall did not show typical syphilitic features , obliterative endarteritis and fibrosis at vasa vasorum , the severely changed tunica media could be detected ( fig . 1preoperative computed tomography showing giant ascending aortic aneurysm with the dimension of 79 mm in size and severely compressed svcfig . 2histological examination . a low - power magnification of tunica media showing medial necrosis with destruction of elastic fibers ( h&e staining , 20 ) . b low - power magnification of adventitia showing no endarteritis obliterans within the adventitia ( elastica masson staining , 10 ) preoperative computed tomography showing giant ascending aortic aneurysm with the dimension of 79 mm in size and severely compressed svc histological examination . a low - power magnification of tunica media showing medial necrosis with destruction of elastic fibers ( h&e staining , 20 ) . b low - power magnification of adventitia showing no endarteritis obliterans within the adventitia ( elastica masson staining , 10 ) a 52-year - old man had suffered from severe edema of his face and upper extremity . he was diagnosed with svc syndrome and referred to our institution for the treatment of svc syndrome . computed tomography scan revealed giant ascending aortic aneurysm with the dimension of 79 mm in size and svc was compressed severely by ascending aorta ( fig . 1 ) . although we had checked serodiagnostic tests for syphilis , treponema pallidum hemagglutination reaction ( tpha ) and fluorescence test assay absorption ( fta - abs ) , the results of their examination were not revealed preoperatively . we performed surgical repair of the aortic aneurysm via median sternotomy in a usual manner . cardiopulmonary bypass ( cpb ) was established between bilateral vena cava drainage and right femoral artery perfusion . the patient was cooled to the core temperature of 25 c , the aneurysm was opened under deep hypothermic circulatory arrest and selective cerebral perfusion was established . the marked adhesion was detected around distal aortic arch suggesting the inflammatory process around the aorta . the distal anastomosis was performed at 6 cm distal site from left subclavian artery . after the distal anastomosis under open distal technique , three neck vessels were reconstructed individually . when we started systemic rewarming , we detected the bleeding from the hole of the opened aortic wall . the hole was slit shape and 8 1 mm in size . we diagnosed the fistula between ascending aorta and svc , and closed the fistula with 4 - 0 polypropylene in a running fashion . postoperatively , we suspected syphilitic aneurysm because we could find that the preoperative serodiagnostic tests were all positive . although postoperative histological examination of the aneurysmal wall did not show typical syphilitic features , obliterative endarteritis and fibrosis at vasa vasorum , the severely changed tunica media could be detected ( fig . 1preoperative computed tomography showing giant ascending aortic aneurysm with the dimension of 79 mm in size and severely compressed svcfig . a low - power magnification of tunica media showing medial necrosis with destruction of elastic fibers ( h&e staining , 20 ) . b low - power magnification of adventitia showing no endarteritis obliterans within the adventitia ( elastica masson staining , 10 ) preoperative computed tomography showing giant ascending aortic aneurysm with the dimension of 79 mm in size and severely compressed svc histological examination . a low - power magnification of tunica media showing medial necrosis with destruction of elastic fibers ( h&e staining , 20 ) . b low - power magnification of adventitia showing no endarteritis obliterans within the adventitia ( elastica masson staining , 10 ) syphilitic aortitis manifests clinically as aortic regurgitation , coronary ostial stenosis and aortic aneurysm more than 1020 years after the primary infection [ 1 , 2 ] . the spirochetal invasion of the aortic adventitia causes an obliterative endarteritis of the vasa vasorum . blood supply to the aortic wall is impaired , which results in weakening of the tunica media and formation of the aneurysm . in our case , we could not detect typical obliterative endarteritis , but severely changed tunica media could be detected . the most frequently involved segment is the ascending aorta , next is the aortic arch and last is the descending aorta . the prognosis for patients with syphilitic aneurysms is extremely poor with the 2-year mortality rate of unrepaired syphilitic aneurysm of 80 % . the majority of death due to aortic aneurysms are caused by rupture [ 4 , 5 ] . since cardiovascular syphilitic infection has disappeared in developed countries since 1990 , we rarely encounter tertiary syphilis today . but we should consider syphilitic aneurysm in the differential diagnosis of thoracic aneurysms especially when the patient does not have manifestations of connective tissue diseases and has no risk factors for atherosclerosis . giant syphilitic aortic aneurysm may compress around structures including the trachea , esophagus , pulmonary artery and svc as in our patient . although the reports of the arterial fistula to pulmonary artery and the svc syndrome can be found , our case of the arterial fistula to svc is first reported case [ 810 ] . we misdiagnosed as the atherosclerotic aneurysm complicated with simple svc syndrome when we checked preoperative - enhanced computed tomography . the most recommended antibiotics is benzylpenicillin potassium ( pcg ) of 1.22.4 million units daily for 3 weeks . although the patient remains asymptomatic 1 and half years later , we should follow the patient carefully for a long time . although syphilitic aortitis has become extremely rare today , syphilis should still be considered as a differential diagnosis of the etiology of aortic aneurysm , especially when the patient does not have manifestations of connective tissue diseases and any risk factors for atherosclerosis .

syphilitic aortitis is usually associated with thoracic aortic saccular aneurysm , aortic regurgitation and coronary ostial stenosis . however , syphilitic aneurysms have rarely been reported today . here , we report a patient with ascending aortic aneurysm with aorta - superior vena cava ( svc ) fistula with positive syphilitic test . a 52-year - old man was admitted to our institution with a giant ascending aortic aneurysm complicated with svc syndrome . computed tomography revealed a giant ascending aneurysm 79 mm in diameter . the result of serodiagnostic tests for syphilis had not been judged yet preoperatively . total arch replacement concomitant with elephant trunk was performed . intraoperatively , we detected the ascending aorta to svc fistula . postoperatively , we suspected the syphilitic aneurysm strongly , because preoperative serodiagnostic test was concluded to be positive . however , histological examination did not show typical syphilitic features . the patient remains asymptomatic 1 year later . although extremely rarely today , syphilitic aneurysm should be still considered in the differential diagnosis of ascending aortic aneurysm .

Introduction Case Discussion Conclusion

PMC3156997

vascular dementia ( vd ) is the third most common cause of dementia ( 8 to 10% ) , following alzheimer 's disease ( ad ) ( 60 to 70% ) and dementia with lewy bodies ( dlb ) ( 10 to 25% ) , but these numbers vary considerably according to the different criteria used for vad 1,2 . subcortical ischemic vascular dementia ( sivd ) is an important subtype of them caused by the cerebral vessels factors ( ischemia , hypoxia , etc . ) which lead to brain tissue damage . it is characterized by lacunar infarcts and deep white matter changes and affects the inner parts of the brain consisting of deep white matter made up of nerve cell fibers . the term the syndromes consist of loss of memory , judgment , reasoning , and changes in mood , behavior and communication abilities 3 . the proportion of vascular dementia ( vad ) attributed to sivd ranges from 36 to 50% , with higher rates in african americans and asian americans 4 than whites 5,6 . for ad patients , brain 's anatomical structures have been analyzed using the vbm ( voxel - based morphometry ) technique recently , demonstrating that the gray matter volume of the hippocampus , parahippocampal gyrus , insula , superior / middle temporal gyrus , thalamus , cingulate gyrus , and superior / inferior parietal lobule is decreased . volumetric analysis of the entorhinal cortex can even distinguish subjects who were destined to develop dementia from normal controls with high accuracy 7 . recently it has been found subcortical vascular dementia ( sivd ) patients has notable cortical thickness and volume alterations in frontal and tempral cortices , and the conversion from mci associated with small - vessel disease ( svmci ) to sivd is associated with cortical atrophy in dorsolateral prefrontal and temporal cortices 8 - 9 . until now alterations of cortical gray matter density in subjects with cognitive impairment have not been reported . the aim of the present study was to prove the possibility of gray matter density detection using fsl - vbm ( http://www.fmrib.ox.ac.uk/fsl/fslvbm ) 10 - 11 , to characterize the cerebral morphology and atrophy patterns of the cortex , and to provide a new method and biomarker for the diagnosis of sivd . nine sivd patients were recruited by physician referral from the department of neurology in southwest hospital , chongqing , china ( table 1 ) . the clinical diagnosis of sivd was made by a senior neurologist according to the criteria suggested by erkinjuntti 12 and dsm - iv ( diagnostic and statistical manual of mental disorders , iv ) . ad patients with subcortical ischemia or mixed dementia who can also meet dsm - iv vad criteria and the patients with large territorial infarction involving cortex or single strategic infarction such as anterior nucleus of thalamus were excluded . all patients have visible white matter hyperintensities ( wmh ) ( grade 3 or more by a reported rating scale 13 ) addressed with t2/flair imaging data in mri scanning . cognitive performance was evaluated through neuropsychological tasks that included : mini mental state examination ( mmse ) , clinical dementia rating ( cdr ) , neuropsychiatric inventory ( npi ) , activities of daily living scale ( adl ) , hachinski and hamilton test . nine age- and sex - matched old people with normal - cognition were recruited from surrounding regions by advertisement . they underwent a comprehensive brain examination to ensure that they had normal brain structure and no neurological lesions . exclusion criteria included patients with aphasia or who otherwise could not complete psychological testing because of a language disorder ; diagnosed chronic or degenerative disease affecting the central nervous system ; active substance abuse disorders ; intracranial hemorrhage ; difficult to control epilepsy that could present with cognitive impairment ; or temporal lobe epilepsy . all of those subjects who with brain trauma , brain tumor , psychiatric disorders , systemic disease or other mri contraindication such as claustrophobia were excluded from this study . present or past use of medication to treat cognition and infarction was not an exclusion criterion . all subjects provided informed consents and the ethics committee at the third military medical university approved the study protocols . the mri brain scanning was performed on a siemens avanto 1.5 tesla scanner with head matrix coil at the department of radiology in southwest hospital , chongqing , china . during the examination , scanner noise was attenuated with earplugs and head motion was restricted with foam padding ( provided by the manufacturer ) around the head and the necessity of head immobility was emphasized to each subject . anatomical image datasets were acquired with a standard t1-weighted high - resolution anatomic scan of magnetization - prepared rapid gradient echo ( mprage ) sequence : tr=1900ms , te=3.37ms , ti=1100ms , flip angle=15 , fov=256mm192 mm , nex=1 , matrix=256192 , 1.001.00 mm in - plane resolution , horizontal slices with slice thickness of 1.3 mm and no gap . the mprage acquisition of each study was ensured the same slice orientation ( paralleled to the anterior commissure ( ac ) and commissural posterior ( pc ) line ) . subsequently , t2-weighted ( t2wi ) images with 36 axial slices , 3 mm thick and 1 mm gap , oriented to ac - pc line were obtained . before further analysis of the 3d brain images the 3d data were copied from the mri scanner and imported to a personal computer running a linux operating system , on which off - line analysis was carried out by anatomical structure image analysis software fsl - vbm . first , by using mri_convert command , dicom files were completely converted to nii.gz format and then these structural images were brain - extracted using bet 14 in order to remove skin and skull . next , tissue - type segmentation was carried out using fast4 15 package , after that gray matter , white matter and csf were produced . the resulting gray - matter partial volume images were then aligned to mni152 standard space using the affine registration tool flirt 16,17 , followed optionally by nonlinear registration using fnirt , which uses a b - spline representation of the registration warp field 18 . the resulting images were averaged to create a study - specific template in order to reduce the effect of inter - subject brain variability during the registration procedure , to which the native gray matter images were then non - linearly re - registered . the registered partial volume images were modulated ( to correct for local expansion or contraction ) by dividing using the jacobian of the warp field . the modulated segmented images were then smoothed with an isotropic gaussian kernel with a sigma of 3 mm for the tfce - based analysis 19 . finally , differences in cerebral gray matter density between the patient group and control group were assessed by voxelwise glm applied using permutation - based non - parametric testing ( 5000 permutations ) 20 . cluster - size shareholding at p - values<0.05 , fully correcting for multiple comparisons across space , was used . nine sivd patients were recruited by physician referral from the department of neurology in southwest hospital , chongqing , china ( table 1 ) . the clinical diagnosis of sivd was made by a senior neurologist according to the criteria suggested by erkinjuntti 12 and dsm - iv ( diagnostic and statistical manual of mental disorders , iv ) . ad patients with subcortical ischemia or mixed dementia who can also meet dsm - iv vad criteria and the patients with large territorial infarction involving cortex or single strategic infarction such as anterior nucleus of thalamus were excluded . all patients have visible white matter hyperintensities ( wmh ) ( grade 3 or more by a reported rating scale 13 ) addressed with t2/flair imaging data in mri scanning . cognitive performance was evaluated through neuropsychological tasks that included : mini mental state examination ( mmse ) , clinical dementia rating ( cdr ) , neuropsychiatric inventory ( npi ) , activities of daily living scale ( adl ) , hachinski and hamilton test . nine age- and sex - matched old people with normal - cognition were recruited from surrounding regions by advertisement . they underwent a comprehensive brain examination to ensure that they had normal brain structure and no neurological lesions . exclusion criteria included patients with aphasia or who otherwise could not complete psychological testing because of a language disorder ; diagnosed chronic or degenerative disease affecting the central nervous system ; active substance abuse disorders ; intracranial hemorrhage ; difficult to control epilepsy that could present with cognitive impairment ; or temporal lobe epilepsy . all of those subjects who with brain trauma , brain tumor , psychiatric disorders , systemic disease or other mri contraindication such as claustrophobia were excluded from this study . present or past use of medication to treat cognition and infarction was not an exclusion criterion . all subjects provided informed consents and the ethics committee at the third military medical university approved the study protocols . the mri brain scanning was performed on a siemens avanto 1.5 tesla scanner with head matrix coil at the department of radiology in southwest hospital , chongqing , china . during the examination , scanner noise was attenuated with earplugs and head motion was restricted with foam padding ( provided by the manufacturer ) around the head and the necessity of head immobility was emphasized to each subject . anatomical image datasets were acquired with a standard t1-weighted high - resolution anatomic scan of magnetization - prepared rapid gradient echo ( mprage ) sequence : tr=1900ms , te=3.37ms , ti=1100ms , flip angle=15 , fov=256mm192 mm , nex=1 , matrix=256192 , 1.001.00 mm in - plane resolution , horizontal slices with slice thickness of 1.3 mm and no gap . the mprage acquisition of each study was ensured the same slice orientation ( paralleled to the anterior commissure ( ac ) and commissural posterior ( pc ) line ) . subsequently , t2-weighted ( t2wi ) images with 36 axial slices , 3 mm thick and 1 mm gap , oriented to ac - pc line were obtained . before further analysis of the 3d brain images , we confirmed that all images were uncontaminated with major head motion . the 3d data were copied from the mri scanner and imported to a personal computer running a linux operating system , on which off - line analysis was carried out by anatomical structure image analysis software fsl - vbm . first , by using mri_convert command , dicom files were completely converted to nii.gz format and then these structural images were brain - extracted using bet 14 in order to remove skin and skull . next , tissue - type segmentation was carried out using fast4 15 package , after that gray matter , white matter and csf were produced . the resulting gray - matter partial volume images were then aligned to mni152 standard space using the affine registration tool flirt 16,17 , followed optionally by nonlinear registration using fnirt , which uses a b - spline representation of the registration warp field 18 . the resulting images were averaged to create a study - specific template in order to reduce the effect of inter - subject brain variability during the registration procedure , to which the native gray matter images were then non - linearly re - registered . the registered partial volume images were modulated ( to correct for local expansion or contraction ) by dividing using the jacobian of the warp field . the modulated segmented images were then smoothed with an isotropic gaussian kernel with a sigma of 3 mm for the tfce - based analysis 19 . finally , differences in cerebral gray matter density between the patient group and control group were assessed by voxelwise glm applied using permutation - based non - parametric testing ( 5000 permutations ) 20 . cluster - size shareholding at p - values<0.05 , fully correcting for multiple comparisons across space , was used . the two groups did not differ significantly in age , sex ratio , or education . the scores include mmse , npi , cdr and adl , as well as those key excluded measuring scales such as hachinski score and hamilton score were summarized in table 1 . the cerebral gray matter density in sivd patients were found significant decreasing than that of the normal controls in brain regions of thalamus , parietal lobe , frontal lobe and temporal lobe ( p<0.05 ) . to date , limited studies of cortical gray matter density have been performed due to the lack of efficient methods . before the methods for automatic calculation of cortical thickness were established , measurement of cortical thickness was performed primarily on the basis of manual contouring , which inevitably incurred significant inter - operator variability . manual measurement also has the drawback of inability to achieve sub - millimeter precision ; therefore the accuracy of the measurements is quite questionable . fsl - vbm is more accurate than spm - vbm and can found more significant cluster 10 , because this method depends on the degrees of freedom of the non - rigid registration 21 and non - linear registration the alteration in cortical thickness have been well studied in several neurological diseases , such as multiple sclerosis 11 , schizophrenia 10 and so on , by using the fsl - vbm analysis . svmci patients have been reported cortical thinning in inferior frontal and orbitofrontal gyri , anterior cingulate , insula , superior temporal gyrus , and lingual gyrus , while cortical thinning in sivd patients involved all these areas plus dorsolateral prefrontal and temporal cortices 22 . to our best knowledge , the present study represents the first attempt to adapt this method to analyze the cerebral gray matter density of vascular cognitive impairment . in this study , the mri technique and anaphase analysis method provide a new way to study the structure of the brain . gray matter density in brain regions of thalamus , parietal lobe , frontal lobe and temporal lobe were found decreasing greatly , which mri can detect . these findings suggest that these brain regions may be the most affected in sivd , that the alterations in gray matter density are a possible characteristic of this type of dementia , and that the measurement of gray matter density in the above regions might help its detection . temporal lobe has been found closed correlation with memory and emotion . based on experimental evidence from both monkeys as well as humans , the prefrontal cortex has been proved to be important not only for attention , emotion and execution processing , but also for inhibition of " prepotent " habitual 23 . some human diseases , such as schizophrenia , involving prefrontal abnormalities result in impairments in attention conflict paradigms 24 . for example , the region of the left inferior parietal has been reported to become active when processing words compared to letter strings . the lateral left inferior region has been reported to be more active when viewing or naming words compared to pictures 25 . thalamus lesion was found associate with memory damage in animal 26 and other human studies 27 . previously , by using tc - hmpao spect method , regional cerebral blood flow in thalamus was found decreased significant in vascular dementia when compared with that in the controls 28 . there is no cure for vascular dementia until now and the focus is currently on early diagnosis and preventing further brain damage . diagnostic research criteria for sivd are based on magnetic resonance imaging ( mri ) findings including substantial white matter lesions ( wml ) , multiple lacunar infarcts , and neuropsychological tests . efforts to relate mri - measured lacunes and white matter changes to cognitive impairment have not been straightforward 26,30 . in this study we found a new biological marker of sivd patients , which can be detected conveniently by fsl -vbm . as a new atraumatic technology , we believe fsl -vbm has much potential value and could promote the diagnosis of sivd in future .

background and purpose : the present study was designed to detect the abnormalities of the cerebral grey - matter density in subcortical ischemic vascular dementia patients by fsl - vbm method to promote the early diagnosis of it.methods : nine subcortical ischemic vascular dementia patients and nine age - matched normal controls underwent mri brain structure scanning that was performed on a siemens avanto 1.5 tesla scanner and standard t1-weighted high - resolution anatomic scans of mprage sequence were obtained . the 3-demensional mprage images were processed with fsl - vbm package and the cerebral gray matter density was compared between the subcortical ischemic vascular dementia patients and normal controls.results : compared with the normal control group , the cerebral gray matter density of subcortical ischemic vascular dementia patients was found significantly decreasing , including brain regions of thalamus , parietal lobe , frontal lobe and temporal lobe ( p<0.05).conclusions : the cerebral gray matter density alterations have closed correlation with cognitive dysfunction in subcortical ischemic vascular dementia patient and can be detected by mri . mri has some potential value in the diagnosis of them .

Introduction Materials and methods Subjects MRI data acquisition MRI data analysis Results Discussion

PMC4061146

the amount of microorganisms in the oral fluid ( saliva ) ranges from 4 millions to 5 billions per 1 milliliter , and in dental plaque between 10 and 1000 billions per 1 gram . their resistance to non - specific oral protective factors and environmental factors ( e.g. , antiseptic solutions ) depends on the cellular membrane , a thick layer of mucus , a capsule , or other biological characteristics . for this reason , standard bacterial cultures with differing biological characteristics were selected for this study : staphylococcus aureus are gram - positive cocci found in the nostrils of up to 60% of humans ; they form microcapsules , and are resistant to high temperatures ( up to 70c ) , disinfectant , and antiseptic solutions , and cause suppurative inflammatory processes . staphylococcus aureus is an important pathogen because of its virulence , antimicrobial resistance , and association with various diseases , including fatal systemic infections , intoxication , cutaneous infectious , and opportunistic diseases . klebsiella pneumoniae are capsule - forming bacteria found in the respiratory tract and are capable of causing atypical pneumonia . candida albicans is a fungus and is part of the normal microflora of the oral cavity , but it can cause an opportunistic infection in immuno - depressed subjects . escherichia coli is part of the normal microflora of the large intestine ; however , due to poor hygiene it can enter the oral cavity and cause suppurative inflammatory processes . the procedures that a prosthodontist performs may cause damage to the patient s oral mucosa and gingiva . the saliva mixes with blood . during the manufacturing of the dental impressions , blood and saliva enter the setting impression material , and it becomes contaminated with bacteria and viruses [ 47 ] . for this reason , according to general hygiene norms , such impressions should be disinfected ; yet disinfection does not always yield the desirable results . when producing gypsum casts using non - disinfected or incompletely disinfected dental impressions , the microorganisms travel from the surface of the impression material into the cast . the infected material then is taken to the manufacturing facilities and may pose threat to dental technicians . dental casts are subject to several forms of contamination , ranging from direct contact with a patient s saliva , to procedures involving measurement , planning , manufacture , laboratory shipping , and storage . dental technicians touch the impressions or the casts with their hands , and their skin is frequently damaged due to their work . when cutting the dental casts , bacteria , fungi , or particles of the disinfectant may travel with the plaster dust from the material to the respiratory tract of personnel , causing infections , and may also accumulate on the clothing or open surfaces in the room . the aim of study was to evaluate the survival of staphylococcus aureus , klebsiella pneumoniae , and escherichia coli bacteria , and candida albicans fungus in non - disinfected dental casts . the results of the study could be used to determine how long dental casts may pose threat to the health of the personnel and the patients . perhaps disinfection of dental impressions would be sufficient , skipping additional disinfection of the dental casts in order to reduce exposure of the personnel to possible allergens the residues of disinfectant solutions in the plaster . although extensive research has been carried out , there are no published studies in which similar issues were addressed . during this experimental study we investigated the survival of the selected bacteria and fungi in dental casts . the study was conducted using standard bacterial ( staphylococcus aureus atcc 25928 , klebsiella pneumoniae atcc 13883 , and escherichia coli atcc 25922 ) and fungal ( candida albicans atcc 60193 ) cultures . saline suspensions ( mcfarland turbidity standard 0.5 ) were prepared using standard bacterial cultures cultivated in trypticase soy agar for 24 hours at 3537c , and candida albicans culture cultivated in sabouraud agar for 3 days . we evaluated and compared changes in the number of the microorganisms in gypsum casts at different time intervals . gypsum casts ( an equivalent to dental cast ) were manufactured in a sterile laminar - flow hood under sterile conditions , using standard alabaster plaster class 2 moldabaster the plaster was sterilized ; therefore , there was no need to form a control group . the dental casts were prepared by pouring 65 g sterile plaster powder , 5 ml sterile distilled water , and 1 ml of a specific bacterial or fungal suspension ( mcfarland turbidity standard 0.5 ) into a sterile mixing container . a sterile spatula was used to stir the contents until an integral mass was formed , which was subsequently placed into a sterile closed container . during the test period the closed containers with the samples were placed on a dental lab shelf to simulate casual cast storage conditions . the number of the aforementioned standard microorganisms per 1 g of the substance was evaluated after 1 , 2 , 24 , 48 , 72 , 96 , and 120 hours . sterile clamps were used in the making of each sample . the sample a piece of the gypsum cast of a known weight ( ca . 1.0 g ) was ground to powder in a sterile mortar , and then placed into a sterile glass with 5 ml of sterile saline solution . the suspension of plaster powder was diluted at the ratio of 1:5 , 1:25 , 1:125 , and 1:625 . one ml of the suspension from each test tube with different dilution ratios was poured into 10 ml of 45c trypticase soy agar ( or sabouraud agar , in the case of candida albicans ) in 2 petri dishes that were then stored at 3537c ( in case of candida albicans , sabouraud agar was stored at 2025c ) . after the cultivation of the indicated duration , colonies of the respective bacterium or fungus in the 2 petri dishes were evaluated , calculating the mean number of the colonies ; multiplying this number by the degree of dilution revealed the number of bacteria or fungus per 1 g of plaster . the data were considered to be statistically significant if the probability of an error was p<0.05 . the study showed that k. pneumoniae survived the longest in gypsum casts up to 4 days compared to the other studied microorganisms ( figure 1 ) . a statistically significant reduction in the number of k. pneumoniae ( from 837911935 to 44120845 ) in gypsum casts was observed after 1 day ( p<0.05 ) . the number of k. pneumoniae colonies was also the highest , compared to other bacteria . after the first and the second hour in the gypsum cast , the number of bacteria remained nearly unchanged . it was only after 2 days that a statistically significant difference was detected ( a reduction from 59154676 to 9285102 , p<0.05 ) . gram - positive bacteria ( staphylococci ) remained viable for up to 3 days in the gypsum casts , presumably due to their cell membrane composed of a thick peptidoglycan layer , a microcapsule ( a thin layer of mucus over the cell membrane ) , and high resistance to environmental factors ( figure 2 ) . no gram - negative bacteria ( e.g. , e. coli ) were found in gypsum casts after 2 days , and a statistically significant reduction in their numbers was observed just after 2 hours ( from 19483336 to 470574 , p<0.05 ) ( figure 3 ) . microorganisms with a eukaryote structure ( e.g. , the fungus c. albicans ) died within 2 days , and a statistically significant reduction was observed after 1 day ( from 31 3662759 to 12 2911234 , p<0.05 ) ( figure 4 ) . figure 5 presents the comparison of the standard microorganisms amounts ( the bacteria and the fungi ) at different time intervals , and the quantitative reduction in their numbers . the quantitative evaluation of the viability of standard microorganisms in gypsum casts showed that physical - chemical characteristics of gypsum casts have different effects on the survival of the standard microorganisms . structural and physiological characteristics of k. pneumoniae and s. aureus affect their survival ( their quantity ) with respect to that of e. coli and c. albicans . the findings of the study showed that the microorganisms did not multiply in the gypsum casts ; instead , their numbers significantly dropped . the study was conducted using the most common oral bacteria and fungus : staphylococcus aureus , klebsiella pneumoniae , candida albicans , and escherichia coli . studies performed in japan showed that all the microorganisms used in our study staphylococcus aureus , klebsiella pneumoniae , and escherichia coli were detected in the oral cavities of 13.6% of patients with no health complaints . according to other authors , staphylococcus aureus is detected in the oral cavities of 15.8% of patients , and candida albicans in 63.0% of patients . the findings of our study showed that the duration of the survival of microorganisms in gypsum casts varied depending on the types of the studied cultures . these bacteria have a thick layer of peptidoglycan and a microcapsule , which is a mucous layer of polysaccharide origin . the gram - negative bacteria klebsiella pneumoniae and escherichia coli differ from staphylococci in their cell membrane structure . however , klebsiella bacteria have a thick mucous layer of polysaccharide origin a capsule that ensured their longest survival in gypsum casts compared to other microorganisms . escherichia coli have no capsule and thus are more susceptible to unfavorable environmental conditions , thus these bacteria survived in gypsum casts for only 12 days . the fungus candida albicans , although it has a eukaryote cell structure , survived in gypsum casts longer than escherichia coli did because fungi have a chitin membrane . the oral cavity may contain pathogenic as well as relatively pathogenic microorganisms that can be transferred into the environment through impressions . in patients with damaged oral mucosa , staphylococcus aureus , klebsiella pneumoniae , and escherichia coli may cause suppurative inflammatory processes . escherichia coli may cause not only a local inflammatory process , but bacteremia as well . candida albicans , as part of the normal oral microflora , is found in 4060% of individuals ; however , this fungus can cause candidiasis in immuno - compromised states or in cases of antibiotic overuse . most commonly , alginate and silicone dental impressions are used in clinical practice . due to their composition , structure , and the hydrophilic setting mechanism , alginate impressions are easily infected by oral microorganisms . a more monolithic structure of the silicone impression mass and its non - hydrophilic setting mechanism significantly reduce the attachment of microorganisms to the impression surface . selecting a suitable impression mass may reduce the spreading of the infective agents as early as the first stages of prosthesis manufacturing . alginate impressions are more frequently contaminated with oral bacteria and fungi , compared to silicone impressions , and they are also more difficult to disinfect . alginate impressions pose a significantly higher threat of contaminating dental casts with bacteria and fungi , compared to silicone impressions , thus silicone impressions are safer with respect to the transmission of the infective agents . previous studies have shown that dental impressions delivered at dental laboratories contain bacteria irrespectively of whether they were disinfected or washed under running water . this suggests that dental casts produced using well - disinfected impressions require no additional disinfection . if the dental casts are stored under normal conditions of a dental technician s office , the number of microorganisms should change more rapidly than it did during our study due to frequently changing temperature and humidity , and because of the plaster ph of 6.1 . an additional disinfection of dental casts would not yield significant results , compared to the risk that the dental technicians would face because of exposure to a potential allergen the disinfectant on the surface of the dental cast . although studies have shown that the effect of the disinfectant on the precision of the dental casts is not significant , the risk remains that the disinfectant may alter the physical characteristics of the dental cast surface , making it more porous , brittle , and more susceptible to wear and tear . rinsing of the oral cavity with an antiseptic solution prior to odontological procedures may reduce the presence of microorganisms in the oral cavity . the most popular oral antiseptic , chlorhexidine , is not always effective against fungi or bacterial spores . thus , mouth rinsing with an oral antiseptic prior to taking impressions followed by full disinfection of the impressions would reliably reduce the contamination of dental casts with microorganisms . as our results show if the impression is disinfected and pre - impression mouth rinsing is used consistently , the dental casts would pose less threat because of the poor survivability of bacteria and fungi in gypsum . gram - positive bacteria staphylococcus aureus and gram - negative bacteria klebsiella pneumoniae demonstrated the longest survival in dental casts ( up to 4 days ) , compared to escherichia coli and candida albicans , which remained viable during the first 2 days ) . dental casts are not a suitable medium for the multiplication of the colonizing microorganisms . individual protective measures ( gloves , protective goggles , and respiratory protection measures ) should be used in order to avoid possible contamination of dental impressions and casts with microorganisms , and to prevent their transmission and spread in environments such as dental laboratories .

backgroundthis study evaluated the survival of the most prevalent oral bacteria and fungi ( staphylococcus aureus , klebsiella pneumoniae , escherichia coli , and candida albicans ) in dental casts , and compared changes in the amounts of these microorganisms at different time intervals to determine how long dental casts may pose threat to the health of dental personnel and patients.material/methodswhen manufacturing the casts , regular water was replaced with sterile distilled water , where suspensions of the studied bacteria or the fungus at certain concentrations were prepared . when the dental casts were fully set ( solidified ) , plaster shavings were examined immediately after the contact of the studied microorganism with the plaster , as well as after 1 , 2 , 24 , 48 , 72 , 96 , and 120 hours . following that , we measured how the amount of the studied bacteria and fungi in 1 gram of the plaster changed within the studied period of time.resultsklebsiella pneumoniae survived in plaster for up to 4 days , and the reduction in the number of these bacteria became statistically significant after 1 day ( p<0.05 ) . staphylococcus aureus remained viable in plaster for up to 4 days , and the number of these bacteria dropped after 1 day ( p<0.05 ) . escherichia coli disappeared after 2 days , and a reduction was already observed after 2 hours ( p<0.05 ) . candida albicans in plaster models died within 2 days , and a reduction in their number was observed after 1 day ( p<0.05).conclusionsthe microorganisms did not multiply in the gypsum casts and their number significantly dropped instead of increasing .

Background Material and Methods Results Discussion Conclusions

PMC2376233

bacterial strains and plasmids plpq2 ( 12 ) is a psc101-derived plasmid in which expression of the e. coli phop - phoq operon is driven by the lacuv5 promoter . pnl3 ( 12 ) is a pbr322-derived reporter plasmid for assaying phop - mediated transcriptional activation that contains the phon promoter fused to lacz . paed4q ( 12 ) is a puc19-derived plasmid in which the phoq sensor domain ( residues 43190 ) is expressed from the phage t7 10 promoter ( 13 ) . e. coli strain csh26q has an internal deletion in the chromosomal phoq gene , which results in a null phenotype ( 12 ) . e. coli strains bl21(de3 ) and b834(de3 ) were used to express phoq for purification . -galactosidase assays-galactosidase assays were performed as described ( 14 ) in n - media with additions of mgcl2 as indicated using strain csh26q / f lacikan / pnl3 containing derivatives of plpq2 with wild - type phoq or mutant variants . variants of phoq in which single amino acids at residues 50 , 54 , or 179 were individually substituted were constructed by site - directed pcr mutagenesis . primers were synthesized in which the appropriate codons were substituted to produce mutant phoq variants as indicated , and the resulting amplified phoq fragments were then cloned into a plasmid vector using nearby restriction enzyme sites . / f lacikan / pnl3 strains containing derivatives of plpq2 with wild - type phoq or mutant variants were grown to mid - log phase in n - media with appropriate antibiotics as described previously ( 14 ) and then induced for 2 h at 37 c with the addition of 1 mm isopropyl--d - thiogalactopyranoside . cells were harvested by centrifugation , resuspended in resuspension buffer ( 20 mm tris - hcl , ph 8.0 , 100 mm nacl ) , and lysed by sonication on ice . cell membranes were recovered from the supernatant by centrifugation at 100,000 g for 15 min and were washed once with resuspension buffer . proteins from equal amounts of membrane were separated by sds - page and were subsequently transferred to immobilon - p transfer membrane ( millipore ) . western analysis was performed using an antibody with specificity for the e. coli phoq cytoplasmic domain as described previously ( 14 ) . the e. coli phoq wild - type sensor domain construct ( residues 43190 ) , and the acid mutant construct ( residues 43190 ) in which residues 148154 were changed from eddddae to qnnnnaq were expressed and purified from e. coli strain x90(de3 ) as previously described ( 12 ) . selenomethionyl ( semet)5 phoq 43190 ( wild type ) was expressed from cells grown in selenomethionine minimal media , and native phoq 43190 ( acid mutant ) was expressed from cells grown in luria - bertani ( lb ) media . crystals of semet phoq 43190 ( wild - type ) protein were grown by the hanging drop , vapor diffusion method in a buffer containing polyethylene glycol 3400 , tris ( ph 8.5 ) , sodium acetate , and 5 mm nicl2 . crystals of the native phoq 43190 ( acid mutant ) protein were grown by the same method in a buffer containing 1.8 m ammonium sulfate , polyethylene glycol 4000 , and sodium acetate . data collection and structure determination data from a four - wavelength multiwavelength anomalous dispersion experiment at the selenium k - edge were collected from a single frozen semet wild - type phoq 43190 crystal at beamline x4a of the national synchrotron light source at brookhaven national laboratory . diffraction to bragg spacings of 2.5 ( 280-mm detector distance ) was collected using 1.5 rotations . data from a single frozen crystal of native acid mutant phoq 43190 were collected at a home cuk radiation source with a raxis - iv detector . the native crystals diffracted to bragg spacings of 2.0 ( 140-mm detector distance ) , and data were collected using 1 rotations . denzo and scalepack of the hkl program suite ( 15 ) were used to index and merge all data sets . selenium sites were found from the multiwavelength anomalous dispersion data after inspection of harker sections of bijvoet- and dispersive - difference patterson maps generated using ccp4 ( 16 ) , and they were refined in sharp ( 17 ) in space group p21 . a partial model of semet phoq 43190 ( wild type ) was generated by automatic model building using arp / warp ( 18 ) , and this model was used in a molecular replacement search to determine phases of the native phoq 43190 ( acid mutant ) in space group p41 . the rest of the native phoq 43190 ( acid mutant ) model was built manually in o ( 19 ) , and the completed model was refined to a resolution of 2.0 using cns ( 20 ) for iterative cycles of simulated annealing , conjugate gradient energy minimization , temperature - factor refinement , and manual rebuilding . an additional density feature was found to be best fit with a sulfate ion , and this was included in the model and refined as well . the complete native phoq 43190 ( acid mutant ) model aided the manual rebuilding of the semet phoq 43190 ( wild type ) model , which was then refined by the same methods as before to a resolution of 2.5 against the low energy remote dataset . three locations of additional electron density could be best fit with one acetate and two nickel ions , and these were added to the model and included in the refinement . data collection and refinement statistics for both structures are listed in tables 1 and 2 . the atomic coordinates and structure factors for both wild - type and acid mutant e. coli phoq sensor domains have been deposited with the protein data bank as accession codes 3bq8 and 3bqa , respectively . 2.5 0.9879 ( low remote ) 10480 3.8 18.6 95.2 ( 93.2 ) 6.1 ( 26.5 ) sephoq wt 2 2.5 0.9793 ( edge ) 10401 3.7 19.4 94.6 ( 92.6 ) 6.2 ( 25.4 ) sephoq wt 3 2.5 0.9790 ( peak ) 10460 3.7 18.6 95.0 ( 93.3 ) 7.0 ( 27.9 ) sephoq wt 4 2.5 0.9641 ( high remote ) 10539 3.7 19.7 95.4 ( 94.3 ) 6.5 ( 23.4 ) phoq mutant 2.0 1.5418 19615 3.3 22.4 99.0 ( 99.3 ) 3.5 ( 8.9 ) avalues in outermost shell are given in parenthesesbrmerge = ( |ii ii|)/|ii| , where ii is the integrated intensity of a given reflection phoq 43190 diffraction data values in outermost shell are given in parentheses rmerge = ( |ii ii|)/|ii| , where ii is the integrated intensity of a given reflection table 2phoq 43190 refinement statisticsparameterwild - type phoqacidic mutant phoq bragg spacings ( ) 20 - 2.5 20 - 2.0 space group p21 p41 cell parameters 33.59 , 113.96 , 45.53 44.42 , 44.42 , 151.86 a , b , c ( )/ , , ( ) 90 , 109.46 , 90 90 , 90 , 90 r 20.3% 16.8% rfree 30.1% 23.0% number of reflections 10,255 19,573 number of total atoms ( non - hydrogen ) 2,421 2,829 number of protein atoms 2,233 2,488 number of ligand atoms 6 ( 1 acetate molecule , 2 nickel ions ) 5 ( 1 sulfate ion ) number of waters 184 336 average b factor 44.8 27.3 r.m.s . bonds ( ) angles ( ) 1.4 1.3 ar = ( ||fo| |fc||)/|fo| , where fo and fc denote observed and calculated structure factors , respectivelybrfree was calculated using 5% of data excluded from refinement phoq 43190 refinement statistics r = ( ||fo| |fc||)/|fo| , where fo and fc denote observed and calculated structure factors , respectively rfree was calculated using 5% of data excluded from refinement 1 ) was determined by multiwavelength anomalous dispersion from a single semet crystal at the selenium k - edge . two molecules were found forming an apparent dimer in the asymmetric unit in space group p21 . clear electron density allowed residues to be traced in for residues 45134 and 137188 of molecule a and for residues 4575 , 83135 , and 138186 of molecule b. missing residues correspond to n- and c - terminal segments and to loops . a total of 275 ordered residues , 184 water molecules , 2 nickel ions , and 1 acetate ion was refined against the low remote data to a resolution of 2.5 with r and rfree values of 20.3% and 30.1% , respectively ( tables 1 and 2 ) . a , the overall structure in complex with nickel is shown as a ribbon diagram . the arg-50 asp-179 salt bridge is shown with residue side chains depicted in a stick representation ( carbon colored yellow , nitrogen colored blue , and oxygen colored red ) . a line of black dots marks interacting atoms of the salt bridge . colored dots are drawn to represent a hypothetical path of the protein backbone through disordered regions corresponding to residues 135 and 136 of molecule a ( light purple ) , and residues 136 and 137 of molecule b ( light red ) . b , the structure of one subunit ( molecule a ) of the wild - type phoq 43190 dimer is shown from a 90 rotation about the vertical axis of the dimer as shown in a. secondary structure elements are labeled in black . a , the overall structure in complex with nickel is shown as a ribbon diagram . the arg-50 asp-179 salt bridge is shown with residue side chains depicted in a stick representation ( carbon colored yellow , nitrogen colored blue , and oxygen colored red ) . colored dots are drawn to represent a hypothetical path of the protein backbone through disordered regions corresponding to residues 135 and 136 of molecule a ( light purple ) , and residues 136 and 137 of molecule b ( light red ) . b , the structure of one subunit ( molecule a ) of the wild - type phoq 43190 dimer is shown from a 90 rotation about the vertical axis of the dimer as shown in a. secondary structure elements are labeled in black . 2 ) was determined by molecular replacement starting from a partial model of wild - type phoq 43190 . two molecules were found in the asymmetric unit in space group p41 . as for the wild - type structure , these molecules are related by a quasi - diad axis of symmetry , but the arrangement is very different in this case ( see below ) . residues at positions 43188 and 45190 of molecule a and b , respectively , were found to be ordered . a total of 292 residues , 336 water molecules , and 1 sulfate ion was refined to r and rfree values of 16.8% and 23.0% , respectively , at a resolution of 2.0 against data collected from a single native crystal ( tables 1 and 2 ) . both the wild - type and acidic cluster mutant phoq 43190 structures adopt a mixed /-fold containing a central five - stranded anti - parallel -sheet flanked by a long n - terminal -helix and additional loops and helices on each side . this type of fold has been reported previously in the structures of other histidine kinase sensor domains such as dcus ( 21 ) and cita ( 22 ) in addition to the s. typhimurium phoq sensor domain ( 9 ) . pairwise superimposition of all molecules using lsqman ( 23 ) , based on a structurally invariant core of 47 c positions determined at the 5 level of escet ( 24 , 25 ) comparison , yields r.m.s.d . values ranging from 0.25 to 0.42 . small differences in the rest of the structure may be attributed to differences in crystal packing and a certain amount of intrinsic structural lability that may be related to protein function . the e. coli and s. typhimurium sensor domains are very similar in sequence ( 81.3% identity ) , and their structures are also similar . pairwise superimposition of the wild - type e. coli molecules with those of the s. typhimurium phoq yield r.m.s.d . values ranging from 0.89 to 0.97 over an average of 129 c positions , and matches for acidic cluster mutant phoq likewise yield r.m.s.d . because the acidic cluster mutant phoq 43190 was refined to a higher resolution and contains a greater number of ordered residues , we define the secondary structure elements based on dssp ( 26 ) assignments of molecule a of this structure . a , the overall structure of the acid mutant phoq 43190 ( molecule a ) is shown as a ribbon diagram with secondary structure elements labeled in black . b , a stereo plot of the c trace of the same acid mutant phoq 43190 molecule . a , the overall structure of the acid mutant phoq 43190 ( molecule a ) is shown as a ribbon diagram with secondary structure elements labeled in black . b , a stereo plot of the c trace of the same acid mutant phoq 43190 molecule . 2 , taking the acidic cluster mutant phoq 43190 structure for reference , and secondary structure assignments are specified in fig . 3 . the phoq sensor domain begins with n - terminal helix h1 ( residues 4662 ) , located on the backside of the central -sheet . helix h1 connects up to a pair of short antiparallel -strands s ( residues 6466 ) and s ( residues 6971 ) that loop above the sheet . the peptide backbone then runs back down through helix h2 ( residues 7679 ) and connects into the first strand s1 ( residues 8589 ) of the sheet . edge strand s2 ( residues 9598 ) follows s1 and connects into helices h3a ( residues 103108 ) and h3b ( residues 111113 ) , which cross over the front of the central sheet to join the other edge strand , s3 ( residues 118125 ) . strand s3 runs down the far side of the sheet and leads to helices h4 ( residues 126133 ) and h5 ( residues 137148 ) , which are angled below the sheet . strand s4 ( residues 154164 ) , the longest strand in the structure , follows h5 , and runs up the length of the central sheet . the peptide backbone then loops back down into the middle strand s5 ( residues 173178 ) of the sheet , and the structure ends with helix h6 ( residues 183185 ) , which follows s5 and continues to the c - terminal end . electron - density features corresponding to non - protein moieties were found in both the wild - type and acidic cluster mutant phoq 43190 structures . in the wild - type structure , one nickel ion is coordinated by carboxylate oxygen atoms of asp-151 and asp-152 from the acidic cluster of subunit a and by asp-128 from symmetry mate b , and the other nickel ion is coordinated by asp-151 and asp-152 of b and asp-128 of a. asp-128 is located on h4 , and asp-151 and asp-152 are located in the eddddae acidic cluster in the loop between h5 and s4 , which had previously been implicated in divalent cation binding ( 12 ) . the presence of nickel at the two sites was confirmed in a bijvoet difference fourier synthesis generated from a dataset collected above the nickel k - edge but below the selenium k - edge . an additional feature of nonprotein density was found in a pocket formed between the two molecules of the asymmetric unit ; this was modeled as an acetate ion coordinated by side chains of residues gln-81 of molecule a , and arg-53 and asn-117 of molecule b. arg-53 , gln-81 , and asn-117 are found in h1 , in the loop between h2 and s1 , and in the loop between h3b and s3 , respectively . nickel was a necessary component in the crystallization buffer to obtain crystals of wild - type phoq 43190 , and the addition of acetate was found to improve crystal size and appearance . in the acidic cluster mutant structure , a single region of unexpected electron density was modeled as a sulfate ion , which is complexed with water molecules coordinated by guanidinium groups of arg-50 and arg-53 from h1 of molecule a , and arg-169 from the loop between s4 to s5 of molecule b. ammonium sulfate was the main precipitant in the crystal buffer used to grow the acidic cluster mutant phoq 43190 crystals . secondary structure elements of e. coli phoq are also shown and labeled above the alignments , with helices in red and strands in blue.inthe top set , the pdc sensor domains phoq and cita are aligned with the pyp pas domain . regions in cita and pyp having c positions structurally aligned with those in e. coli phoq are shaded in light gray.inthe bottom set , the pas domains clk-6 , fixl , and ncoa-1 are aligned with pyp . regions in clk-6 , fixl , and ncoa-1 having c positions structurally aligned with those of pyp are shaded in light gray . organism names are abbreviated in italics ( ec for e. coli , st for s. typhimurium , kp for klebsiella pneumoniae , eh for ectothiorhodospira halophila , dm for drosophila melanogaster , bj for bradyrhizobium japonicum , and mm for mus musculus ) . secondary structure elements of e. coli phoq are also shown and labeled above the alignments , with helices in red and strands in blue.inthe top set , the pdc sensor domains phoq and cita are aligned with the pyp pas domain . regions in cita and pyp having c positions structurally aligned with those in e. coli phoq are shaded in light gray.inthe bottom set , the pas domains clk-6 , fixl , and ncoa-1 are aligned with pyp . regions in clk-6 , fixl , and ncoa-1 having c positions structurally aligned with those of pyp are shaded in light gray . organism names are abbreviated in italics ( ec for e. coli , st for s. typhimurium , kp for klebsiella pneumoniae , eh for ectothiorhodospira halophila , dm for drosophila melanogaster , bj for bradyrhizobium japonicum , and mm for mus musculus ) . comparison between pdc sensor domains and pas domains a dali ( 27 ) search comparing phoq 43190 to proteins in the pdb structural data base reveals great similarity to other histidine kinase sensor domains such as s. typhimurium phoq ( 9 ) , luxq ( 28 , 29 ) , and dcus ( 21 ) ( z scores = 22.3 , 6.3 , and 6.2 , respectively ; r.m.s.d . these periplasmic sensor domains have been described by others as belonging to the pas ( per - arnt - sim ) domain superfamily ( 30 ) , and dcus and cita have been directly compared with pyp ( 31 ) . pas domains are found in a wide range of organisms and include a diversity of intracellular signaling domains , including fixl , nifl , ntry , yntc , ntrb , and nifu ( 32 ) . dali ( 27 ) also finds similarities between e. coli phoq 43190 and pas domains such as the transcriptional coactivator ncoa-1 ( 33 ) and the drosophila clock protein clk-6 ( 34 ) , although these matches score lower than those with other sensor domains ( z scores = 5.2 and 2.5 , respectively ; r.m.s.d . values = 2.6 and 4.4 , respectively ) , and pyp is not listed . a structure - based alignment of the phoq and cita sensor domains with the pyp , clk-6 , fixl , and ncoa-1 pas domains illustrates the similarities and several important differences between these classes of proteins ( fig . we have defined the criteria for the alignments such that corresponding structurally aligned segments have at least three contiguous c positions lying within 2.5 of each other . pairwise sequence identities between phoq and these pas domains range from 6% to 8% , and structural alignments are very restricted ( 3039 c positions , table 3 ) . in all cases , more residues can be superimposed in alignments between different sensor domains or between different pas domains than in alignments between sensor domains and pas domains . only the -sheets are in common between pas domains and the / sensor domain ; differences primarily lie in residues between the second and third -strands and in the n - terminal helix , present only in sensor domains . these structural differences distinguish these sensor domains from pas domains and have led us to term this the pdc ( phoq - dcus - cita ) domain . thus , pdc proteins are distinguishable both from pas domains and from sensor domains that form four helical bundles such as tar ( 35 ) and narx.6 phoq is distinguished from dcus and cita in having the h4-h5 helix pair and acid cluster as an insertion . values of pdc and pas domain superimpositionspypphoq cita 1.46 over 49 c pyp 1.02 over 39 c clk-6 1.34 over 65 c 1.21 over 37 c fixl 1.07 over 66 c 1.01 over 36 c ncoa1 1.22 over 41 c 1.28 over 30 c r.m.s.d . values of pdc and pas domain superimpositions dimerization of phoq 43190the wild - type phoq 43190 dimer ( rotation = 158.6 , screw translation t = -3.11 ) ( 36 ) buries 1528 of total accessible surface area between the two subunits . the dimer is quasi - symmetric about an interface formed primarily by interactions between the h1 helices of each protomer that include hydrophobic contacts , hydrogen bonding , and electrostatic interactions . there are also additional interactions between residues on helix h1 and residues in the loops between s4 and s5 , h2 and s1 , h3b and s3 , and h2 and s1 . the structure reveals a salt bridge formed between asp-179 of molecule a and arg-50 of molecule b in which the corresponding c positions are separated by 11.3 . a symmetrically related salt bridge between arg-50 of molecule a and asp-179 of molecule b is not formed due to the asymmetric nature of the dimer , which places those c positions 16.9 apart . the orientation of the wild - type phoq monomers is such that both the n - terminal h1 and c - terminal h6 helices are aligned in the same direction in a manner where the ends would be poised to enter into the transmembrane segments tm1 and tm2 of a full - length molecule . the roughly parallel arrangement of the n- and c - terminal helices in the wild - type phoq 43190 dimer is similar to that observed in the dimeric structure of the aspartate receptor tar ( 35 ) . two acid mutant molecules interact in their crystal environment through similar surfaces as those in the wild - type dimer , and they bury a substantial interface ( 2350 ) , but orientations are radically different . this seems to be a non - physiological arrangement , because n- and c - terminal helices point in opposite directions and the arg-50 asp-179 salt bridge , seen below to be functionally important , does not form . measurements of activity in vivo to examine the importance of the dimer interface , and in particular the arg-50 asp-179 salt bridge , to the function of phoq , r50d and d179r single mutants , which would disrupt the salt bridge , and an r50d / d179r double mutant , which could recreate the salt bridge in the opposite direction , were constructed . an additional g54d mutant was constructed in which we reasoned the insertion of a charged residue in the hydrophobic interface would be disruptive . the abilities of such mutants to activate phop - mediated transcription of a phon - lacz reporter gene in vivo were compared with the wild - type protein . as shown in fig . 4a , phon - lacz expression is induced to roughly 3400 units by mgcl2 limitation when wild - type phoq protein is present , but it is only induced to only 900 and 245 units , respectively , when the phoq - r50d and phoq - d179r mutants are present . the effect of the double mutant is indeed compensatory rather than additive ; reporter expression is activated to 2225 units for phoq - r50d / d179r , > 50% of the wild - type level . the effect of the dimer - disrupting phoq - g54d mutant results in reporter gene induction to roughly 142 units in the magnesium limiting condition , an effect that most closely matches that of the phoq - d179r mutant . these results indicate that the salt bridge observed in the crystal structure is physiologically important in the function of the protein and suggest that the integrity of the interface is crucial for the formation of the active state . a , e. coli strain csh26q / flacikan , containing the pnl3 reporter plasmid and plpq2 for expressing the wild - type and mutant phoq proteins , were assayed for -galactosidase activity following growth in n - media ( 0 m mgcl2 ) or as supplemented with 40 m or 10 mm mgcl2 . standard deviations are < 10% of the mean , calculated from at least three individual experiments . b , the relative expression levels of the wild - type and mutant phoq proteins are shown by western blotting , probed using an antibody specific for the cytoplasmic domain of phoq . plpq2 vector lacking the phop - phoq operon . in vivo activity and relative expression levels of phoq . a , e. coli strain csh26q / flacikan , containing the pnl3 reporter plasmid and plpq2 for expressing the wild - type and mutant phoq proteins , were assayed for -galactosidase activity following growth in n - media ( 0 m mgcl2 ) or as supplemented with 40 m or 10 mm mgcl2 . standard deviations are < 10% of the mean , calculated from at least three individual experiments . b , the relative expression levels of the wild - type and mutant phoq proteins are shown by western blotting , probed using an antibody specific for the cytoplasmic domain of phoq . the sensor domain of phoq is clearly similar in structure to dcus ( 21 ) and cita ( 22 ) sensor domains despite being very dissimilar in sequence ( 7.4% and 4.5% identity , respectively ) . this phoq - dcus - cita ( pdc ) fold represents a different structural class ( / ) of sensor domains from the all - helical sensor domain classes such as tar ( 35 ) and narx . the pdc domain fold characteristically begins with a long n - terminal -helix that leads into a central -sheet core flanked by short helices on both sides . the central five - stranded antiparallel -sheet has the same topology as that of pas domains , and this feature dominates in structure similarity searches . this , in turn , has led others to identify pdc sensor domains as pas domains ; however , there are no actual similarities outside the -sheet , whereas pdc and pas domains each have their own distinguishing characteristics . the functionally important n - terminal helix is unique to pdc domains , and the segment connecting the two -strands at the edges of the sheet is distinctive for pdc and pas domains . in pdc domains , the residues between the second and third strands transverse across the front of the sheet in a single span , often helical . phoq residues in this region align with those in cita and in other x - ray structures of pdc sensor domains , such as dcus , lisk , and dctb . in pas domains , the segment between the second and third strands takes a longer and more tortuous path than in pdc domains , comprising multiple helices and typically ending in a helix running antiparallel to the third strand . such differences between pdc domains ( dcus and cita ) and pas domains were also noticed previously ( 37 ) . certain sequence similarities have been established among pas domain family members ( 30 ) , but pas - conserved sequences are not found in the phoq sensor domain . it appears that , despite sharing topologically identical five - stranded -sheets , pdc and pas domains do nevertheless belong to separate protein superfamilies . in the wild - type structure , the n - terminal helices that form the dimer interface lie in a parallel orientation , and we believe that the dimer interface observed in the wild - type phoq structure is biologically relevant . the orientation of the phoq protomers allows for the formation of the arg-50 asp-179 salt bridge , and the physiological importance of the salt bridge to function was confirmed by our mutagenesis studies ( see above ) . other studies characterizing phoq mutants ( d179a or d179l ) are also consistent with importance of the salt bridge to phoq function ( 38 ) ; however , the difference in phenotypes obtained between the two sets of mutational studies on the same location may be a result of differences in charge and size of the mutant residues . such results suggest that normal phoq function is dependent and sensitive to the energetics of the dimer interface . it is believed that histidine kinase transmembrane regions form four - helical bundles as observed in the structure of the natronobacterium pharaonis phototaxis srii - htrii complex ( 39 ) . the structures of the narx sensor domain and the aspartate receptor tar ( 35 ) have parallel n- and c - terminal helical regions that would be compatible with connection to such membrane - spanning regions in an intact molecule . the arrangement of the wild - type phoq protomers orients the n- and c - terminal helices in a parallel manner also suitable for connection with a four - helical bundle membrane - spanning region , and a similar orientation of terminal helices has also been observed in a dcus dimer . it must be noted that , in contrast to the wild - type e. coli phoq sensor domain , the s. typhimurium phoq sensor ( 9 ) shows a different putative dimeric arrangement . distances between the two n- and c - terminal residues within the s. typhimurium phoq dimer are reported to be 27 and 55 , respectively ( 9 ) , whereas the distances between n- and c - terminal residues within the wild - type e. coli phoq are each 18 apart , as measured between c positions corresponding to residues 45 and 186 in each protomer . we believe that the dimeric arrangement of protomers in the e. coli phoq structure places the n- and c - terminal ends in a position more suitable for connection into a putative four - helical bundle arrangement of transmembrane helices . the physiologically relevant arg-50 asp-179 salt bridge observed in the wild - type phoq dimer is not present in the s. typhimurium phoq dimer , which places the c positions of residues arg-50 and asp-179 , and arg-50 and asp-179 , 28.1 and 27.5 apart , respectively . in order for the salt bridge to form , a large rearrangement would be required at the dimer interface of the s. typhimurium phoq structure . asp-179 in the structure of the s. typhimurium phoq ( 9 ) sensor domain participates in hydrogen bonding with the side chains of thr-48 and lys-186 ( 9 ) , and mutations of asp-179 and lys-186 result in impaired phoq function ( 9 ) . equivalent residues in the e. coli phoq sensor domain are within weak van der vaals contact distance but are not hydrogen - bonded . it is possible that these differing arrangements of residues along the interface between the c - terminal helix and the central sheet in the two phoq structures may reflect different conformational signaling states . such residues may be involved in mediating conformational changes between regions of the protein involved with ligand binding to the membrane - spanning helices . additional experiments will be required to address such possibilities . in an attempt to learn more about the role of the acidic cluster in phoq function , we solved the phoq 43190 structure containing the eddddae to qnnnnaq mutations ( amino acid residues 148154 ) , which render the protein defective in divalent cation binding and activation . these crystals were obtained in a buffer that lacked divalent cations , and nothing is bound to the replacement sequence , although it adopts essentially the same conformation as for the wild - type structure . the acidic cluster mutant also forms a quasi - diad dimer in the crystal ; however , unlike in the wild - type dimer , here the n - terminal helices lie in an antiparallel orientation at the dimer interface and the arg-50 asp-179 salt bridge does not form . we believe this arrangement to be non - physiological , because the n- and c - terminal pairs are too far apart to connect to the putative membrane - spanning four - helix bundle . self - association propensities of membrane proteins are diminished by orders of magnitude when freed from membrane tethering ( 40 ) . thus , even though sensor domains are expected to be functionally dimerized in situ on the membrane , they may exhibit quite low dimer affinity when isolated in solution . the e. coli phoq sensor domain has been shown to be monomeric in solution by analytical ultracentrifugation with a lower limit of 600 m measured for the kd of dimerization ( 12 ) , and likewise both the cita and dcus sensor domains have also been reported to be mostly monomeric in solution ( 21 , 41 ) . it follows that a certain lability may exist in the manner by which sensor domains self - associate , and therefore dimeric interactions within in a crystal - lattice environment may not necessarily be biologically relevant . such lability has been observed in studies involving the cita sensor domain ( 22 ) and in our unpublished studies of the dctb and lisk sensor domains . in such cases , non - physiological interactions between sensor domain protomers can be found in the crystal lattice , sometimes mediated by required ions in crystallization buffers . we suggest that the dimers formed in the e. coli acid mutant phoq and s. typhimurium phoq structures may simply be the result of crystal packing interactions that have overridden the relatively weak affinity for the formation of a physiologically relevant dimer . does suggest that alteration of the acidic cluster by mutagenesis , and perhaps by divalent cation binding as well , might also affect the energetics of the dimer interface . if such effects are applicable to the intact membrane - bound molecule , then signal transduction mediated by cation binding at the acidic cluster would seem to employ a mechanism that involves a dynamic dimer interface and may not require quaternary interactions with additional proteins or with the membrane . mg and ca are thought to act in part as physiologically relevant signaling ligands by directly binding the phoq sensor domain and producing a conformational change that influences the enzymatic activities of the cytoplasmic domain ( 68 ) . although we were unable to obtain suitable crystals in the presence of either mg or ca ions , the crystallization buffer contained 5 mm nicl2 , which was required for the formation of crystals . the structure of wild - type phoq 43190 contains two ni ions , each associated with one of the two molecules in the asymmetric unit , forming lattice contacts between the side chains of asp-151 and asp-152 , and asp-128 of a symmetry mate . asp-151 and asp-152 are part of the acidic cluster ( eddddae ) that had previously been implicated in divalent cation binding ( 12 ) . divalent cations ( ca ) are also associated with acid clusters of two of four salmonella phoq sensor domains ( 9 ) , but that site is displaced from our ni site and coordinated peripherally by asp-149 . there do not seem to be ion - associated conformational distinctions in these two structures . divalent cations thermodynamically stabilize the sensor domain fragment to denaturation in a fashion expected for a direct binding model , and this mode of binding requires the presence of the acidic cluster . in addition , substitution of the cluster with uncharged isosteres ( qnnnnaq ) results in a protein that is defective for in vivo activation when extracellular mg concentrations are low ; however , substitution of the cluster with alanine residues ( aaaaaaa ) results in a protein that functions normally in response to extracellular mg.7 one possible explanation for these paradoxical results is that the sensor domain possesses one or more additional ligand binding sites , and neutralization of the acidic cluster site by conservative substitution with non - charged isosteres mimics divalent cation binding at this site to favor formation of the repressed state . when the acidic cluster site is substituted with alanines , the side chains necessary to form the repressed state are absent , and the protein can respond normally to binding at the other site(s ) . interaction between a ni ion and asp-128 in the crystal structure suggests that this residue might be part of such an additional physiologically relevant divalent - cation binding site . but the d128a mutation , both in the context of the wild - type acidic cluster and the alanine - substituted cluster , results in a protein that still responds normally to extracellular mg and ca ( data not shown ) . perhaps asp-128 is not absolutely necessary for binding at the second site , asp-128 either may not interact with mg or ca , or such interactions do not affect protein function . close inspection of the structure does not reveal any other potential divalent cation binding sites . the acidic cluster resides in the lobe of phoq formed by the connection from h5 to s4 . alignment of phoq from various organisms indicates that the cluster and lobe are present in the enteric versions of phoq but not in non - enteric versions ( 42 ) , suggesting that the acidic cluster may play a specialized role in enteric bacteria . the position of the lobe with respect to the dimer interface and the rest of the structure suggests that it might lie very close to the membrane in the intact molecule . it has been suggested that from the s. typhimurium phoq structure that acidic regions may interact with the membrane phospholipids in a manner to allow chelation of divalent ions such as ca ( 9 ) ; however , such a mechanism has not been established in vivo . further research may be required to elucidate the specific role of the acidic cluster in its relation to divalent binding and signaling , and any involvement of membrane phospholipids in biological function .

the phop - phoq two - component system is a well studied bacterial signaling system that regulates virulence and stress response . catalytic activity of the histidine kinase sensor protein phoq is activated by low extracellular concentrations of divalent cations such as mg2 + , and subsequently the response regulator phop is activated in turn through a classic phosphotransfer pathway that is typical in such systems . the phoq sensor domains of enteric bacteria contain an acidic cluster of residues ( eddddae ) that has been implicated in direct binding to divalent cations . we have determined crystal structures of the wild - type escherichia coli phoq periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues ( qnnnnaq ) . the phoq domain structure is similar to that of dcus and cita sensor domains , and this phoq - dcus - cita ( pdc ) sensor fold is seen to be distinct from the superficially similar pas domain fold . analysis of the wild - type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between arg-50 and asp-179 and with nickel ions bound to aspartate residues in the acidic cluster . the physiological importance of the salt bridge to in vivo phoq function has been confirmed by mutagenesis . the mutant structure has an alternative , non - physiological dimeric association .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

PMC3304287

a 28-year - old woman , with primary infertility of three years was referred for laparoscopic myomectomy with a history of severe dysmenorrhea and a diagnosis of posterolateral wall myoma . on pelvic examination , a palpable myoma was noted on the posterior uterine surface , and the uterus was felt to be approximately 10 gestational weeks in size . a well - circumscribed mass measuring 5.16 6.0 4.25 cms was seen in the posterolateral fundus and was found to be impinging on the endometrial stripe . the patient was given a choice between conventional laparoscopic myomectomy and the single incision approach . a 2 cm vertical incision was made at the base of the umbilicus and peritoneal access was gained . , norwalk , ct ) , [ figure 1 ] was inserted through the incision . the device , made from an elastic polymer , was slightly hourglass shaped and could be deployed through 2 cm fascial incision . it contained four openings : one for insufflation via a right - angled tube and three that could accommodate trocars 5 to 12 mm in size . the compressibility of the elastic polymer allowed for the access ports to expand and form fit the space in which they resided , and the ports also passed through the working channels . the sils tm port has the capacity of up to three laparoscopic instruments of 5 to 12 mm after careful survey of the abdomen and pelvis , dilute vasopressin , 20 u , in 100 ml of saline solution , was injected subserosally over the posterior myoma . once the correct plane was entered , the myoma was dissected out of the uterus using a 5 mm myoma cork screw and blunt scissors . using mostly blunt dissection , the enucleation of the myoma was done [ figure 2 and 3 ] . hemostasis was ensured and a piece of adhesion barrier ( gynecare interceed ; ethicon inc . , west somerville , new jersey ) was then cut in half , introduced into the abdomen through one of the 5 mm trocars , and placed over the hysterotomy incision . the myoma was then grasped with a 12 mm claw forceps and electric morcellation was done using rotocut g1 morcellator morcellation ( rotacut ) the advantages obtained by electronic morcellation over manual morcellation from umbilical incision site are : reduction in operative timedecreased risk of hernia formation due to absence of tearing or stretching of fascialower risk of injury to the surrounding tissues . reduction in operative time decreased risk of hernia formation due to absence of tearing or stretching of fascia lower risk of injury to the surrounding tissues . as laparoscopic myomectomies are performed routinely in our center , rotocut is preferred over gynecare morcellex for its cost effectiveness and speed . it is reusable and can be used indefinitely with periodic replacement of the cutting blade . the fascia was closed with a running 0 polyglactin 910 ( vicryl , somerville , nj ) suture . the skin was then approximated with a series of interrupted 3/0 monocryl sutures ( ethicon inc . ) . ten milliliters of 0.5% bupivacaine hydrochloride ( sensorcaine ) were injected into the incision site , and dermabond adhesive ( ethicon inc . ) was applied . the total procedure time ( time from first incision to end of procedure , d and c ) single - incision surgery has been reported to offer patients improved cosmetic outcomes as compared to multiport laparoscopic surgery , and possibly less postoperative pain , although these potential benefits have yet to be demonstrated in a well - designed prospective trial . a number of advantages have been proposed including cosmesis , less incisional pain , less blood loss , and the ability to convert to standard multiport laparoscopic surgery . an additional morcellation port is avoided as specimen retrieval / morcellation can be done through the umbilical incision . the primary limitations of sils are the restricted degrees of freedom of movement , lack of triangulation , the number of ports that that can be used , and the proximity of the instruments to each other during the operation all of which increase the complexity and technical challenges of the operation . many of these difficulties may be related to the technique of port placement and utilization during single incision laparoscopic surgery . a number of methods have been described for port access to perform sils , including multiple fascial punctures through one skin incision and use of novel port access devices . to further overcome the technical challenges of sils , different instruments that provide angulations and small profile trocars , endostitch , are being developed . the barbed suture greatly facilitates myometrial closure because there is no need to tie knots and there is no backsliding of the suture , which enables continuous wound closure with even distribution of tensile strength throughout the repair . these benefits of barbed suture are especially valuable in single - incision surgery , because intracorporeal knot tying can be more challenging here than in the multiport approach . currently , careful case selection is paramount , so that these procedures can be explored safely , with a low threshold to convert to standard laparoscopy , as indicated , for safety and quality of care . the use of novel port access devices , articulated instruments , and endostitch self - retaining sutures , makes the procedure easier , with a potential for saving time .

single port laparoscopic surgery ( spls ) , also called sils is the natural extension of multi - incisional laparoscopic surgery , in the quest for reduction of traumatic insult and residual scarring to the patient . today with the evolution of newer instruments , bidirectional self - retaining sutures , and surgical experience we are able to perform many surgeries in gynecology .

INTRODUCTION SURGICAL TECHNIQUE DISCUSSION

PMC5014762

chromatin is the complex of dna and histone proteins that packages eukaryotic dna into chromosomes . the nucleosome is the repeating structural subunit of chromatin and consists of 147 bp of dna wrapped around a histone octamer core . translational positioning of nucleosomes along the dna sequence influences the accessibility of regulatory sequences to the transcriptional machinery and can thereby regulate gene expression levels ( for review , see hughes and rando , 2014 , radman - livaja and rando , 2010 ) . the average nucleosome - positioning profile over all yeast genes consists of a nucleosome - depleted region ( ndr ) of 150 bp , with well positioned 1 and + 1 nucleosomes upstream and downstream of the ndr , respectively . the transcription start site ( tss ) is located in the + 1 nucleosome , there is a regularly spaced nucleosomal array over the first kb of the gene body , and nucleosome positions become fuzzier toward the middle and end of the coding sequence ( brogaard et al . , 2012 , because ndrs are thought to facilitate transcriptional activation by enabling access of regulatory proteins to their binding sequences , ndr formation or loss can lead to gene activation or silencing , respectively . the distribution of nucleosomes along the genome depends in part on the underlying dna sequence , with promoter regions enriched in poly a tracts mostly excluding nucleosomes ( kaplan et al . , 2009 , yuan et al . , 2005 ) . in addition to poly a tracts that passively disfavor nucleosome assembly , ndrs can also be formed through active nucleosome removal from promoter regions by remodelers , such as rsc ( parnell et al . , 2008 ) , or nucleosome displacement by general transcription factors ( tfs ) , such as abf1 and rap1 ( yarragudi et al . , 2004 , whereas dna sequence composition contributes to nucleosome occupancy in yeast , it is the action of chromatin remodelers and the transcriptional machinery that establishes precise nucleosome positioning over genes ( gkikopoulos et al . , 2011 , hughes et al . , 2012 , , 2015 , pointner et al . , 2012 , weiner et al . , 2010 ) . indeed , in vitro assembly of nucleosomes onto purified yeast genomic dna results only in nucleosome depletion over poly a tracts but little evidence for nucleosome positioning , whereas addition of yeast extract to such reconstitutions yields a more - accurate recapitulation of nucleosome positioning patterns observed in vivo ( zhang et al . , 2011 ) . purified atp - dependent remodelers , such as chd1 and swi / snf family members , can generate ndrs at promoters and regularly spaced nucleosomal arrays over gene bodies similar to those seen in vivo , even in the absence of transcription ( lieleg et al . , however , such in vitro nucleosome reconstitutions do not perfectly match nucleosome positions observed in vivo ( hughes et al . , 2012 ) . in vivo , the process of transcription plays a key role in nucleosome positioning , due both to the direct effects of rna polymerase on nucleosomes and to the effects of remodelers that are recruited to target genes during transcriptional activation or elongation ( bintu et al . , 2011 , radman - livaja et al . , 2011 , studitsky et al . , 1997 , weiner et al . , 2010 ) . contrary to the steady - state landscape of nucleosome positioning , chromatin structure dynamics over the cell cycle , during which chromosomes are subject to dramatic perturbations caused by replication and mitosis , are not well characterized . dna replication initiates the disassembly of maternal nucleosomes ahead of the replication fork and their reassembly in its wake on one or the other daughter chromatid ( alabert and groth , 2012 ) . as nucleosomes can influence transcription , depending on their precise locations , replication provides an opportunity for the cell either to re - establish the same nucleosome - positioning profiles or to rearrange the nucleosomal landscape and thereby maintain or change its gene expression program , respectively . the process of nucleosome re - positioning after the disruption caused by replication is not well understood . specifically , it is not known how and where nucleosomes re - position themselves on newly replicated dna and how long it takes them to reconstitute the canonical mid - log positioning pattern . this is related to the questions of when transcription resumes after the disruption caused by dna replication , whether both new gene copies are transcribed , and whether transcription re - activation is a cause or a consequence of nucleosome positioning maturation . in order to address these questions , we have developed a method for genome - wide mapping of nucleosome positions on recently replicated dna in budding yeast : nascent chromatin avidin pull - down ( nchap ) . in this method , we isolate newly synthesized dna at varying times after a pulse of the nucleotide analog 5-ethynyl-2-deoxyuridine ( edu ) , which , along with micrococcal nuclease ( mnase ) digestion , allows us to follow genome - wide nucleosome - positioning dynamics after the passage of the replication fork on both leading and lagging dna copies . we find that nucleosomes assume their mid - log positions with varying rates at different genomic loci . because we find that highly transcribed genes exhibit a rapid return to their canonical chromatin architecture , we hypothesized that transcription participates in the regular phasing of nucleosomes in the gene body minutes after the passage of the fork . consistent with this , treatment with the rna polymerase inhibitor thiolutin interfered with chromatin maturation over coding regions . experiments in deletion mutants reveal a role for chd1 and isw1a in nucleosome phasing relative to the tss and a role for hir in determining the linker length between nucleosomes . in contrast to transcription - dependent maturation of gene body chromatin , aspects of promoter packaging , such as the ndr midpoint and the position of the + 1 nucleosome , appear to be determined earlier , possibly in the absence of transcription elongation . together , our data illuminate the genomic landscape of chromatin maturation following replication , and our methodology enables future genetic interrogation of the mechanisms responsible for chromatin maturation . in order to map the positions of nucleosomes on recently replicated dna , we developed nchap , which combines edu labeling of nascent dna ( sirbu et al . , 2011 , wirges et al . , 2007 ) with mnase digestion of chromatin . edu is a thymidine analog that is incorporated during the synthesis of new dna , and mnase is an endonuclease that preferentially cleaves dna in the linker regions between nucleosomes . cells are arrested in g1 with factor and released into s phase in the presence of edu . aliquots are taken at regular intervals following release into s phase , and cells are fixed with formaldehyde . fixed cells are then treated with mnase , and nucleosome - protected dna is isolated after cross - link reversal . edu from purified 150-bp fragments is conjugated to biotin azide in a click reaction , and biotinylated dna fragments are isolated using streptavidin - coated magnetic beads . illumina paired end sequencing libraries are prepared ( adapted from borodina et al . , 2011 ) from the nascent dna attached to beads . in order to differentiate between the lagging and the leading strand copies , the edu - containing nascent strand is separated from its template using primer extension from one end of the adaptor - ligated fragment ( figure 1a ) . upstream of efficient replication origins will originate from the lagging strand copy , whereas the complementary crick reads will be from the leading copy . several controls validate the ability of nchap to identify newly replicated dna in relatively unperturbed cells . first , flow cytometry profiling of dna content shows that s phase progression is not impaired in the presence of edu ( figure s1a ) . second , we tested the ability of our protocol to specifically capture the single edu - bearing strand of dna , using in - vitro - generated fragments that have incorporated edu in only one strand ( figure s2 ) . this fragment was then subjected to the same procedure that we used to generate nchap libraries as outlined in figures 1a , s2a , and s2b . following streptavidin pull - down of these test libraries , qpcr with strand - specific primers showed that the fraction of fragments with the edu - containing strand in the expected orientation was 70%85% . finally , we also generated dna fragments in which only one strand has incorporated edu in vivo , taking advantage of the 53 resection and gap - filling steps that occur during the repair of a double - strand break at the matalpha1 locus ( reviewed in haber , 2012 ) , which we induced in the presence of edu ( figure s2c ) . here , we observe a 10-fold enrichment of the edu - containing strand in the expected orientation . overall , our tests show that our strand - specific library construction protocol can efficiently isolate the nascent dna strand and thus differentiate between leading and lagging strand copies of the genome . we next applied nchap to cells released from g1 arrest into s phase for varying lengths of time . nchap data at early time points reveal strong peaks surrounding known origins of replication ( autonomously replicating sequence [ ars ] ; nieduszynski et al . , 2007 , 2002 ) , validating the ability of our protocol to specifically map nucleosomes assembled on newly synthesized dna . the enrichment of nchap data ( blue peaks ) around origins is not due to mnase bias toward replicated regions , because read density distributions from mnase - digested input fractions ( before biotinylation and streptavidin pull - down ) exhibit the relatively even occupancy expected of the nucleosome landscape in yeast ( pink peaks ) . average nucleosome profiles from the nascent chromatin fraction resemble classic average nucleosome profiles , indicating that edu incorporation does not interfere with mnase digestion or nucleosome assembly ( figure s3a ) . together , these data validate the ability of nchap to accurately identify nucleosome - protected regions of recently replicated dna in a strand - specific manner . during cell - cycle arrest and release , asynchrony among individual cells in the timing of g1 release and entry into s phase results in heterogeneity in the location of the replication fork in any given cell , meaning that , even for early firing regions of the genome , nchap will capture dna that has been replicated from 1 to as many as 20 min prior . consequently , in order to characterize the dynamics of chromatin maturation following replication , we carried out nchap across a time course in which asynchronous yeast was subject to a brief pulse of edu followed by a thymidine chase for varying lengths of time . an asynchronous population by definition contains cells at all stages of the cell cycle , including 15%40% of cells that are in s phase and which , as a population , will have replication forks located at every location along the genome . as a result , a relatively short pulse of edu will label short stretches of replicating dna covering the entire genome over the whole cell population . the thymidine chase stops further incorporation of edu , and subsequent fixation at regular time intervals provides snapshots of simultaneous nucleosome positioning changes shortly after replication in all replicating cells . in order to capture times as close as possible to the moment immediately after the passage of the replication fork , we sought to identify the minimal duration of the edu pulse that provided appreciable incorporation into replicated dna . we used flow cytometry profiling of cells labeled with fluorescein ( fam)-conjugated edu to monitor the kinetics of edu incorporation in asynchronous cells ( figure s4 ) . edu labeling is detectable within 15 min of its addition to the culture ( figure s4a ) , and the majority of replicating cells have incorporated edu after 2530 min ( figure s4 ) . edu incorporation could be delayed and slowed by growing yeast at suboptimal temperatures to extend the length of the cell cycle ( figure s4b ) . at 30c , an initial lag phase of 15 min is followed by a gradual increase in the numbers of cells that have incorporated edu , as well as an increase in edu - fam fluorescence intensity per cell as genome replication progresses and more edu is incorporated in each s phase cell ( figure s4c ) . a slower increase in the average cellular edu - fam intensity over time at 25c and 37c compared to 30c is consistent with slower or stalled replication forks , delayed s phase entry , and/or slower edu uptake and processing ( figure s4c ) . because the fraction of edu - positive cells in the asynchronous cell population increases gradually and cells need to be incubated with edu for 25 , 40 , or 50 min ( if grown at 30c , 37c , or 25c , respectively ) before all cells that were in s phase at the moment of edu addition become edu positive ( figure s4b ) , we conclude that the rates of edu import and processing can vary widely among different cells in the population , possibly due to variable expression of the edu transporter ( hent1 ) and thymidine kinase ( tk ) that were introduced into our yeast strain . all subsequent experiments were performed at 30c . to assess whether we could effectively halt edu incorporation using an excess of cold thymidine , we pulsed asynchronous yeast with edu for 20 or 5 min and then assayed edu - fam at varying times after thymidine addition ( figure s5 ) . both the levels of edu - fam across the population and the fraction of edu - fam - positive cells stay constant up to 25 min after the thymidine chase ( figures s5b and s5c ) , and then as cells enter a new round of replication , the fraction of edu - positive cells decreases , as expected for a successful thymidine chase . moreover , the average edu - fam intensity per cell remains low throughout the chase , consistent with edu being incorporated into only a small fraction of the genome , as intended ( figure s5e ) . we carried out two separate pulse - chase time course experiments : one with a 20-min and another with a 5-min edu pulse ( figure s5 ) . we calculated the pearson correlation between the nucleosome - positioning profile for each gene in the yeast genome ( from 500 bp upstream of the tss to the stop codon ; xu et al . , 2009 ) in our nchap data and the corresponding profile from a mid - log total chromatin standard ( weiner et al . , 2010 ) for each time point ( figure 2a ; table s1 ) . for each gene , we define its maturation index as the average correlation over the time course between the nucleosome profiles from nascent chromatin and the total chromatin standard , with individual genes exhibiting a wide range of maturation indices ( figures s3b and s3c ) . importantly , data for total chromatin consistently exhibit higher correlations to the standard than do nascent chromatin data ( figure 2a ; plot below the heatmap ) , demonstrating that the wide range of maturation indices in nascent chromatin is not an artifact of variability in mnase digestion across the time course . progressive changes in nucleosome positioning on nascent dna are also evident from whole - genome pairwise correlation analysis ( figure s6a ; table s2 ) . what distinguishes genes that rapidly adopt their mature chromatin state following replication ? sorting genes according to their maturation index from the 20-min edu pulse experiment , we find that genes that have the highest maturation index are also generally highly transcribed during active growth in rich medium ( kim et al . , 2010 ) . this trend is shown via a 50-gene running window average of rna pol2 occupancy and is even more evident when genes are divided into quintiles ( 1,245 genes each ) of maturation indices : the average rna pol2 occupancy in the highest quintile ( 5 ) is 10-fold higher than in the lowest quintile ( 1 ; figure 2a , top middle and right panels ) . this suggests that the process of transcription plays a role in re - establishing nucleosome positions over genes after chromatin disruptions caused by dna replication . box plot and pairwise t test analysis of correlations to the standard for quintiles 1 and 5 from two biological replicates of the 20-min edu pulse experiment show that the two quintiles are significantly different in all time points from both replicates ( p value of t test 0.05 ) and that most of the variability between replicates comes from early time points in the first quintile ( figures s6b and s6c ) . although data from the 5-min edu time course differ quantitatively from data from the 20-min edu time course ( figure 2b ) , in both datasets , nascent chromatin increasingly matches the mid - log standard as the time course progresses ( figures 2a , bottom , and s7 ) , and this trend is also replicated in the second 20-min edu pulse experiment ( figure s7 ) . moreover , the correlation of maturation indices in each quintile ( defined above ) with average rna pol2 occupancy is also preserved in both time courses when focusing on robustly measured genes ( figure 2b ) . to examine the process of chromatin maturation more closely , we compared the average tss - aligned nucleosome profiles from nascent chromatin to the same profiles obtained from mid - log yeast , averaging data according to quintiles of genes grouped by maturation index . visual inspection of these averaged profiles reveals that nucleosomes become better defined over the first kb of the gene body over time ( figures 2c2e ) . this improvement in nucleosome phasing can be quantitated using a measure of peak to trough for nucleosomes a low peak - to - trough ratio can either be due to a low average nucleosome occupancy at that position across the cell population or fuzzy positioning ( nucleosomes are not placed at the same distance from the tss in all genes and in all cells ) . the average peak - to - trough ratios for nucleosomes + 2 to + 7 on nascent chromatin after a 20-min edu pulse reach a plateau between the 4- and 8-min time points for rapidly maturing ( fifth quintile ) genes , whereas , in the first quintile , the ratios are stabilized only 10 min later ( figure 2d , top ) . similar rates of average peak / trough increase were confirmed in a second biological replicate of the 20-min edu pulse experiment ( figure s8 ) . a quadratic fit to the curves from figure 2d ( top ) reveals that genes in the first and fifth quintiles reach their half - maximal peak / trough 20 and 7 min after the chase , respectively . consistent results were obtained using the data from the 5-min edu pulse ( figure 2e ) , albeit with somewhat more - rapid apparent rates of nucleosome phasing over gene bodies ( figures 2d , bottom , and 2e ) . as the genes that exhibit comparatively rapid phasing of nucleosomes over gene bodies ( the fifth quintile ) are relatively highly transcribed , we propose that the regular phasing of nucleosomes downstream of the tss is a consequence of transcription elongation . in contrast to the relatively slow chromatin maturation observed over gene bodies , the + 1 nucleosome and the midpoint of the ndr are already in place at the beginning of our time course for the majority of genes ( figures 2c and 2e ) . thus , the positions of the + 1 nucleosome and the ndr are determined early after the passage of the replication fork . indeed , promoter chromatin architecture is established so rapidly that it is impossible to pinpoint its exact kinetics using our assay , as even for the 5-min edu pulse time course , there is a 10-min window between edu addition and the first recorded time point , during which time edu is incorporated into dna at different moments in different cells ( figure s4 ) . to directly test the involvement of transcription in nucleosome positioning maturation , we treated cells with the rna polymerase inhibitor thiolutin in a parallel experiment with the 5-min edu pulse time course shown in figure 2 ( figure s9 ) . chromatin maturation is greatly impaired upon treatment with the inhibitor ( figures 3 , s6 , and s7 ) , thus providing experimental support to our hypothesis that transcription elongation is involved in the reordering of nucleosomes on nascent dna . importantly , thiolutin specifically affects nascent chromatin maturation , as total chromatin fractions from thiolutin - treated and untreated cells are nearly indistinguishable ( figures 3 , s6 , and s7 ) . note that thiolutin has been added after the edu pulse to avoid negative effects of the inhibitor on replication and edu incorporation . as expected , the average peak / trough ratios in thiolutin - treated cells are lower than in non - treated cells . we can still detect slow nucleosome phasing maturation , with peak / trough ratios increasing at a similar rate in the first and fifth quintiles , even in the absence of transcription ( figure 3c ) , suggesting that there is also a transcription - independent mechanism responsible for nucleosome reorganization . still , we can not exclude the possibility that some residual transcription occurs in thiolutin - treated cells and accounts for the observed slow chromatin maturation . in any case , we conclude that transcription plays a central role in establishing chromatin architecture over gene bodies . in order to better understand the mechanisms involved in nucleosome positioning maturation , we repeated the 5-min edu pulse - chase experiment in mutants with deletions of hir1 , chd1 , or ioc3 chd1 and isw1a are both atp - dependent chromatin remodelers that associate with the gene body during transcription and are involved in nucleosome array organization over coding sequences ( gkikopoulos et al . , 2011 , radman - livaja et al . , 2012 , smolle et al . , hir1 is a subunit of the hir nucleosome assembly complex , which participates in histone turnover and replication - independent nucleosome assembly ( green et al . , 2005 , lopes da rosa et al . , 2011 , ray - gallet et al . , 2002 ) . nucleosome phasing is globally reduced in all three mutants ( although to a somewhat lesser extent in hir1 ) compared to wild - type ( wt ) profiles ( figure 4b , left ) . the level of disorganization of nucleosomal arrays in gene bodies is comparable between nascent and total chromatin profiles , suggesting that perturbations caused by dna replication persist after s phase in the absence of these chromatin remodelers . more striking , however , is the difference in linker lengths between nascent and total chromatin profiles ( figure 4b , right ) . nucleosomes appear to be less densely packed shortly after replication , with an average linker length of 20 bp compared to 13 bp in total chromatin in wt , chd1 , and ioc3 cells alike . in hir1 cells , total and nascent chromatin have the same 20-bp linker length , suggesting that hir activity tightens the spacing between nucleosomes after replication , consistent with a recent report on hir1 effects on nucleosome positioning in nascent chromatin ( fennessy and owen - hughes , 2016 ) . as detailed above , the strand separation step in our library - generation protocol enables us to distinguish between nucleosome - positioning profiles on the leading and lagging daughter chromatids . the leading or lagging copy annotation is assigned according to the position of a gene relative to its closest replication origin and whether its reads map to the watson or the crick strand watson reads upstream and downstream of an origin will be lagging and leading copies , respectively . however , due to varying efficiencies of yeast origins ( every origin is not activated in every s phase cell ; yang et al . , 2010 ) , leading and lagging annotations can only be unambiguously assigned to genes located near efficient origins of replication , leaving us with a set of 1,064 genes ( figures s10a and s10b ; experimental procedures ) . nucleosome profiles of lagging and leading copies of each gene in this set and from every time point were compared to the corresponding profiles from the final ( 22 min ) time point in the 20-min edu pulse - chase experiment ( figure 2 ) . the resulting correlation profiles were ordered according to the average difference ( for all three experiments ) in maturation indices between the lagging and the leading strand copies of each gene ( figure 5a ; table s3 ) . we could detect comparable differences in nucleosome positioning maturation on leading and lagging copies in the three datasets at 433 genes out 1,064 , with 200 genes showing significant differences between leading and lagging copies ( figures 5b and s11 ) , suggesting that nucleosome re - positioning can occur independently on the two daughter chromatids . there is no global effect of leading or lagging strand replication on nucleosome re - positioning because maturation indices are higher on the leading or the lagging copy in equal proportions . lagging and leading profiles from total chromatin also show asymmetry in their correlations , albeit to a lesser extent than the nascent profiles ( figures 5a and 5e ) . this is not due to mnase sequence bias toward the watson or the crick strands of individual genes , as all genes were compared to the corresponding watson or crick profiles from the 22-min time point standard . it is more likely that the asymmetry we observe in total chromatin profiles comes from the substantial fraction of s phase cells , which in this experiment represents 40% of the population ( figure s9b ) . the relatively slower - maturing copies in the top and bottom quartile of the heatmap in figure 5a are enriched for genes in which the newly synthesized strand also serves as the template for transcription ( figure 5c ) . this observation is consistent with at least two hypotheses : ( 1 ) edu incorporation on the template strand potentially interferes with rna pol2 initiation or elongation , thus delaying transcription - coupled chromatin maturation ( figures s10c and s10d ) or ( 2 ) asymmetric recruitment of chromatin remodeling enzymes and/or tfs to one copy , resulting in the preferential transcription of that gene copy . for example , asymmetric transcription following replication could result from transcription preferentially occurring on the leading strand copy when the newly synthesized copy of a gene s promoter and the replication fork are oriented in the same direction , potentially as a result of the underassembly of chromatin on the lagging strand immediately behind the fork . several observations suggest that asymmetric chromatin maturation results from differential expression of the two gene copies after replication . first , differences between maturation indices of the leading and lagging copies are substantially reduced in the presence of thiolutin , suggesting that differences in chromatin maturation dynamics on the two copies of a gene may be due to differences in transcription rates of the two copies , i.e. , when neither gene copy is transcribed , chromatin maturation is equally slow on either copy ( figures 5d and s11 ) . second , to test whether edu interferes with transcription when it is incorporated in the template strand , we compared steady - state mrna levels in mid and late s phase of edu - treated and untreated synchronized cells using gene expression microarrays in two independent biological replicates ( figure 6 ) . consistent with recent studies ( voichek et al . , 2016 ) , we find that rna levels of 95% of cell - cycle - independent genes do not change from mid to late s phase , which could be a consequence of either a 2-fold decrease in transcription rates on both copies or the expression of only one gene copy . for this group of genes , edu also had no effect on rna levels ( figures 6a and 6b ) , arguing against the hypothesis that asymmetric chromatin maturation is an artifact of edu effects on transcription . moreover , although a small group of 343 genes ( whose expression was not buffered after s phase ) exhibited edu - dependent inhibition of transcription ( figures 6b and 6c ) , these genes are not enriched for genes that show differences in chromatin maturation between the leading and the lagging gene copies . the non - buffered gene set is enriched for ribosomal genes and genes involved in translation ( figure 6d ) . we propose that , for the majority of genes , rna production is buffered after genome replication by the suppression of transcription in one of the two copies ( probably the one with the nascent strand as the transcription template ) , which we detect as a difference in transcription - dependent chromatin maturation between the two gene copies ( figure 5 ) . future studies will focus on the mechanisms that regulate gene expression levels in the genome after replication , which should clarify whether the two gene copies are differentially transcribed following replication . chromatin features change throughout the cell cycle , with the biggest perturbations occurring during dna replication and mitosis . histone proteins on the maternal genome are removed from the dna ahead of the replication fork and are recycled on one or the other daughter chromatid , with newly synthesized histones restoring a full complement of nucleosomes to both daughter genomes . here , we describe a method for following nucleosome positioning dynamics on newly replicated dna , which we call nchap . nchap allows us to isolate nascent chromatin and follow in parallel changes in nucleosome positioning on the leading and lagging strand chromatids shortly after the passage of the replication fork . whereas other studies have concentrated on proteomic analysis of bulk nascent chromatin ( alabert et al . , 2014 , 2011 ) , we provide a genome - wide locus - specific timeline of nucleosome footprint changes after replication . an earlier study , which mapped okazaki fragments , reported an enrichment of okazaki fragment ends at the positions of nucleosome dyad axes ( smith and whitehouse , 2012 ) . however , it is not clear whether nucleosome assembly on the lagging strand precedes okazaki fragment ligation , as proposed . given that we find that nucleosome maturation is not significantly faster on leading or lagging strand genomes ( figure 5 ) , we conclude that any lagging strand - specific nucleosome deposition processes must occur rapidly relative to the 10-min time resolution achieved here and that the nucleosome positioning maturation process described here takes place after okazaki fragment maturation . we propose that gene expression programs are maintained from one cell generation to the next by the formation of ndrs on daughter chromatids very early after the passage of the replication fork ( figure 7a ) . due to the heterogeneous rates of edu import in the population , we can only conclude that promoter maturation happens within 10 min after replication fork passage , and this process most likely occurs independently of transcriptional elongation , because transcription - dependent nucleosome phasing is detected later on in our time course . it may involve a dna sequence that repels nucleosomes ( such as poly a tracts ) and consequently favors the ( re)binding of tfs , chromatin remodelers , and other components of the transcription machinery that were probably present at the locus before replication . or an initially bound nucleosome may be evicted through the action of a chromatin remodeler recruited to the site by a sequence - specific tf , or the bound tf itself may prevent nucleosome binding . these tfs / remodelers presumably then help establish the positions of the + 1 and 1 nucleosomes . a similar model was recently proposed for promoter architecture re - establishment after replication in drosophila cells ( ramachandran and henikoff , 2016 ) . in regions without nucleosome - repelling sequences or without sequences for available tfs , nucleosomes are deposited at regular intervals shortly after the passage of the replication fork . at this stage , nucleosomes are slightly delocalized ( i.e. , not exactly in the same position ) , both on the two nascent copies within the same cell and in different cells in the population , resulting in average nucleosome profiles with low peak - to - trough ratios . remodelers that accompany the elongating rna pol2 ( chd1 or isw1a ) reorder nucleosomes in its wake and position them at more - regular intervals . consequently , nucleosomes on genes with higher rna pol2 occupancy will be better phased over the whole population . concomitantly , hir reduces the spacing between nucleosomes from on average 20 bp ( found in nascent chromatin ) to 13 bp ( measured in total chromatin ) . we nevertheless found a significant number of genes with low rna pol2 occupancy and high maturation indices , which , as suggested by our results with thiolutin - treated cells , is probably due to a transcription - independent chromatin maturation process . this could be a consequence of the action of transcription - independent chromatin remodelers , although it is not clear why these remodelers only act on a subset of poorly transcribed genes . it is also possible that , in the absence of transcription , nucleosomes are reorganized passively through statistical positioning within discrete domains located between boundaries akin to chromosomal interaction domains ( cids ) recently observed in yeast ( hsieh et al . , 2015 ) . alternatively , a transient burst of transcription right after replication could also be responsible for high maturation indices on these genes . a detailed analysis of rna pol2 occupancy kinetics on nascent dna will be necessary to distinguish between these possibilities . because the nascent strand in the slower - maturing copy tends to be the transcription template strand , observed differences in maturation rates between leading and lagging gene copies could be a consequence of gene expression buffering . whereas rna pol2 progression may be impaired when it encounters edu in the template strand , transcription of both copies of the gene after replication in the absence of edu is only seen at 343 mostly highly transcribed genes ( note that these are not the same genes that exhibit copy - specific maturation behaviors ) . the reason there is no detectable edu effect on steady - state rna levels in most genes could be a consequence of recently reported buffering effects of h3k56 acetylation by rtt109 ( voichek et al . , 2016 ) that potentially operate via the preferential expression of only one gene copy after replication . we propose that the suppressed copy would generally be the one with the nascent strand as the transcription template strand . consequently , differences in maturation rates between the two gene copies might be caused by asymmetric h3k56 acetylation and recruitment of chromatin - remodeling enzymes and/or tfs to one of the copies , which would cause differences in transcription rates and nucleosome positioning maturation rates . interestingly , when the lagging nascent strand is the transcription template , the replication fork and rna polymerase advance in the same direction . conversely , replication and transcription travel in opposite directions when the transcription template strand is the leading nascent strand . it is therefore possible that tfs bound to promoters ahead of the fork rebind preferentially to the leading or lagging copy after replication as a consequence of the differential rates of okazaki fragment ligation and fork speed . as illustrated in figure 7b , it is less likely that rna pol2 ( on the yet un - replicated promoter ) and the replication fork will collide when replication and transcription travel in the same direction . as the fork travels unhindered , okazaki fragment ligation may lag behind the fork and tfs bound to the promoter ahead of the fork are more likely to rebind to the leading copy , thus favoring transcription and consequently chromatin maturation of the leading copy as observed . on the other hand , when the two travel in opposite directions , a head - on collision of the fork and rna pol2 that is ahead of the fork is more likely . the fork then possibly stalls and slows down , and okazaki fragment ligation now happens almost simultaneously with synthesis , which allows tfs to bind to the leading or lagging copy of the promoter . it is , however , difficult to imagine why there would be a bias toward the lagging copy as our results predict . future studies will test whether only one gene copy is transcribed after replication and consequently find out which copy is suppressed . taken together , future studies should further illuminate the mechanisms responsible for rapid establishment of nucleosome positions and could potentially identify subtle consequences of slow maturation on genome function . detailed protocols are available in the supplemental experimental procedures . all experiments ( except those in figure s2c ) were done with the strain pv1 ( mata ade2 - 1 trp1 - 1 can1 - 1000 leu2 - 3,112 his3 - 11,15 gal psi+ rad5 + ura3::ura3/gpd - tk(7x ) aur1c::adh - hent1 bar1::kanr ) . the experiment in figure s2c was done with the cvy61ho strain ( mata ade2 - 1 his3 - 11,15 trp1 - 1 leu2 - 3,112 can1 - 100 bar1::hisg trp1::brdu inc [ brdu = hsv - tk + hent1 ] pjh132 [ gal::ho ura3 ] ) . mutant strains from figure 4 are as follows : hir1 : strain ac5 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 ura3 - 1 trp+ can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 hir1::nat bar1::kanr);chd1 : strain rz12 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 chd1::leu2 ) ; andioc3 : strain rz15 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 ioc3::kanr ) . hir1 : strain ac5 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 ura3 - 1 trp+ can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 hir1::nat bar1::kanr ) ; chd1 : strain rz12 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 chd1::leu2 ) ; and ioc3 : strain rz15 ( mata ade2 - 1 his3 - 11,15 leu2 - 3,112 trp1 - 1 ura3 - 1 can1 - 100 gal psi+ rad5 + ura3::gdp - tk(7x ) aur1c::adh - hent1 ioc3::kanr ) . for the synchronization experiment in figure 1 , cells were arrested in g1 with factor and then transferred into preheated media containing 10 m edu and pronase . aliquots were taken and fixed with 1% formaldehyde right before release and then at regular intervals after release starting at 25 min from media change . in the edu - thymidine pulse - chase experiments , exponentially growing cells were transferred to media with 10 m edu preheated at 30c . thymidine was added after 5 or 20 min and incubated for another 5 or 10 min . cells were then pelleted and transferred into fresh media with thymidine ( and thiolutin when indicated ) , and aliquots were taken at indicated time points and fixed as above . cells from frozen pellets were spheroplasted by bead beating in the bullet blender ( next advance ) . spheroplasts were treated with mnase , which was adjusted to the cell density in each tube in order to obtain 80%90% mononucleosomal - sized fragments after 20 min at 37c . after cross - link reversal , dna was extracted with phenol- chloroform - iso amyl alcohol ( pci ) , and 150-bp mono - nucleosomal sized fragments were subsequently purified from 2% agarose gels . purified 150-bp fragments were mixed with biotin azide in a cubr solution . after a 2-hr incubation at 37c , biotinylated dna was incubated with streptavidin - coated magnetic beads ( blocked with salmon sperm dna ) . following ligation with illumina genome sequencing adaptors with in - line barcodes , dna was subjected to primer ( illumina pe primer 2.0 ) extension with 2deoxyuridine 5-triphosphate ( dutp ) to separate the nascent strand from its complement . after degradation of the dutp - containing strand with user enzyme , the nascent dna strand was pcr amplified . paired - end sequencing was done on a hiseq 2000 ( illumina ; cnag ) or on a next seq sequencer ( illumina ) in o.j.r.s laboratory . measurements were made with facscalibur ( bd biosciences ; fl-1 filter ; forward scattered light [ fcs ] size cutoff : 70 ) . genomic dna was isolated from g1-arrested flash - frozen cell pellets and sonicated with the bioruptor pico cup sonicator . cells were released into s phase in media with or without 10 m edu , as above . fifty - milliliter aliquots were flash frozen in liquid nitrogen 32 and 40 min after release for rna isolation . total rna was isolated from frozen pv1 cell pellets with trizol and treated with dnase i. rna was reverse transcribed using oligodt as primers . the resulting cdna was dye coupled with cy5 or cy3 n - hydroxysuccinimide ( nhs ) esters and purified as described previously ( liu et al . , 2005 ) . the cy5- or cy3-labeled cdna was mixed with cy3- or cy5-labeled genomic dna , respectively , and hybridized to agilent 8x15k yeast gene expression arrays . images were scanned with the innoscan 710 microarray scanner ( innopsys ) and processed with the mapix software . data were normalized by dividing the cy5/cy3 ratio for each probe with the average cy5/cy3 ratio for the whole array . sequences were aligned to the s. cerevisiae genome using blast - like alignment tool ( blat ) . we kept reads that had at least one uniquely aligned 100% match in the paired - end pair . read count distribution was normalized to one by dividing each base pair count with the genome - wide average base - pair count . genes that are replicated from efficient origins were filtered as follows : ( 1 ) efficient origins were defined as origins whose read density peak heights were 0.6 at the 25-min time point in the experiment from figure 1 . ( 2 ) genes that were within the boundaries of the read density area around efficient origins at the 25-min time point were considered as being replicated from that particular origin in most cells . analysis was done using in - house perl and r scripts ( available upon request ) . the analysis in figures s6 , s7 , and s11 was performed using perl ( statistics::ttest ) and r scripts as detailed in the supplemental experimental procedures and figures s6 , s7 , and s11 . m.r .- l . developed nchap , designed the experiments , performed the experiments in figure s2 , developed analysis tools , analyzed the data , and wrote the manuscript . o.j.r . initiated the development of nchap , advised on experimental design and data analysis , and wrote the manuscript .

summarychromatin is thought to carry epigenetic information from one generation to the next , although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication . here , we measure a key aspect of chromatin structure dynamics during replication how rapidly nucleosome positions are established on the newly replicated daughter genomes . by isolating newly synthesized dna marked with 5-ethynyl-2-deoxyuridine ( edu ) , we characterize nucleosome positions on both daughter genomes of s. cerevisiae during chromatin maturation . we find that nucleosomes rapidly adopt their mid - log positions at highly transcribed genes , which is consistent with a role for transcription in positioning nucleosomes in vivo . additionally , experiments in hir1 mutants reveal a role for hir in nucleosome spacing . we also characterized nucleosome positions on the leading and lagging strands , uncovering differences in chromatin maturation dynamics at hundreds of genes . our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template .

Introduction Results Discussion Experimental Procedures Author Contributions

PMC3862396

lyme disease ( ld ) , caused by the spirochete bacteria of the genus borrelia , is the most common vector - borne infectious disease in north america . more than 38,000 new cases were reported in the us in 2009,1 but underreporting is estimated to be six- to twelvefold , making the true number likely over 200,000 cases per year.2 ld is caused by five species of spirochete bacteria of the genus borrelia . borrelia burgdorferi sensu stricto is the main cause of ld in north america , while borrelia afzelii , borrelia garinii , b. burgdorferi , borrelia spielmanii , and borrelia bavariensis are the cause of most cases of ld in europe.3 ld was named after two towns in connecticut lyme and old lyme where the disease was recognized as a separate entity with the investigation of a cluster of children who experienced uncommon arthritic symptoms preceded by a characteristic skin rash during 19751976 . this rash , termed erythema migrans ( em ) , had been linked in europe to the bite of ixodes ticks . b. burgdorferi was first identified in 1982.4 ld manifests itself as a multisystem inflammatory disease that affects the skin in its early , localized stage , and then spreads to the joints , nervous system , heart , and other organ systems in its later disseminated stage.5 in the us , ld is transmitted by the bite of vector ticks of the genus ixodes , primarily by the deer tick ixodes scapularis.6 ixodid ticks are responsible for transmitting the spirochetes from mammals to humans . ixodid ticks have a 2-year life span , in which they pass through three developmental stages larva , nymph , and adult feeding only once per stage.4 ixodid ticks acquire b. burgdorferi by ingesting a blood meal from an animal reservoir like the white - footed mouse ( peromyscus leucopus).2,7,8 in more urban areas , the reservoir is more diverse due to host availability , and will include chipmunks , shrews , squirrels , and even birds and lizards.9 the tick larvae overwinter and emerge the following spring in the nymphal stage , which is the stage of the tick that is most likely to transmit the borrelia infection.10 nymphs are responsible for 90% of human disease transmission ; nymphal stage ixodid ticks are rarely noticed because of their small size ( < 2 mm).4,6 ld is endemic in north america , europe , and asia , and the distribution of the vectors directly affects the incidence of the disease.7 most cases of ld in the us occur in southern new england , southeastern new york , new jersey , eastern pennsylvania , eastern maryland , delaware , and parts of minnesota , wisconsin , and michigan.10 the centers for disease control and prevention ( cdc ) national notifiable diseases surveillance system lists ld as a notifiable disease within its summary of notifiable diseases.8 the incidence of ld in virginia has increased almost threefold from 2002 to 2011 , with virginia ranking 15th in the nation for reported ld infections.11 ld is the fastest - growing vector - borne disease in the us.6 this growth is in part due to the geographic spread of zoonotic hosts like the white - tailed deer southward and westward from northern regions of virginia.11 while the white - tailed deer are required for the widespread distribution of the ixodid tick , they are not the most suitable hosts for b. burgdorferi.12 a 50-year - old caucasian female presented to the urgent care clinic with extreme headaches , fever with intermittent shivering cold spells , 10/10 generalized joint pain , excessive thirst and fluid intake , and a progressing rash on her back . the patient described the headaches as tremendous pressure pushing inwards as though she was wearing a heavy helmet , such pressure that sneezing and coughing gave her the perception that her head would explode . three days before her visit to the urgent care clinic ( day one ) , the patient noticed an initial small , slightly raised lesion resembling an insect or spider bite . the patient did not examine her back , but described what she thought to be a scab covering the bite while she scratched . on day two , the patient s fever reached 39.3c ( 102.8f ) , and she had an increasing burning sensation at the site of the bite . a family member examined the bite , and recognized that the bump had developed into a circular rash . the patient took over the counter ibuprofen and benadryl , and was able to sleep through the night . on day three , the patient felt poorly , but completed a normal working day . by day four , the patient awoke feeling worse , with 10/10 joint pain , high fever , and excessive thirst and fluid intake ; this led her to seek medical attention . she had a large circular red rash on her back with bulls - eye appearance , 16 18 cm in diameter ( figures 1 and 2 ) . the patient recalled a walk in the woods 3 weeks prior to her exam , but denied finding a tick on her body . the patient s white blood cell count and platelet count , hemoglobin , and hematocrit were within normal ranges . her serum chemistry was unremarkable , except for elevated glucose , borderline low alkaline phosphatase , and slightly depressed osmolality . a serologic test for ld ( antibody response to b. burgdorferi by enzyme - linked immunosorbent assay [ elisa ] ) was ordered and showed negative results ( table 3 ) . the patient gradually began feeling better , and was able to return to work after 4 days of antibiotic medication . on day nine , her body temperature was within normal limits , the polydipsia and headaches had dissipated , and the rash had decreased to 11 14 cm . the patient expressed concern that a 10-day treatment was inadequate in proportion to her symptoms and to the size of her rash . her doxycycline prescription was extended to 30 days by her primary care physician , continuing at 500 mg twice daily . a second set of serologic tests were ordered on day 28 , which demonstrated the presence of immunoglobulin ( ig)-m without igg antibodies to b. burgdorferi . a steiner stain failed to reveal spirochetes . on day 48 , 2 weeks after completing the antibiotics , the patient began experiencing lower - extremity neuralgia as well as tingling in her hands . the patient visited her primary care provider , who started a second 30-day treatment of doxycycline ( 500 mg twice daily ) and gabapentin ( 900 mg daily before bed ) . the patient has been referred to a local physician who has extensive experience with ld for continued management of her medical therapy . at the time of this report , a year later , this patient reports that her symptoms include chronic fatigue and intermittent arthralgia that has required a cane to mobilize at times . the large circular red rash on our patient s back with bulls - eye appearance is em , the characteristic rash of early course ld ( figures 1 and 2 ) . em begins as a red macule or papule that expands to a large annular area of erythema up to 87 cm in diameter.10,13,14 as the rash border expands , it may leave a partial central clearing . in the us , less than 35% of patients with em will demonstrate central clearing.15 less commonly , the center of the rash may appear vesicular or necrotic , as is demonstrated in this case report.16 in endemic areas , the presence of em is considered a reliable indicator of ld . em arises within 1 month at the tick - bite site , and is present in as many as 70% of patients.4 em typically improves dramatically , and will often resolve following the initiation of appropriate oral antibiotic therapy with either doxycycline or amoxicillin.6 ld occurs in stages , with different clinical manifestations that reflect the immune response to b. burgdorferi.4 the hallmark of a stage i , early localized infection , is characterized by em accompanied by systemic viral - like symptoms , including fever , malaise , headache , and joint pain.7,17,18 stage ii , early disseminated infection , develops within 19 months following an untreated infection , and is due to hematogenous spread of bacteria to sites distant from the original em lesion.4,16,19 early disseminated infection affects the skin , heart , and the nervous system.16,19 fifty percent of patients develop multiple em lesions that are typically small and can occur anywhere on the body.4 cardiac involvement is seen in 0.3%4% of cases,7,20 and is usually characterized by varying degrees of transient atrioventricular block.2023 less than 10% of untreated patients develop cranial nerve palsy or lymphocytic meningitis.7,4,24,25 cranial nerve vii is often affected , resulting in unilateral or bilateral facial palsy . spinal fluid examination may reveal lymphocytic pleocytosis and elevated levels of protein and b. burgdorferi - specific antibody.2427 stage iii , late ld , occurs months to years after the original tick exposure , and includes neurologic manifestations that may progress to encephalopathy , peripheral neuropathy , encephalomyelitis , and arthritic symptoms.28 the typical arthritic pattern is monoarticular and oligoarticular arthritis that is chronic relapsing.29 chronic arthritis typically involves one or two large joints , with preference for the knees , and occurs in 11% of untreated patients with erythema.4,16,30,31 on examination , inflamed joints are warm with large effusions . synovial fluid is inflammatory , with white blood cell counts averaging 24,000/mm with a predominance of neutrophils.4,16 if untreated , the arthritis may persist or may resolve spontaneously . a few of these patients will have persistent arthritis after antibiotic therapy , and are classified as antibiotic - refractory lyme arthritis.19 synovial biopsy specimens reveal exaggerated proliferation of synovial tissue , which covers articular cartilage , causing cartilage destruction and permanent deformities.16,20 arthritis of late ld is similar to rheumatoid arthritis , with its characteristic pannus development and consequences.4,32 the diagnosis of early ld can be made on the clinical presentation of the classical em and on a history of known tick bite or probable exposure , considering that serologic testing is too insensitive in the early acute phase of ld.7,33,29 supporting serologic evidence is necessary to secure the diagnosis for disseminated stages.4,33 the laboratory diagnosis of ld is rarely based on the microscopic visualization of the borrelia spirochete in clinical samples using polymerase chain reaction assay of biological samples or a culture of the skin biopsy , because these tests are not sensitive enough.3,4,6,10 the cdc recommends a two - tier approach for detection of b. burgdorferi - specific antibodies . detection of igm antibody , produced during early weeks of ld , can help identify recent infections of b. burgdorferi . igg antibodies , present > 6 weeks after onset of illness , rise to higher concentrations than igm antibodies , and can persist for months or years.34 first - generation elisas for the detection of anti - borrelia antibodies lack specificity . the inclusion of a second , more specific , serological method ( western blotting ) is used to exclude false - positive elisa samples.35,36 in the elisa assay , wells of plastic microwell plates are sensitized by passive absorption with b. burgdorferi antigen . any antigen - specific antibody in the sample will bind to the antigen coating the well.34,37 peroxidase - conjugated goat antihuman igm / igg antibody is added to the wells , and the plate is incubated . the conjugate will react with the human igm / igg antibody immobilized in the solid phase in step one.34,37,38 the microwells containing immobilized peroxidase conjugate are incubated with peroxidase substrate solution.37,38 hydrolysis of the substrate by peroxidase produces a color change . the color intensity of the solution depends on the antibody concentration in the original sample . the color intensity of the solution is measured photometrically , and an optical density value is calculated . then , an immunoglobulin - status ratio ( isr ) is calculated for each specimen by dividing the specimen optical density value by the cutoff optical density value . a positive isr means that antibodies to b. burgdorferi are presumptively detected in the serum specimen.34,3740 in western blotting , an antigen mixture prepared from b. burgdorferi strain b31 is separated by sodium dodecyl sulfate if antibodies to b. burgdorferi are present in the serum specimen , they will bind to antigens on the nitrocellulose membrane.42 bands in which a reaction has occurred will be detected by the addition of an enzyme - labeled antihuman igg or antihuman igm reagent that allows for the visualization of bands on the nitrocellulose membrane.41,42 practically speaking , the test produces something similar to a bar code with several lines or bands . each band represents antibodies to a different component of the b. burgdorferi bacteria.43,44 these specific band patterns have been internationally recognized . it is recommended that igm western blot is considered positive if at least two bands are present out of a total of five . it is recommended that igg western blot is considered positive if at least five bands are present out of a total of ten.38,4345 there has been laboratory variation reported that results in the spread of the sensitivity of the elisa assay . the sensitivity of the elisa assay varies from 29%40% in patients with em during the acute phase to 28%78% during the convalescent phase.4648 early ld and em are treated with antibiotics on an outpatient basis . doxycycline ( 100 mg twice daily by mouth ) , amoxicillin ( 500 mg twice a day by mouth ) , or cefuroxime ( 250 mg twice a day by mouth ) for 1014 days are equally efficacious.7,4,49 doxycycline is often the drug of choice , because it is also considered the treatment for the gram - negative bacterium anaplasma phagocytophilum , a potential tick - borne coinfection.29 macrolides like azithromycin , clarithromycin , and erythromycin should not be selected , due to possible antibiotic resistance that has been identified.50,51 approximately 10%20% of patients treated for ld with a recommended 2-week course of antibiotics will have lingering symptoms of fatigue , headache , musculoskeletal pain , and lethargy.11 thirty - four percent of a population - based , retrospective cohort study in massachusetts were found to have arthritis or recurrent arthralgia , neurocognitive impairment , and neuropathy or myelopathy , for a mean of 6 years following treatment for ld.52 sixty - two percent of a cohort of 215 consecutively treated ld patients in westchester county , new york were found to have arthralgia , arthritis , and cardiac or neurologic involvement for an average of 3.2 years after treatment.53 this complication is commonly known as post - treatment ld syndrome ( ptlds).54 however , in the absence of additional tests to rule out the eradication of the initial infections , others would argue that the more appropriate diagnosis is chronic ld.55 coinfections are a troubling complication for patients with chronic ld . the reported prevalence of coinfections in the us ranges between 4% and 28% . in the us , coinfections can include anaplasma phagocytophilum , which causes human granulocytic anaplasmosis , and babesiosis.56 the risk for coinfections is increased , due to the ixodid tick being a vector for all three.5658 symptoms of a babesiosis infection include fatigue , malaise , weakness , fever ( > 38c ) , myalgia , arthralgia , and anorexia.56 severe babesiosis infections may progress to acute respiratory distress syndrome , disseminated intravascular coagulation , congestive heart failure , renal failure , myocardial infarction , splenic infarcts or splenic rupture , and death.56 records of 139 patients with babesiosis between 1982 and 1993 were analyzed , and nine patients ( 6.5% ) died , 35 ( 25.2% ) were admitted to the intensive care unit , and 35 ( 25.2% ) required hospitalization for more than 14 days.59 rashes in patients with babesiosis are often reflective of concurrent infection with ld.60 we report the case of a patient who presented to a local primary care treatment facility with flu - like symptoms , headache , an expanding em rash , and later serological studies supporting the diagnosis of ld . following an antibiotic regimen twice the length and double the recommended treatment dose outlined by the infectious diseases society of america ( idsa ) there is a need for diagnostic tests sensitive enough and specific enough for identifying ld in all stages of infection . some clinicians will find controversy in the diagnosis and treatment of patients presenting with signs and symptoms of ld but lacking any dermatological presentation of the em rash and presenting with negative serological tests . current tests may prevent clinicians from being able to diagnose patients accurately who may not have obvious symptoms like em . those with clinical presentations distorted by coinfections will also likely experience a delay in their treatment . clinicians who hesitate to treat patients who do not display all of the diagnostic criteria required by the idsa may see their patients continue to progress from a subclinical phase to a more advanced phase of ld .

a 50-year - old woman from pulaski , virginia , presented to a local clinic with headaches , fever , generalized joint pain , excessive thirst and fluid intake , and a progressing rash on her back . on physical examination , she had a large circular red rash on her back with a bulls - eye appearance , 16 18 cm in diameter . serologic tests confirmed a diagnosis of lyme disease . the patient could recall a walk through the woods 3 weeks prior , although she never noticed a tick on her body . following a prolonged course of antibiotics , this case report presents a patient with ongoing symptoms consistent with post - treatment lyme disease .

Introduction Case report Discussion Conclusion

PMC3806166

endogenous peptides , protein , and oligonucleotides are among the main drugs which attract much attention because of their great potentials in treating chronic diseases . however , the extreme in vivo environment of human body has always limited the therapeutic applications of these substances [ 2 , 3 ] . polymeric nanoparticles have attracted much attention as delivery systems due to their ability in overcoming the physiological barriers and protecting and targeting the loaded substances to specific cells [ 4 , 5 ] . naturally occurring polymers such as chitosan ( cs ) cs is a biodegradable polysaccharide , and it is derived from deacetylation of chitin . apart from its biocompatibility , the low toxicity , hemostatic , and bacteriostatic properties also contribute to its various applications in pharmaceutical field [ 911 ] . several anions have been investigated to crosslink cs like sodium sulphate and dextran sulphate ( ds ) . ds is able to modify protein and sirna entrapment efficiency ( ee ) without the use of hardening agents and control the rate of drug release due to its high charge density . besides ds is a cheap material , it produces mechanically more stable nanoparticles compared to the pentasodium tripolyphosphate ( tpp ) [ 16 , 17 ] . several studies had reported the unique features of chitosan nanoparticles ( cs nps ) using ds . however , the modulation of preparative parameters on their physical characteristics is still not fully investigated , for example , the influence of ds steric hindrance on the electrostatic attraction between cs and bsa . furthermore , the determinant of a successful drug delivery system is dependent on its physical characteristics and stability . therefore , the objectives of present study were to modulate preparative parameters to obtain nanosized particles of cs nps and to determine their colloidal stability at different storage temperatures and in various suspending mediums . low molecular weight chitosan ( 70 kda with the degree of deacetylation 75%85% ) , acetic acid glacial , phosphate buffered saline ( pbs ) , bovine serum albumin ( bsa , 46 kda ) , and bradford reagent was purchased from sigma - aldrich inc . , usa . double - stranded sirna ( sense : 5-gauuauguccgguuauguauu-3 , antisense : 3-uacauaaccggacauaaucuu-5 ) was purchased from thermoscientific dharmacon , usa . protein ladder ( high range ) , laemmli sample buffer , 10x tris / glycine / sodium dodecyl sulfate buffer , ammonium persulfate , tetramethylenediamine ( temed ) , 2% bis solution , and 40% acrylamide solution were purchased from bio - rad , usa . tris - hcl buffer was obtained from invitrogen , usa . all other chemicals used were of analytical grade . cs and ds solution were dissolved in 1% v / v acetic acid and distilled water , respectively . ph of cs solution was adjusted to ph 4 by adding 1 m naoh or 1 m hcl . ds solution ( 0.05% , 0.1% , 0.15% , 0.2% , and 0.25% w / v ) was added dropwise into cs solution ( 0.1% w / v ) under magnetic stirring ( wisestir digital multipoint magnetic stirrer ms - mp8 , daihan scientific , korea ) at 250 rpm for 15 min to form nanoparticles . the resultant nanoparticles were washed and harvested by ultracentrifugation ( optima l-100 xp ultracentrifuge with a rotor nv 70.1 , beckman - coulter , usa ) twice at 12 500 rpm for 15 min at 10c . for bsa association into cs nps , bsa was dissolved in cs solution ( 0.1% w / v , ph 4 ) to produce a final concentration of 1 mg / ml . for sirna association into cs nps , 3 l of sirna ( 15 g/l ) in deionized water was added to ds solution ( 0.05% , 0.1% , 0.15% , 0.2% , and 0.25% w / v ) before adding this dropwise to cs solution ( 0.1% w / v ) . electrophoretic mobility measurements ( e ) of cs nps were performed with a zetasizer nano zs ( malvern instruments , uk ) and e was measured against waiting time . particle size , surface charge , and polydispersity index ( pdi ) of freshly prepared cs nps were measured using a zetasizer nano zs ( malvern instruments , uk ) , based on the photon correlation spectroscopy ( pcs ) techniques . morphological characterization of unloaded cs nps , bsa / sirna loaded cs nps ( ds : cs weight ratio of 0.5 : 1 , 1 : 1 ) was carried out by using transmission electron microscopy ( tem ) , tecnai spirit , fei , eindhoven ( the netherlands ) . bsa / sirna loaded cs nps were separated from the solution by ultracentrifugation ( optima l-100 xp ultracentrifuge with a rotor nv 70.1 , beckman - coulter , usa ) at 14000 rpm for 30 min . bsa content in the supernatant was analyzed by a uv - vis spectrophotometer at 595 nm ( u.v-1601 ; shimadzu , japan ) using the bradford protein assay as per manufacturer instruction . sirna content in the supernatant was analyzed by a uv - vis spectrophotometer at 260 nm . the bsa / sirna entrapment efficiency ( ee ) was calculated using the following equation:(1)ee(%)=(total amount of bsa / sirna added)(free amount of bsa / sirna)(total amount of bsa / sirna added ) 100 . freshly prepared cs nps ( made from 0.05% and 0.1% w / v of ds and cs solution , resp . ) were centrifuged at 12 500 rpm for 15 min prior to storing . after ultracentrifugation , the obtained pellets were resuspended in either distilled water ( measured ph of 6.6 ) or pbs ph 7.4 . the particle size and surface charge were measured at predetermined storage time durations ( 0 , 1 , 2 , 3 , 5 , 8 , and 14 days ) , and at either ambient temperature or 4c . the release of bsa / sirna was determined from cs nps with the highest ee ( ds : cs ratio 1 : 1 , ee = 98% 0.2 and 95 4 , resp . ) . bsa / sirna loaded cs nps were suspended in tris - hcl buffer solution ( ph 7.4 , 4 ml ) and placed on a magnetic stirrer with a stirring speed of 100 rpm at 37c ( ms mp8 wise stir wertheim , germany ) for 48 h at 37c . at predetermined time intervals ( 0 , 0.5 , 1 , 2 , 4 , 6 , 12 , 20 , 24 , and 48 h ) , samples were centrifuged at 14 000 rpm for 30 min at 10c . then , the supernatant was decanted and replaced with an equivalent volume of fresh buffer solution . the amount of released bsa / sirna in the supernatant was analyzed by a uv - vis spectrophotometer ( u.v-1601 ; shimadzu , japan ) at a wavelength of 280 and 260 nm , respectively . the integrity of bsa released from cs nps was determined by sds - page ( 12% resolving and 10% stacking gel ) using mini - protein system ( bio - rad , usa ) . bsa samples were mixed with laemmli sample buffer in 1 : 1 ratio and heated at 95c for 5 min . samples ( 15 l ) were loaded into the wells and the gel was run using a mini - protein system tetra cell at a constant voltage of 150 v for 90 min with a running buffer containing 25 mm tris , 192 mm glycine , and 0.1% sds at ph 8.3 . the sample bands were stained for 40 min with 0.1% coomassie blue solution containing 40% acetic acid and 10% methanol , followed by staining overnight with a solution of 40% acetic acid and 10% methanol . all the data were presented as mean standard deviation ( sd ) . statistical analysis ( anova test and tukey 's posthoc analysis ) was performed by using the statistical package for the social ( spss ) programme version 15 . figure 1(a ) demonstrates the results of electrical mobility ( e ) against waiting time . from the graph , it could be observed that the e remained plateau and constant after 30 min . this demonstrates that the formation of stable electrical double layer ( e.d.l . ) was not instantaneous but required some moments . the effects between cs concentration and ds final concentrations on the size of cs nps are presented in figure 1(b ) . it was observed that most of the cs nps with the size of less than 500 nm were obtained at a low cs concentration ( 0.1% w / v ) . an increasing trend in particle size could be observed with increasing the ds concentration from 0.05 to 0.25% w / v . in general , ds concentration of 0.05% w / v ( low concentration ) produced nanoparticles with particle size less than 500 nm . contrary to that , large nanoparticles ( > 1000 nm ) were obtained when concentration of both polymers was increased to 0.25% or above . based on the results , ds concentrations from 0.05 to 0.20% w / v were selected for the following studies . furthermore , an increase in the ds : cs weight ratio ( higher density of negative charges from ds present in the system ) led to an increase in particle size but a decrease in particle surface charge ( table 1 ( above ) ) . as the cs weight exceeded the mass of ds , a positive value of + 56.2 1.5 mv was obtained . however , particle surface charge decreased to 34.7 4.34 mv when more negatively charged ds was added . it was continuously decreasing when the ds : cs weight ratio had reached to 2.5 : 1 . this was expected to be due to an excess of ds molecules accumulated on the surface of nanoparticles . table 1 ( below ) shows that ds 0.2% w / v possessed the largest particle size after being loaded with bsa . particle size for ds at concentration of 0.1 and 0.15% w / v was also larger than the empty ones ( p < 0.05 ) . on the other hand , higher positive values of surface charge were observed for bsa loaded nanoparticles compared to the empty ones . moreover , higher ee values could be achieved by increasing the ds : cs weight ratio above 0.5 : 1 . the ee of nanoparticles at ds : cs weight ratio of 1 : 1 , 1.5 : 1 , and 2 : 1 was 98.6 0.2% , 88.5 5.0% , and 91.5 3.1% , respectively . the highest ee was obtained at a ds : cs weight ratio 1 : 1 ( table 1 ( below ) ) . table 2 shows that ds 0.2% w / v possessed the largest particle size ( 900 60 nm ) after being loaded with sirna . sirna loaded cs nps at different ds concentrations ( 0.05 , 0.1 , 0.15 , and 0.2% w / v ) showed smaller particle size . the ee of nanoparticles at ds : cs weight ratio of 0.5 : 1 , 1 : 1 , 1.5 : 1 , and 2 : 1 was 94 3% , 95 4% , 92 2% , and 90 2% , respectively . the images of the cs nps were obtained by tem ( figure 2 ) . figures 2(a ) and 2(b ) show that unloaded cs nps exhibited a spherical structure . the images demonstrated that nanoparticles generated from sirna ( figures 2(e ) and 2(f ) ) showed irregular morphology ; however , bsa loaded nanoparticles displayed elongated morphologies ( figures 2(c ) and 2(d ) ) . both nanoparticles made from 0.05 and 0.10% w / v ds were increased in size over time as shown in figure 3(a ) when stored at ambient temperature . a significant increase in particle size this finding corroborated with the results of surface charge which showed a decrease in surface charge to nearly neutral . in contrast , when they were stored at 4c , their particle size and surface charge remained unchanged up to 14 days for nanoparticles made from 0.10% w / v ds . a slight change was observed for 0.05% w / v ds ( figures 4(a ) and 4(b ) ) . on the other hand , when these nanoparticles were suspended in pbs ph 7.4 , all formulations were aggregated to larger sizes of more than 1 m with pdi values more than 0.5 . their particle surface charges were also nearly neutral , ranging from + 0.2 to + 2.5 mv . figure 5(a ) illustrates that the release of bsa could be divided into two stages based on the release rate . in the first stage , the bsa was rapidly released from the cs nps and showed a burst release in the first 6 h. this resulted in a 45% 5 cumulative release of bsa . in the second stage , bsa was slowly released from 6 h up to 48 h , resulting in a cumulative bsa release of more than 60% . the integrity of bsa released from cs nps was evaluated by sds - page and is shown by figure 5(b ) . the bands observed confirmed that bsa that had endured the loading and release processes at 37c after days 1 and 2 were not different from that of freshly prepared bsa standards . therefore , it could be concluded that bsa remained in its native form in the cs nps under the experimental conditions . figure 6 illustrates that the release of sirna could be divided into two stages based on the release rate . in the first stage , the sirna was rapidly released from the cs nps and showed a burst release in the first 6 h. this resulted in a 58% 5 cumulative release of sirna . in the second stage , sirna was slowly released from 6 h up to 48 h , resulting in a cumulative bsa release of more than 85% . the method used to produce cs nps in the present study is a mild process , and it enables control of the particle size by varying certain parameters for example , concentration of added salts , viscosity , quantity of nonsolvent , and molecular weight of polymer . this study was started with the investigation to obtain information regarding electrical state of ionizable groups of cs nps by determining the stabilization time of e.d.l . the data obtained suggested that formation of stable e.d.l . during nanoparticles preparation required some moments after stopping the stirring . these moments were needed in order for the electrolytes to penetrate towards the particles nucleus . thus , waiting time of 40 min was needed before e of cs nps could be accurately measured . this finding was similar to the cs - tripolyphosphate ( cs - tpp ) nps which suggested the same waiting time . a study was also carried out to determine the influence of polymer concentration on particle formation . the study was aimed at establishing the range of polyelectrolytes concentration to produce nanoparticles with the desired size . to study the effects of the varying concentrations of cs and ds on the formation of nanoparticles , cs and ds solution of 0.1 , 0.25 , and 0.5% w / v were prepared . variable volumes of ds solution ( 1 , 2 , 3 , 4 , 5 , 5.8 , and 10 ml ) were mixed with 5 ml of each cs concentration ( 0.10.5% w / v ) . the final concentration of cs and ds was calculated , and sizes of samples were categorized either as 100500 , 5011000 , or more than 1000 nm . it was found that particle size was affected by the ds concentration . this finding corroborated with the results of cs - tpp nps . in general , the desired size of nanoparticles confined between 100 and 1000 nm . however , previous studies [ 19 , 20 ] have shown that the loaded nanoparticles would normally produce a larger size than the empty ones . furthermore , the results revealed that only ds concentration of 0.05% w / v was able to produce nanoparticles with particle size less than 500 nm as shown in table 1 . it was expected as when both polymers were in low concentrations , the addition of ds to the cs resulted in small coacervate nuclei . contrary to that , large coacervates which exceeded 1000 nm in size tended to form when both polymers concentration increased to 0.25% or above . chitosan 's ability of spontaneously forming coacervate is due to the interaction of oppositely charged polyelectrolytes to form a polyelectrolyte complex with reduced solubility . the mixture of high concentration of ds with cs is therefore more likely to affect the entanglement of the cs chains and solubility of the resulted complex . as a result , a high degree of complexation and coacervate will be produced . this allowed for a more efficient interaction between the cationic cs and oppositely charged ions , and thus a smaller particle size was produced . in addition , an increase and excess in the molar mass of the polyanion used resulted in larger particles because highly neutralized complexes were formed and they tended to flocculate . in this study , particle surface charge of the nanoparticulate system was dependent on the weight ratio of ds and cs . this relationship could be useful in obtaining the desired particle surface charge density to facilitate adhesion and transport properties of the nanoparticles . in present study , the incorporation of bsa into cs nps was achieved by simply mixing the acidic cs solution containing dissolved bsa molecules with the ds solution at room temperature without addition of stabilizer . bsa is frequently used as a model protein because it embraces the general characteristic of other proteins and it is biocompatible to humans . it was found that cs nps were comparatively larger in size after loading with bsa . this trend may be possibly due to the molecular weight and size of the added bsa molecules . nanoparticles of 150300 nm are found mainly in the liver and the spleen . besides , according to some reports , the ideal size requirement for nanoparticles developed for cancer treatment is between 70 and 200 nm . although nanoparticles should be not greater than 150 nm to cross the endothelial barrier , the literature always reports the penetration of particles larger than the limits of fenestrations . indeed , fenestration and the vasculature can undergo modification under various pathological conditions . for instance , tumor growth will induce the development of neovasculature characterized by discontinuous endothelium with large fenestrations of 200780 nm . besides , it was observed that the particle surface charge of bsa loaded nanoparticle was higher than the empty ones . this may be due to the cationic characteristic possessed by bsa when present in acidic condition . the positive charges from cs and bsa molecules therefore have contributed to a higher value of particle surface charge for the loaded nanoparticles . positively charged cationic polymers can effectively bind to and protect nucleic acids such as dna , oligonucleotides , and sirna . in this study , the incorporation of sirna into cs nps was achieved by simply mixing the acidic cs solution with the ds solution containing sirna at room temperature . it was found that particle size of cs nps was comparatively smaller in size after loading with sirna . the smaller size of cs nps loaded with sirna could be due to neutralization of negative charges of nucleic acid by cationic polymer resulting in condensed smaller sized nanoparticles . the sirna loaded cs nps also showed higher zeta potential than blank cs nps , following the same trend as that of bsa loaded cs nps . the sirna loaded cs nps showed higher entrapment efficiency ( < 90% ) for all ds : cs weight ratios . the entrapment efficiency of nanoparticles at ds : cs weight ratio of 1 : 1 , 1.5 : 1 , and 2 : 1 was higher than the weight ratio of 0.5 : 1 . this phenomenon was most probably due to higher proportion of ds presented in the nanoparticles . as more ds added , it would facilitate more bsa to be entrapped into nanoparticles . this could be explained by the fact that bsa is a zwitterionic molecule . at the ph of formulation medium of 3.54.0 , the solubility of bsa thus , bsa would be able to electrostatically attach and stably load into the nanoparticles . in acidic solution , bsa could possess positive charge and compete with cs to interact with ds electrostatically . this finding was corroborated with the increased positive surface charges of bsa loaded cs nps compared to unloaded ones . moreover , there are multi - ionic sites on bsa , and this feature could facilitate the incorporation of bsa into nanoparticles . this finding differs from the finding with cs - tpp nps . in the study , the electrostatic interaction was present between bsa and cs , instead of bsa and tpp . it was also suggested that bsa should dissolve in a solution with ph higher than its isoelectric point in order for bsa to possess negative charge and interact with the positively charged cs molecules . this finding therefore demonstrated that electrostatic interaction is the main contributing factor to promote the incorporation of bsa into nanoparticles either via cs - protein interaction or ds - protein interaction . tem allows nanoscale visualization of individual nanoparticles and provides information of both size and morphology . the particle morphology is an important factor for the colloidal and chemical stability as well as the bioactivity of nanoparticles . sirna loaded cs nps showed irregular morphology ; however , bsa loaded cs nps showed elongated morphology . this could be due to larger size of bsa which may entangle or act like a shield to cs , thus limiting the overall exposure of cs within structure . this information could provide a view about the stability of nanoparticles under different media and temperature . the stability of nanoparticles was investigated by assessing their variation in mean particle size and surface charge over time . at first , the nanoparticles were resuspended in distilled water at ph 6.6 which was filtered by 0.2 m filter to remove possible contaminants present in water . for this study , only nanoparticles made from 0.05 and 0.10% w / v of ds were tested . the particle size was up to 1643 442 nm and 2218 587 nm for 0.15% and 0.20% w / v ds , respectively . the increment of particle size may due to cs nps themselves erode and lose their spherical shape in an aqueous environment , and consequently the mean diameter of particle would rise as response to this erosion . furthermore , particle surface charges for the nanoparticles made from both concentrations were decreasing over time . it was suspected that cs may be degraded in aqueous media even though in absence of lysozymes . the results showed that cs nps were more stable when stored at 4c as their particle size and surface charge were unchanged or slightly changed up to 14 days . the results also suggested that cs nps should not be stored at ambient temperature as they are prone to degradation . the results therefore suggested that cs nps stored at room temperature are more prone to degradation than those that were stored in cool environment . it was probably due to the cool environment which may slow down the kinetic motion of nanoparticles . thus , the nanoparticles could maintain their spherical shape and erosion would be less likely to occur . moreover , it was observed that these nanoparticles were aggregated in pbs at ph 7.4 . this may attribute to lower particle surface charge of nanoparticles in pbs , near to neutral . particle surface charge which dropped to zero may indicate that cs nps had undergone charge cancellation by phosphate groups of pbs . the neutral charged status of these nanoparticles may cause losing of intra- and intermolecular forces , important to maintain the nanoparticles individually . as a result in contrast to pbs , distilled water can provide numerous hydrogen ions to form hydrogen bonds which can assist in breaking aggregation of nanoparticles by interacting with ionizable groups of cs nps . the in vitro release study of bsa and sirna from cs nps was carried out in tris - hcl buffer . the release of bsa and sirna could be divided into two stages based on the release rate . in the first stage , the drug was rapidly released from cs nps . the release of bsa and sirna at this stage might involve the diffusion of bsa / sirna bound at the particle surface . in the second stage , bsa / sirna the remaining bsa / sirna in cs nps would not completely be released until the particles were completely eroded or dissolved in release medium . this might have been due to the interaction between the remaining bsa / sirna and free amino group on the cs segments [ 28 , 29 ] . in addition , the synthesized system which has been previously described as being able to be formulated under mild conditions assured that the stability of the proteins loaded into cs nps was intact as determined by sds - page . in summary , this study shows that cs and ds concentration as well as ph were the parameters controlling particle size and surface charge of cs nps . nanoparticles less than 500 nm could be obtained at ds : cs weight ratio of 0.5 : 1 at ph 4 . in the case of bsa entrapment , nanoparticles with higher ds : the highest percentage of entrapment efficiency achieved was at 0.10% w / v ds ( ds : cs ratio of 1 : 1 ) . however , cs nps loaded with sirna showed high entrapment efficiency ( > 90% ) for all ds : cs ratios . storage temperature and suspending medium were found to be the factors that could influence the stability of cs nps . cs nps were labile and tend to destabilize at ambient temperature but withhold this labile behavior when cool environment ( 24c ) was provided . in addition , cs nps had better stability in distilled water than in pbs which might be due to hydrogen bonds that formed between water molecules and ionizable groups of cs nps .

chitosan nanoparticles ( cs nps ) exhibit good physicochemical properties as drug delivery systems . the aim of this study is to determine the modulation of preparative parameters on the physical characteristics and colloidal stability of cs nps . cs nps were fabricated by ionic interaction with dextran sulphate ( ds ) prior to determination of their storage stability . the smallest cs nps of 353 23 nm with a surface charge of + 56.2 1.5 mv were produced when cs and ds were mixed at ph 4 and with a ds : cs mass ratio of 0.5 : 1 . an entrapment efficiency of 98% was achieved when bsa / sirna was loaded into the nanoparticles . the results also showed that particle size and surface charge of cs nps were slightly changed up to 2 weeks when stored at 4c . greater particle size and surface charge were obtained with increasing the concentration of ds . in conclusion , nps were sufficiently stable when kept at 4c and able to carry and protect protein .

1. Introduction 2. Materials and Methods 3. Results 4. Discussions 5. Conclusions

PMC3016820

current use of laparoscopy in the evaluation and management of trauma patients has been a natural extension of this trend . although utilized for both blunt and penetrating injuries , laparoscopy has gained the most widespread acceptance as a useful tool in the management of patients with penetrating abdominal injuries . its ability to accurately determine anterior peritoneal penetration from stab and others have expanded its role beyond simply a screening tool for injury , to its current use in some centers as a diagnostic and therapeutic modality . the purpose of this study was to evaluate the role of laparoscopy in the trauma setting at our urban level ii trauma center . more specifically , we examined its use in the management of hemodynamically stable trauma patients with penetrating anterior abdominal injuries . we also sought to determine how our results compared with those of other centers regarding the widely accepted laparoscopic advantages of reduced morbidity , decreased rates of negative laparotomy , and shortened length of hospital stay ( los ) . we conducted a retrospective chart review of 4541 trauma patients who were admitted to our level ii trauma center between july 1 , 1999 and july 31 , 2002 . penetrating injuries accounted for 817 ( 18% ) of these trauma admissions . there were 209 penetrating injuries isolated to the abdomen , of which 116 were gunshot wounds ( gsw ) and 93 were stab wounds ( sw ) . the first group consisted of hemodynamically stable patients without evidence of abdominal peritoneal penetration based on physical examination , local wound exploration , or further confirmatory studies , including focused abdominal sonography for trauma ( fast ) , computed axial tomography ( ct ) , or diagnostic peritoneal lavage ( dpl ) , or all of these . the additional confirmatory studies were not applied in a standard fashion to all patients , but rather selected based on clinical indication and attending physician preference . in addition , stab wounds with peritoneal penetration , but no peritoneal signs , also underwent a course of observation . the second group of patients was those with peritoneal signs or confirmed evidence of abdominal peritoneal penetration based on physical examination , local wound exploration , or further confirmatory studies . the third group of patients was taken to the operating room for laparoscopic evaluation due to questionable peritoneal penetration . criteria for laparoscopy was a hemodynamically stable patient with an injury trajectory suggestive of peritoneal penetration that could not reliably be ruled out based on physical examination or further confirmatory studies . no attempt was made to laparoscopically assess the extent of the intraabdominal injuries or make therapeutic interventions . laparotomy was negative if it was discovered after the abdomen was opened that no peritoneal penetration had occurred . a nontherapeutic laparotomy was one that confirmed peritoneal penetration with either no intraabdominal injuries or injuries so minor that they did not require surgical repair . the patients in these 2 groups were closed without therapeutic intervention and admitted to the floor for postoperative recovery . the laparoscopic findings were described as either positive or negative based on evidence of peritoneal penetration . figure 1 details the algorithm used for management of our patients with penetrating abdominal wounds . injury severity score ( iss ) and length of hospital stay ( los ) were calculated for each patient in the study . morbidity and mortality figures were also generated for each patient from our computer database by using standard methodology to define associated complications . penetrating abdominal injuries accounted for 209 of our trauma admissions , of which 116 were gsw and 93 were sw . gender breakdown of these patients was male , 171 ( 82% ) and female , 38 ( 18% ) . thirty - three patients ( gsw=9 , sw=24 ) were observed in the emergency department based on their initial physical examination , radiologic studies , or additional confirmatory studies , or all of these . the majority of the gsw patients received immediate laparotomies due to the higher ballistic forces associated with these injuries . in general , gsw to the anterior abdomen required laparotomy unless the wound was tangential . table 1 provides the negative , nontherapeutic , and therapeutic outcomes in the formal laparotomy group with regards to the number and percentage of procedures and los for patients with gsw and sw . a review of the negative laparotomies revealed that possibly 8 of 10 gsw and all 14 stab wounds could have been done laparoscopically , based on the location of the entrance wound in the anterior abdominal wall . however , 2 of the negative gsw laparotomies assessed tangential wounds that would not have been well visualized by laparoscopy alone . all of these patients had anterior abdominal wounds , and 2 patients had flank wounds in addition to the anterior wounds . of these 22 patients , 7 had an additional confirmatory test prior to going to the operating room ( table 2 ) . eight sw were converted to an open procedure , and 7 had laparoscopy only ( table 3 ) . additional confirmatory test in patients managed with laparoscopy * gsw = gunshot wound ; ct = computed tomography ; fast = focused abdominal sonography for trauma ; sw = stab wounds ; los = length of hospital stay . results of laparoscopy group results of laparotomies after conversion table 5 lists the number and percentage of gsw and sw patients who underwent laparotomy , laparoscopy , or were observed . mortality was 13% ( gsw=20 , sw=0 ) for the laparotomy group , and 0% ( gsw=0 , sw=0 ) for the laparoscopy group . morbidity was 27% ( gsw=42 , sw=7 ) in the laparotomy group and 14% ( gsw=1 , sw=2 ) in the laparoscopy group . these numbers reflect the increased severity of injury with the laparotomy group . however , we found no difference in morbidity or mortality when we compared data for the negative laparotomy group and the negative laparoscopy group . management of gunshot wounds versus stab wounds mean los was 7.9 days for the laparotomy patients and 3.0 days for the laparoscopy patients . this difference can be attributed to the higher severity of injury associated with the laparotomy group . however , a more useful comparison of los is that between the negative laparotomy group and the negative laparoscopy group , which revealed an los of 4.1 and 1.9 , respectively . laparoscopic evaluation of the peritoneal cavity is not a new concept , having first been described by kelling over a century ago . the first reports demonstrating the utility of laparoscopic surgery in the evaluation of trauma patients were published soon thereafter in the 1920s . these early trauma studies investigated laparoscopy as a method to diagnose internal bleeding in patients with traumatic abdominal injuries . reports describing similar experiments were published sporadically throughout most of the last century . however , mainly due to technological limitations , it would be many years before laparoscopic surgical techniques would prove their practical utility and gain widespread acceptance . it is now evident that despite the lack of early enthusiasm in the surgical community for these new techniques , the laparoscopic pioneers correctly recognized the potential benefits of minimally invasive surgery in the trauma patient . recent technologic advances in optics and laparoscopic instrumentation have greatly broadened the applications for minimally invasive surgery . we now have a better understanding of the physiologic changes and complications associated with these procedures . increased training and utilization of minimally invasive procedures in all areas of surgery has come an increase in expertise among trauma surgeons . these techniques have evolved from what was once a novelty into an important part of every surgeon 's practice . for trauma patients , laparoscopy provides clear visualization of the peritoneal space and anterior abdominal wall , and unlike other diagnostic modalities , has the additional benefit of potential for therapeutic intervention . however , despite its increased use in trauma , the optimum role of laparoscopy in this setting has not yet been clearly defined . there continues to be variation among trauma centers on how best to optimize its application , taking full advantage of its benefits while overcoming its limitations . laparoscopy has been used as a screening , diagnostic , and therapeutic tool to evaluate both blunt and penetrating trauma at various centers . villavicencio and aucar authored an extensive review in 1997 in which they compared outcomes collected from 37 separate studies , involving over 1900 patients . a review of the data showed that it was most useful as a screening tool , missing only 1% of injuries and preventing 63% of patients from unnecessary laparotomies . the data were less encouraging when laparoscopy was used as a diagnostic tool , with missed injury rates reported between 41% and 77% . these numbers reflect a diagnostic accuracy that is unacceptable to most surgeons who have used the laparoscope less frequently in this manner . laparoscopy is infrequently used as a therapeutic tool ; however , several reports have demonstrated favorable results with laparoscopic repair of a variety of intraabdominal injuries , including the dia - phragm , liver , gallbladder , spleen and bowel . emergency department evaluation of the injured patient has evolved greatly over the years , based mainly on the technology of the time . trauma surgeons have had available a variety of diagnostics tools to assist with the management of their patients , including dpl , fast , and ct . the increased availability of laparoscopy now offers them even more flexibility during the workup of injured patients . certainly the evaluation of every trauma patient starts with the advanced trauma life support ( atls ) primary survey followed by a thorough physical examination . it is important to carefully inspect the patient 's wounds , because findings on the initial physical assessment usually determine the decision - making algorithm . local wound exploration is limited in its ability to determine specific intraabdominal injuries , but it can often determine peritoneal penetration and thereby avoid the need for further studies . in patients who are obtunded due to central nervous system dysfunction or intoxication as well as in those with distracting injuries to multiple body systems , the physical examination may be less reliable . wounds with tangential trajectories can also be difficult to accurately assess , necessitating additional diagnostic studies . diagnostic peritoneal lavage is simple , inexpensive , and can be quickly performed in the emergency department . however , dpl is limited by poor specificity , inability to accurately assess the retroperitoneum , and potential for missed hollow viscus or diaphragmatic injuries . it is also an invasive test that has some risk , albeit low , for procedural complications . although dpl has been considered the standard modality to assess traumatic intraabdominal injury for many years , its use is now declining in favor of more accurate , less invasive modalities , such as ultrasound ( fast ) and ct . we have significantly decreased the number of dpls performed at our institution in recent years as well , relegating its use to screening for late presenting potential hollow viscus injuries or rapid evaluation of blunt injuries if fast is unequivocal or unavailable . technological improvements have greatly enhanced the capabilities of ct and ultrasound , both of which now have an important role in trauma management . fast is now widely used in most trauma centers ; however , its major role is in blunt trauma . it has a low specificity for organ injury , but can effectively determine the presence of free fluid in the gravity dependent spaces of the peritoneal cavity . some centers have used positive fast examination findings as an indicator for laparotomy in penetrating trauma . improvements in ct speed and resolution have allowed for reliable evaluation of both the peritoneum and retroperitoneum . ct findings can often confirm or rule out peritoneal penetration based on a more clearly delineated wound trajectory or evidence of intraabdominal injury . unlike blunt injuries that can often be managed by observation and monitoring , ct confirmation of penetrating intraabdominal injury warrants laparotomy at our institution . the results of our study were consistent with those of other centers regarding the generally accepted laparoscopic advantages of decreased rates of negative laparotomy , shortened length of hospital stay , and quicker return to normal activity . the laparotomy patients had many more pulmonary complications including atelectasis , pneumonia , and respiratory failure . they also were found to have more wound complications including dehiscence , infection , and abscess formation . the decreased mobility of the laparotomy patients led to one deep vein thrombosis in this group . the results of our study did not show any difference in morbidity or mortality between the negative laparoscopy and the negative laparotomy groups . we found that a decrease occurred in los from 4.1 days in the negative laparotomy group to 1.9 days in the negative laparoscopy group . brandt et al looked at 21 trauma patients who underwent laparoscopic evaluation of both penetrating and blunt injuries , and found that emergency laparoscopy was 100 percent accurate in determining the need for laparotomy . a more recent retrospective review demonstrated a direct correlation between increased use of laparoscopy and decreased rates of negative laparotomy . our study revealed that laparoscopic evaluation spared many patients unnecessary laparotomies . after reviewing the operative reports of patients with negative laparotomies , it was determined that possibly 8 of 10 gsw and all 14 stab wounds could have been done laparoscopically . two of the 10 gsw patients had lateral or posterior injuries that required a more detailed evaluation of the retroperitoneum than would have been possible by laparoscopy alone . although we currently use the laparoscope solely as a screening tool for peritoneal penetration , the next logical progression for us is to conduct a more effective laparoscopic evaluation of specific organ structures in the trauma setting . this could potentially decrease or eliminate the conversions from laparoscopy to laparotomy that were nontherapeutic . this would entail a systematic laparoscopic evaluation of the entire peritoneal cavity , including running the small bowel , visualizing the diaphragm and solid organs , and accurately assessing for evidence of pelvic or retroperitoneal injury . the threshold for conversion would vary among surgeons based on laparoscopic expertise and confidence in the laparoscopic examination . data show that laparoscopy is a useful modality for evaluating and managing trauma patients with penetrating injuries . we have found that it is particularly helpful as a screening tool for anterior abdominal wall wounds and lower chest injuries to rule out peritoneal penetration . increased use of laparoscopy in select patients with penetrating abdominal trauma will decrease the rate of negative and nontherapeutic laparotomies , thus lowering morbidity , decreasing length of hospitalization , and provide for more efficient utilization of available resources . as technology and expertise among surgeons continues to improve ,

background : minimally invasive surgery has become increasingly utilized in the trauma setting . when properly applied , it offers several advantages , including reduced morbidity , lower rates of negative laparotomy , and shortened length of hospital stay . the purpose of this study was to evaluate the role of laparoscopy in the management of trauma patients with penetrating abdominal injuries.methods:we conducted a 3-year retrospective chart review of 4541 trauma patients admitted to our urban level ii trauma center . penetrating abdominal injuries accounted for 209 of these admissions . patients were divided into 3 treatment groups based on the characteristics of their abdominal injuries . management was either observation , immediate laparotomy , or screening laparoscopy.results:thirty-three patients were observed in the emergency department based on their initial physical examination and radiologic studies . after emergency department evaluation , 154 patients underwent immediate laparotomy . in this group , 119 therapeutic laparotomies , 11 nontherapeutic laparotomies , and 24 negative laparotomies were performed . a review of the negative laparotomies revealed that possibly 8 of 10 gun shot wounds and all 14 stab wounds could have been done laparoscopically . twenty - two patients underwent laparoscopic evaluation , 9 of which were converted to open procedures.conclusion:minimally invasive surgical techniques are particularly helpful as a screening tool for anterior abdominal wall wounds and lower chest injuries to rule out peritoneal penetration . increased use of laparoscopy in select patients with penetrating abdominal trauma will decrease the rate of negative and nontherapeutic laparotomies , thus lowering morbidity and decreasing length of hospitalization . as technology and expertise among surgeons continues to improve , more therapeutic intervention may be done laparoscopically in the future .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC5036132

qm / mm methods ( for reviews , see , e.g. , refs ( 17 ) ) have been used extensively in studies of enzymatic reactions . in order to obtain reliable results , it is crucial to perform extensive sampling and also to use a reliable qm model for either calibrating a semiempirical model ( e.g. , the evb model ) or actual determination of ab initio ( ai ) qm ( ai)/mm free energy surfaces . in considering the reliability of such methods , one of the obvious issues is the size of the qm region . the size problem is frequently attributed to the treatment of the boundaries between the qm and mm regions , but this issue is not so serious if one uses the same models in the reference water reaction and in the enzyme active site . nevertheless , one would like to know the limitations imposed by the size of the qm region . here we noted that the great advances in computer power and the use of specialized computers ( e.g. , gpu ) allows one to use very large qm regions and the results of the corresponding calculations can be used to imply that it is essential to use large qm regions to obtain reliable results . thus , it is important to conduct careful analysis of the size dependence of qm / mm calculations . the issue of the size dependence in qm clusters has been a subject of significant interest . similarly , the size dependence of the qm region in qm / mm models has been the subject of recent studies . some studies explored the size dependence of qm / mm calculations of spectroscopic properties . more relevant works ( from the perspective of this paper ) examined the energy difference in enzymatic reactions . however , as much as enzymatic reactions are concerned , the most important issue is the reliability of the calculated activation free energy , that should reflect an extensive averaging , that has not been performed in most of the previous studies . one exception is the study of ref ( 26 ) , which evaluated the free energy of proton transfer ( pt ) in lysozyme for qm regions of different sizes . this work found that for a qm with a radius of less than 6 we can have errors of about 3 kcal / mol . here we note that pt reactions involve a rather small change in charge separation and one can expect a larger dependence on the size of the qm region in reactions that involve a larger change in charge distribution . thus , we focus in this work on the dependence of the calculated activation free energy on the size of the qm region in the reaction of the enzyme comt ( catechol - o - methyl transferase ) that catalyzes the transfer of a methyl group from sam ( s - adenosyl - l - methionine ) to a catecholate ion via an sn2 type reaction , as shown in scheme 1 . the sam unit is truncated to the methyl group for clarification . the issue of the size of the qm region in comt was highlighted recently in a study which implied that the inclusion of large protein residues ( up to 500 atoms ) in the qm region is needed in order to obtain convergent results . while ref ( 11 ) only looked at the convergence of ground state properties including the donor acceptor distance , ref ( 12 ) examined the trend in activation energy , but this was done by evaluating the single point energy change upon moving to the transition state ( rather than the activation free energy ) . nevertheless , such results may discourage further use of qm / mm calculations with medium qm size and thus require further systematic studies that will be performed in this work . in choosing comt as our benchmark it belongs to the general class of methyltransferases that plays an important role in human physiology and several diseases such as cancer , and genetic diseases . comt degrades catecholamines such as dopamine and has thereby attracted a major interest in its probable role in dopamine related pathologies , in particular parkinson s disease . v158 m comt polymorphism is speculated to be responsible for the violent behavior of schizophrenic patients . it is therefore imperative to study such enzymes and understand their function at the molecular level . it should also be noted that the comt catalyzed decomposition of dopamine follows a parallel pathway to the reaction catalyzed by mao b , which was recently studied using the evb method . hopefully , the present study can also be useful by shedding additional light on the results and in some cases limitations of previous qm / mm studies of comt . our study focuses on the dependence of the activation free energy of the reaction of comt on the size of the qm region . for very small sizes where , for example , the mg is treated classically , we find an obvious deviation from the converging results ( although this problem can be fixed by semiempirical approaches with proper parametrization of the qm / mm interactions ) . for larger systems , our study used the semiempirical pm6 method ( evaluated by the mopac2009 package ) to treat the qm region , whereas the rest of the protein was treated classically , using the enzymix force field . the qm / mm interface between the qm and mm surfaces was implemented through the corresponding module of the molaris package . the initial coordinates for the qm / mm dynamics were taken from the crystal structure of the human , soluble form of comt ( s - comt ) . the first step for each system involved a relaxation by slowly heating the protein from 30 to 300 k for at least 45 ps . the subsequent md simulations involved time steps of 1 fs at 300 k. the simulation was divided into 31 frames with each frame simulated for 10 ps . ( o c ) , was constrained using a force constant of 100 kcal / mol and varied from 1.0 to 2.2 . the pmf was obtained by combining the individual simulation windows , using the weighted histogram analysis method ( wham ) approach . further calculations for a few systems ( i , ii , and iii ) were done for a longer time , by dividing the simulation into 51 frames and running each window for 30 ps . this yielded similar results to the shorter runs , and therefore , for all other systems , we only carried out shorter runs . the boundary conditions for the simulations were introduced by immersing the protein in an 18 sphere of water molecules using the surface - constraint all - atom solvent ( scaas ) boundary conditions . the local reaction field method ( lrf ) which is similar to having no cutoff for electrostatic interactions is used to treat the long - range effects . the geometric center of the reacting system was taken as the center of the simulation sphere . the positions of all atoms beyond 20 from the center of the systems were fixed as found in the initial crystal structure , and their nonbonded interactions with the atoms within the simulation sphere were turned off . to determine the protonation state , we used our coarse grained ( cg ) model . subsequently , all residues that lie within 12 from the center of the sam and catecholate anion were explicitly ionized during the simulation . a large charge charge dielectric constant was used to treat the effect of the more distanced ionizable residues . three different starting configurations were generated for the simulations by taking structures from a long trajectory at a time difference of 45 ps . the resulting structures were used in the qm / mm simulation , and the final result was taken as the average of the three runs . obviously , more correct barriers should be obtained with high level qm methods , but the issue here is the size dependence of the barrier and not obtaining an accurate barrier . of course , we hope that the size dependence of the pm6 is related to the size dependence in more rigorous models . our study focused on the activation barrier for the methyl transfer step , which is depicted in scheme 1 . the different residues considered as a part of the qm region are shown in figure 1 . the active site consists of mg ion coordinated to the catecholate ion , two aspartate residues , asparagine residue , and a water molecule . the transition state with the mg ion and the ligands in the primary shell is shown in figure 1c for a representative case . ( b ) a schematic representation of the active site of comt enzyme with some important residues . ( c ) the transition state for the methyl transfer reaction between sam and catecholate ion for a representative case ( distances are in ) . in order to explore the size dependence of the qm region the smallest region consisted of the substrates alone , while the largest region included the metal and seven residues , which were selected on the basis of the distance from the substrates . the first qm region investigated ( system i ) is described in figure 2 . this system includes only the two substrates , sam and the catecholate ion , and consists of 26 atoms in the qm region including the linked atoms . the mg metal ion was treated classically as a point charge with a repulsive van der waals center . system i with only the substrates ( catecholate and sam ) as the qm region and system iii with the substrates , mg ion , and the surrounding ligands as the qm region . the qm part is shown as sticks and part of the mm region as wires ( the rest of the protein is not shown for clarity ) . the next system considered ( system ii ) also included only the substrates in the qm region . however , the mg was treated using the dummy atom approach developed by qvist and warshel . this approach , which involves the inclusion of six dummy atoms around the metal center , as depicted in figure 3 , was shown to yield improved solvation energies . md6 model complex used instead of mg in the classical simulation of system ii , where the ds represent the dummy atoms and the ls are the general ligands around mg . subsequently , the mg ion and the surrounding ligands ( h2o , asp169 , asp141 , and asn170 ) were included in the qm region ( system iii shown in figure 2 ) . it should be noted that only a part of the residues as shown in figure 2 was included in the qm region . inclusion of the complete residue did not alter the result significantly , and hence , in all further calculations , we used chopped residues as the ligands coordinated to the mg ion . the change in the barrier upon moving to system iii reflects qm charge transfer effects , which are discussed below . next , we considered the effect of individual amino acid residues near the active site by including them separately in the qm region , while still retaining the residues in system iii ( figure 4 ) . the different residues considered are glu199 ( system iv ) , lys144 ( system v ) , tyr68 ( system vi ) , trp143 ( system vii ) , and met40 ( system viii ) . lys144 and glu199 form h - bonds with the catecholate ion and result in a charged qm system . the free energy barriers obtained for systems iv and v were 16.4 and 16.5 kcal / mol , respectively . during the simulations , the hydroxyl proton at the catecholate shuttles between the o atom of the catecholate and the o atom of the glu residue . as will be discussed below , this corresponds to another reaction path and should be considered accordingly . the inclusion of tyr 68 ( which is considered to be an important residue as its mutation decreases significantly the activity ) yields a barrier of 15.8 kcal / mol . the inclusion of met40 and trp143 yields activation free energies of 15.9 and 17.1 kcal / mol , respectively . we also included in the qm region tyr147 ( via ) and w38 ( viia ) , which are approximately 10 away from the center of the substrates . this yields barriers of 16.4 and 16.9 kcal / mol , which are similar to the barriers in other systems where the residues are much closer to the active site . the activation free energies in kcal / mol are provided in parentheses . after studying the effect of individual residues , we included both lys144 and glu199 ( system ix ) together with the mg metal and its ligands ( figure 4 ) . these two residues lie closest to the substrates forming weak h - bonding interactions and are therefore included in most of the systems described hereafter . further , we included three residues together keeping the mg and its surrounding ligands , lys144 and glu199 , constant . in system xiii , we studied the effect of another residue , trp43 , on the activation barrier . the different systems created ( figure 5 ) are ( a ) lys144 , glu199 , trp143 ( system x ) ; ( b ) lys144 , glu199 , tyr68 ( system xi ) ; ( c ) met40 , tyr68 , trp143 ( system xii ) ; and ( d ) trp 143 , trp43 , tyr68 ( system xiii ) . in the final and largest qm systems , we included four residues together , namely , lys144 , glu199 , trp143 , and met40 , resulting in system xiv and residues lys144 , glu199 , trp143 , and tyr68 that generated system xv . systems that include in the qm region three residues along with the mg and its surrounding ligands . the activation free energies in kcal / mol are provided in parentheses . in total , 15 different qm systems have been considered and it has been found that the free energy barrier lies in the range 14.519.4 kcal / mol . the largest system including seven residues does not show much improvement over the system containing the mg and ligands alone . on the basis of our results , we propose that mg and its surrounding ligands ( system iii ) are sufficient to be included in the qm region . furthermore , we also carried out qm / mm studies for the y68a mutant , where the experimental free energy barrier for the mutant is 20.0 kcal / mol whereas that of the wild type is 18.4 kcal / mol . in this mutant case , we only investigated systems iii and i. it was found that , while the calculated free energy for wild type with system i is 22.5 kcal / mol , the corresponding barrier for y68a is 26.8 kcal / mol ( leading to an increase of 4.3 kcal / mol in the barrier ) . in the case of system iii , the barriers for the wt and the mutant are 14.5 and 16.6 kcal / mol , respectively ( leading to a 2.1 kcal / mol increase in barrier ) . it can be seen that the trend on increase in barrier upon mutation is reproduced by both systems ( where system i gives a significant overestimate ) . the results of the above study are summarized in figures 6 and 7 as well as table 1 . the analysis of the results can start with the obvious finding that cases that do not include the ion and its ligands in the qm systems ( cases i and ii ) present a major approximation , since the corresponding qm treatment does involve major charge transfer ( ct ) . as we pointed out , the inclusion of the mg ion in the qm region requires inclusion of the ligands of this ion , and such a treatment can be very expensive when one uses a reasonable level of the qm treatment . fortunately , treating the mg ion classically and using the proper parametrization can give reasonable results . the pmfs obtained for different systems for the pm6/mm potential , which have been calculated using the wham procedure . all systems include the substrates : sam and catechol ( shown in wires ) . another case , which requires special attention , is the inclusion of glu199 in the qm system ( in addition to system iii ) without including additional residues . in this case , we find a partial proton transfer ( pt ) from the catecholate to glu199 . however , we actually have here a trivial case of another reaction mechanism ( namely , a pt to glu199 ) . of course , one should examine whether we have a real pt or an artifact of the use of the pm6 method . in fact , the problem disappears already when we add lys144 or additional residues to the qm region . we also find that adding lys144 alone to system iii also leads to a deviation in the reaction free energy . now again we have a residue that is involved in the reaction , where a pt from the catechol to the lys can be considered as an initial step of the reaction ( see also below ) . here a partial pt for the back reaction can appear in small qm regions , but again adding more residues leads to a disappearance of the deviations . we like to note that , although lys144 is the most likely candidate to accept the proton from catechol leading to the formation of catecholate ion , it is still unclear whether the proton stays on lys144 or is transferred to some other residue or water molecules . the close proximity of this residue to mg ( 3.2 ) in the crystal structure results in this ambiguity , as it may lead to the low pka of these residues . in fact , the finding that the lys144ala mutant leads to a minor reduction in kcat indicates that the lys does not play a major role in the rate acceleration . thus , although our previous evb calculations gave a somewhat larger catalytic effect with neutral lys144 , we kept this residue ionized here ( in the cases when it was in the classical region ) . regardless of the actual protonation state of the lys residue during the nucleophilic attack , our analysis of the size dependence is still valid for the model system used . overall , we note that , while the convergence of the activation free energies is encouraging , the convergence for the reaction free energies is not as good as that of the activation free energies ( table 1 ) , for single residues that can be involved in side reactions like pt . nevertheless , once other qm residues were included with the problematic residues , we obtained converging results . this feature was found for glu199 , lys144 , and , for less clear reason , trp143 , where in all cases adding more residues leads to the disappearance of this problem . qm / mm approaches provide a major tool for evaluating the energetics of chemical reactions in complex molecular systems . however , some workers can assume that the results depend drastically on the size of the qm system and thus presumably the qm / mm idea is inherently a major approximation . apparently , what is meant by approximation is not uniformly agreed upon . for example , some may assume that an accurate result can be obtained only when the protein and the solvent are represented by a high level ab initio approach . however , obtaining reliable results also requires a major sampling and satisfying the requirement of both a proper sampling and a reliable qm approach is an extremely challenging task that would be hard to accomplish in the near future . there are ways to use large qm systems , including our constraint dft ( cdft ) approach , or the use of a fast processor or specialized computers that allow running with large qm systems . however , the requirement of extensive sampling makes it crucial to exploit qm / mm approaches with a limited size of the qm region , while exploring systematically what are the limitations of such strategies . this work explored the approximations associated with the use of a relatively small size for the qm region , by a systematic analysis of this issue while modeling the free energy profile of the reaction of comt . as seen from figure 8 , the convergence of the activation free energy is quite reasonable ( except for models i and ii , as discussed below , the reasons for those deviations are obvious ) . convergence of the activation free energy as a function of the number of qm residues . the numbers in brackets are the number of qm atoms for the given system . the deviations between the results with different sizes of the qm region are more pronounced when we compare the calculated exothermicity ( table 1 ) . in analyzing the corresponding results first , we note that the cases that do not include the ion and its ligands in the qm systems present a major approximation , since the corresponding qm treatment does involve charge transfer ( ct ) . thus , we do have here an obvious case where one should try to include the mg in the qm treatment . fortunately , the ct effect can be captured by changing the charge on the mg and the ligands and by proper refinement of the van der waals parameter without the use of computationally expensive methods . the ct can also be represented by including the mg in an evb treatment that can reproduce this effect . furthermore , even without including the mg in the evb region , we can easily reproduce the trend obtained with the mg in the qm region by a standard evb treatment that includes parametrization of the reaction exothermicity . as stated in section iii , the convergence for the reaction free energy is not as good as the activation free energies . this is in particular true for single residues ( i.e. , glu199 and lys144 ) that can be involved in side reactions such as pt reactions . however , once such residues are surrounded by other qm residues , we obtain converging results . such a behavior seems to reflect instability of the qm calculations , which do not follow a simple additive trend . this is reflected in the observation that the problem does not occur when the problematic residues are part of the mm system . it is also likely that the size dependence will be reduced if we use a polarizable force field . it should also be noted that our reaction is not a conventional sn2 reaction , as it involves two charged species that react to give a neutral product , thereby posing difficulties that arise in an sn1 reaction . thus , the solvation free energy of the reactive ionic species might result in a sensitivity of the reaction free energy . some workers tend to attribute the main source of the size dependence problem to the connection between the qm and mm link atom . however , a part of this problem is drastically reduced by using hybrid orbitals . furthermore , the problem is drastically reduced if one uses the same boundaries for reactions in water and reactions in enzyme as is done with the evb method . it is important to point out that even with some dependence on the qm size it is possible to explore quite reliably the catalytic and mutational effect . this issue has been demonstrated here with the wt to y68a mutations in two different qm descriptions . such a finding , for a mutant that is the subject of significant controversy , is clearly important in view of the possible presumption that one must use large qm regions in exploring mutational effects . reference ( 12 ) obtained about 3 kcal / mol deviation from the final result for about 10 residues but then suggested that the convergence requires at least 30 residues ( where the deviation from the converging result is about 23 kcal / mol ) . ignoring the issue that the calculations are based on a single point energy evaluation , we like to address the implication that one needs to get around 2 kcal / mol convergence before exploring catalytic problems . first , obtaining 2 kcal / mol errors due to the size of the system still allows exploring enzyme catalysis , when the difference between the barrier in enzyme and in water is frequently on the order of 10 kcal / mol . of course , it is also allowed ( as shown here ) to explore mutational effects . second , no first principle approach can at present give errors of less than 2 kcal / mol . that is , changing the level of the quantum method can lead to several kcal / mol errors and adding a polarizable force field can change the results by a few kcal / mol . obviously not performing sampling can lead to enormous errors . furthermore , very large errors can also be obtained from evaluation of electrostatic contributions without proper relaxation of the protein . such calculations may produce an unphysical low dielectric that would not provide sufficient screening for charge charge interaction . apparently , even proper free energy calculations still involve several kcal / mol errors due to incomplete sampling , which , for example , can miss water penetration effects . the above - mentioned errors are inherent to current calculations , and once we are aware of such errors , we can better assess the effectiveness of different approximations . for example , the error can frequently be reduced by using a well - defined reference ( e.g. , the wt barrier in mutational studies and the reaction in water in exploring catalysis ) . as usual , the best way of exploring different approximations is to change the length of the run , the system size , and other factors and to explore the stability of the calculations as well as the relationship to the observed quantity . it might also be pointed out here that there are various implications that the catalytic effect deduced from calculations with limited qm size ( mainly the conclusion that the catalysis is associated with electrostatic effects ) might be questionable and can be changed with larger qm sizes . now , not only have evb calculations seemed to give extremely stable conclusions , but also equally importantly , other proposed catalytic factors ( e.g. , dynamical effects , quantum effects , and more ) have never been reproduced by consistent calculations regardless of the size used . overall , the present study indicates that the size dependence is not as crucial as one may suspect . in particular , the size dependence is significantly smaller than the catalytic effect . of course , the residues that are in direct contact with the reacting atoms can be involved in a major charge transfer or even in another reaction path . however , the qm description of residues that are not in direct contact do not change the activation barriers of chemical transformation in a major way .

although qm / mm calculations are the primary current tool for modeling enzymatic reactions , the reliability of such calculations can be limited by the size of the qm region . thus , we examine in this work the dependence of qm / mm calculations on the size of the qm region , using the reaction of catechol - o - methyl transferase ( comt ) as a test case . our study focuses on the effect of adding residues to the qm region on the activation free energy , obtained with extensive qm / mm sampling . it is found that the sensitivity of the activation barrier to the size of the qm is rather limited , while the dependence of the reaction free energy is somewhat larger . of course , the results depend on the inclusion of the first solvation shell in the qm regions . for example , the inclusion of the mg2 + ion can change the activation barrier due to charge transfer effects . however , such effects can easily be included in semiempirical approaches by proper parametrization . overall , we establish that qm / mm calculations of activation barriers of enzymatic reactions are not highly sensitive to the size of the qm region , beyond the immediate region that describes the reacting atoms .

Introduction Computational Methods Results Discussion Conclusion

PMC2773148

natural product phosphonates are in wide use in medicine and agriculture.(1 ) one member of this class of natural products , phosphinothricin ( pt ) , is an unusual amino acid with a phosphinate side chain ( scheme 1 ) . synthetic pt ( glufosinate ) is the active component of the commercially important herbicides liberty , ignite , and basta , which are used in conjunction with transgenic crops such as corn , soybean , cotton , and canola . the mode of action of pt is due to the unique carbonphosphoruscarbon moiety that mimics the -carboxylate of glutamate thereby inhibiting glutamine synthetase resulting in a toxic buildup of ammonia and glutamine starvation in the plant.(2 ) recent genetic and biochemical studies identified 2-hydroxyethylphosphonate ( 2-hep ) and hydroxymethylphosphonate ( hmp ) as intermediates in the pt biosynthetic pathway.(3 ) during pt biosynthesis , the carboncarbon bond of 2-hep is cleaved to afford hmp and formate by the product of the phpd gene , hydroxyethylphosphonate dioxygenase ( hepd , scheme 1 ) . initial characterization has revealed that hepd is a member of the cupin superfamily of proteins and that it only requires molecular oxygen and ferrous iron for activity.(4 ) 2-hep analogues deuterium - labeled at the c1 or c2 positions showed that the hydrogen atoms at c1 are retained in hmp and that one of the hydrogen atoms at c2 is present in formate . in addition , labeling studies with o2 and h2o demonstrated that hepd is a dioxygenase . the active site of hepd contains a 2-his/1-glu facial triad(5 ) characteristic of a large class of nonheme iron dependent enzymes.(6 ) a cocrystal structure , containing 2-hep coordinated to a cd - ion in the active site , demonstrated bidentate metal coordination of the substrate.(4 ) although hepd lacks significant sequence homology to other previously characterized enzymes , the crystal structure revealed that the active site architecture and tertiary structure of hepd are similar to 2-hydroxypropylphosphonate epoxidase ( hppe),(7 ) an enzyme involved in the biosynthesis of the phosphonate antibiotic fosfomycin.(8 ) hepd and hppe not only share similar structures but also act on similar substrates , 2-hep and ( 2s)-hydroxypropylphosphonate ( ( 2s)-hpp ) , respectively . on the basis of the initial characterization , two mechanisms were proposed for the transformation of 2-hep into formate and hmp ( scheme 2 ) . in both mechanisms , catalysis is initiated by bidentate coordination of substrate to the ferrous iron followed by binding of molecular oxygen to form an fe(iii)o2 species , which is postulated to abstract a hydrogen atom from c2 of 2-hep to form fe(iii)ooh ( i ) and a carbon centered radical . although these initial steps are not uniformly accepted for nonheme iron dependent enzymes , elegant studies by the laboratories of bollinger and krebs have provided spectroscopic and kinetic support for the feasibility of these steps for myo - inositol oxygenase ( miox ) . the proposed mechanistic pathways for hepd then bifurcate . in one mechanism , the ferric hydroperoxide and carbon - based radical proceed to form an alkylhydroperoxide and a ferrous iron center ( scheme 2 ) . the peroxy hemiacetal intermediate formed can then undergo a criegee rearrangement to afford o - formyl - hmp ( iv ) . hydrolysis of this intermediate at c1 ( necessary to account for observed isotope washout during the transformation(4 ) ) would afford the products . alternatively , if the substrate radical in intermediate i reacts with the terminal oxygen atom of the ferric - hydroperoxide , the substrate is hydroxylated and an fe(iv)=o intermediate ( ii ) is generated . the hemiacetal in intermediate ii can then undergo a retro - claisen - like carboncarbon bond scission affording formate and a hornerwadsworthemmons - like carbanion ( iii ) that is stabilized by metal coordination . this electron rich intermediate can then attack the oxygen atom of the electrophilic ferryl species to provide the observed products.(4 ) initial support for the hydroxylation mechanism came from the observation that hepd oxidizes 2-hpp to 2-oxopropylphosphonate ( opp ) , with the enzyme being inactivated in the process.(4 ) both proposed mechanisms can account for the oxidation of 2-hpp and the inactivation of hepd , but the mechanisms of enzyme inactivation are different ( scheme 3 ) . the hydroperoxylation mechanism predicts that the enzyme is inactivated via oxidation of ferrous iron by hydrogen peroxide produced in the criegee rearrangement whereas in the hydroxylation mechanism the metal is trapped in the inactive fe(iv ) state . since hydrogen peroxide was not observed in the previous study , the latter mechanism was favored.(4 ) to further investigate the mechanism of catalysis , in this study additional substrate analogues , putative catalytic intermediates , and labeled substrates incubation of 2-hep ( 2 mm ) with hepd ( 10 m ) resulted in the expected appearance of a peak in the p nmr spectrum corresponding to hmp . unexpectedly , after full conversion of 2-hep to hmp , the peak corresponding to hmp slowly decreased in intensity with the concomitant appearance of a new peak at = 2.3 ppm that was shown to correspond to phosphate by spiking with authentic material . when hmp ( 2 mm ) was incubated with hepd ( 10 m ) the recorded p nmr spectrum showed appearance of the same new peak ( figure 1a ) . to identify the carbon - containing product , synthetic h2hmp was incubated with hepd , and aliquots of the reaction organic acids were derivatized to the 2-nitrophenylhydrazide and analyzed via lc - ms , demonstrating time - dependent production of h - formate ( figure 1b ) . leaving out any component from the standard assay conditions ( hepd , hmp , or fe(ii ) ) did not result in production of formate . additionally , when 2-hep was incubated for extended periods of time with stoichiometric amounts of hepd , all substrate was consumed and two equivalents of formate were produced , one from conversion of 2-hep to hmp and formate , and one from the subsequent oxidation of hmp to phosphate and formate . hepd catalyzes oxidation of hmp to afford ( a ) phosphate ( pi ) , as evidenced by p nmr spectroscopy , and ( b ) h - formate from h2hmp as the carbon containing product in a time dependent fashion as evidenced by lc - ms . ofhmp ( iv , scheme 2 ) is a proposed intermediate on the hydroperoxylation mechanistic pathway . therefore , ofhmp was synthesized and presented as a potential substrate to ferrous hepd , the oxidation state of the enzyme that ofhmp would interact with as an intermediate ( scheme 2 ) . ofhmp is not particularly stable and was partially hydrolyzed to hmp and formate during workup of the synthetic procedure . the sample presented to hepd therefore contained both ofhmp and hmp in a 1:1 ratio . a control experiment showed that further hydrolysis is very slow under the hepd assay conditions without hepd . thus , a sample of ofhmp and hmp ( 1:1 ) was incubated with hepd and the reaction was monitored by p nmr spectroscopy revealing peaks corresponding to phosphate and ofhmp , and a hmp peak that had strongly decreased in intensity compared to ofhmp ( figure s1 ) . the ratio of the intensities of the peaks corresponding to phosphate and ofhmp was 1.0:0.9 indicating that most of the hmp present in the original mixture had been oxidized to phosphate , consistent with the data presented in the previous section . however , the experiment also showed that no enzyme catalyzed hydrolysis of ofhmp had occurred . given that hmp is a substrate for hepd , 1-hydroxyethylphosphonate ( 1-hep ) and 1-hydroxy-2,2,2-trifluoroethylphosphonate ( 1-hep - cf3 ) were tested as substrate analogues . under multiple turnover conditions ( 10 m hepd and 2 mm substrate analogue ) , ( 1.0 mm ) was then incubated with 0.5 equiv of hepd and , after acidic workup , two peaks were observed in both experiments . one peak corresponded to the starting material and the other peak at = 2.3 ppm corresponded to phosphate . to determine the other products generated from 1-hep , the substrate analogue ( 0.3 mm ) was incubated with hepd ( 0.1 mm ) for 2 h , followed by analysis of organic acids by lc - ms after derivatization as described in the experimental section . a peak was observed in the chromatogram with a retention time and mass consistent with derivatized acetate . the produced acetate was quantified ( 0.11 mm ) , closely corresponding to the amount of hepd in the assay . the phosphorus - containing product before acidic workup was then determined by incubating the analogue with hepd and analysis by p nmr spectroscopy ( figure 2a ) . the spectrum showed three peaks ; one for unreacted 1-hep , a small peak for phosphate and an additional peak at = 2.2 ppm . sulphuric acid was then added to the assay , the precipitated enzyme was removed by centrifugation , and another p nmr spectrum was recorded . the spectrum showed a peak for 1-hep and a peak for phosphate suggesting the third peak at = 2.2 ppm was associated with an acid labile intermediate that can be converted to phosphate under acidic conditions . this intermediate was then identified as acetyl phosphate ( ap ) by spiking with authentic material . because 1-hep was only converted when incubated with high concentrations of hepd , the enzyme is likely inactivated during the conversion of 1-hep to acetylphosphate . indeed , preincubation of hepd ( 2 m ) with acetylphosphate ( 100 m ) inhibited oxidation of its native substrate 2-hep . similarly , incubation of hepd ( 100 m ) with 1 equiv of ap decreased the rate of formate production by 67% compared to an assay without ap . p nmr spectra after incubation of hepd with ( a ) 1-hep , ( b ) 1-hep - cf3 . the p nmr spectrum for the reaction of 1-hep - cf3 with hepd also showed a peak for the starting material and a peak for phosphate ( figure 2b ) . to determine the identity of any nonphosphorus containing products , 1-hep - cf3 ( 0.3 mm ) was incubated with hepd ( 0.1 mm ) and the f nmr spectrum was recorded . two peaks were observed , with one peak corresponding to unreacted 1-hep - cf3 and a second signal corresponding to trifluoroacetate , as demonstrated with an authentic sample . integration of the peaks in the spectrum showed a ratio of 1:2 for trifluoroacetate:1-hep - cf3 . the amount of trifluoroacetate and phosphate formed was approximately equal to the amount of hepd in the reaction , suggesting that hepd was inactivated after converting 1-hep - cf3 to trifluoroacetate and phosphate . however , no reduction of formate production from 2-hep was observed after preincubation of hepd with trifluoroacetate by itself or with trifluoroacetate and phosphate . we previously reported that 2-hpp is accepted as a substrate for hepd and converted to opp.(4 ) to determine the stereochemistry of this transformation ( 2s)- and ( 2r)-hpp were prepared(12 ) and presented to hepd and a p nmr spectrum was recorded after 2 h. the spectrum showed oxidation for the r but not the s isomer . the stoichiometry of ( 2r)-hpp consumed to opp produced was determined by incubation of hepd with a 3-fold excess of ( 2r)-hpp and quantification of the opp generated via lc - ms . reaction of 0.1 mm , 0.25 mm , or 0.5 mm hepd with 0.3 mm , 0.75 mm , and 1.5 mm ( 2r)-hpp produced opp concentrations of 0.052 0.011 mm , 0.122 0.005 mm and 0.220 0.016 mm , respectively . these three experiments demonstrate stoichiometries of opp produced to hepd of 0.52:1 , 0.49:1 and 0.44:1 , respectively . in the hydroperoxylation mechanism , we previously showed that one equivalent of h2o2 rapidly inactivates approximately two ferrous hepd molecules,(4 ) and hence the observed stoichiometries at first glance appear to agree very well with the hydroperoxylation mechanism ( i.e. , each turnover of hepd results in one opp and 2 oxidized , inactive enzymes ) . one caveat is that complete conversion of ( 2r)-hpp to opp is assumed in this model . to probe if ( 2r)-hpp was cleanly converted to opp , hepd ( 0.5 mm ) was incubated with ( 2r)-hpp ( 1.5 mm ) . edta and dithionite were added to improve signal quality through sharpening of the peaks in the p nmr spectrum ( figure 3 ) that were very broad at the high enzyme concentrations used . the spectrum after 2 h showed peaks corresponding to ( 2r)-hpp , opp , and phosphate in a 1:0.4:0.33 ratio ; this ratio did not change after the initial enzymatic conversion . since all phosphorus - containing products originated from ( 2r)-hpp , the concentrations of ( 2r)-hpp , opp and phosphate are approximately 867 , 346 , and 286 m , respectively . to account for the presence of phosphate , opp was incubated under identical conditions with stoichiometric amounts of hepd and the p nmr spectrum was recorded , but no conversion of opp to phosphate was observed . then ( 2r)-hpp ( 1.5 mm ) was incubated with hepd ( 0.5 mm ) for 2 h and the sample was analyzed for organic acids . both acetate and formate ( 364 and 216 m , respectively ) were observed in significantly higher concentrations than in control reactions ( 62 and 16 m of acetate and formate , respectively ) . collectively , these results are consistent with partitioning of ( 2r)-hpp to generate opp , which is unreactive toward further enzymatic oxidation , in addition to acetate and hmp ( scheme 4 ) . subsequent oxidation of hmp produces the observed formate and phosphate products , as discussed above . p nmr spectrum after incubation of hepd with ( 2r)-hpp demonstrating formation of opp as well as inorganic phosphate ( pi ) . the previously proposed mechanisms(4 ) were restricted by the outcome of studies using either o2 or h2o , which provided complementary evidence that the hydroxyl oxygen in the hmp product was derived in part from molecular oxygen and in part from solvent . a third possibility that was not considered involves partial incorporation of the hydroxyl oxygen from 2-hep into hmp . a possible mechanism for such a process is shown in scheme 5 and in scheme s1 in the supporting information . after generation of the peroxy hemiacetal ( v ) , this species could be converted into a dioxirane intermediate ( vi ) . such a mechanism has precedent in reactions in organic solvents(13 ) and usually involves an activated oxygen leaving group , a role that might be played by an active site acid in the enzyme ( scheme 5 ) . the carbonyloxide resonance structures vii can be drawn for the dioxirane that then illustrate a mechanism for exchange analogous to that proposed for the observed exchange in organic solvents . for hepd , exchange of the hydroxide originating from the peroxy group with solvent and reformation of a peroxy hemiacetal could result in incorporation of oxygen from substrate and/or molecular oxygen ( scheme 5 ) , and/or solvent ( scheme s1 ) into the hydroxyl of the hmp product . to probe the feasibility of such a mechanism , the hydroxyl group in 2-hep was synthetically labeled with o,(14 ) and the labeled substrate analysis of hmp by lc - ms and of formate via derivatization to the tbdms ester and analysis by gcms revealed that the o label was present exclusively in formate and not hmp ( figure 4 ) . in combination with the observed 40% wash - in of oxygen derived from solvent into the hmp product,(4 ) the mechanism in scheme 5 is unlikely ( see also scheme s1 ) . blue circles indicate oxygen from substrate , orange circles indicate oxygen from o2 , and half filled circles represent oxygens with potential solvent wash - out / wash - in . ( a ) extracted ion chromatograms for hmp ( black line ) and o hmp ( gray line ) from reaction of 2-oh - hep . ( b ) mass spectrum of o formate ( 105 m / z ) , the peak at m / z 103 is spurious formate . other analogues of hep tested as potential substrates of hepd are shown in figure 5 . the compounds were incubated with hepd under both catalytic and single turnover conditions and the assays were analyzed via p nmr spectroscopy and lc - ms . both methods showed that none of the tested analogues gave rise to new products when compared to authentic standards . the studies with structural analogues of 2-hep presented here provide new information toward a better understanding of the mechanism of hepd . at present , we do not have direct evidence for the early steps in the proposed mechanistic cycle , but because external reducing equivalents are not required for the overall transformation , and because the substrate does not contain low energy electrons , there appears to be no pathway for formation of more reactive species than a formally ferric - superoxo intermediate . in addition , the results of the current study also strongly suggest the oo bond is not broken prior to hydrogen atom abstraction from substrate . two other nonheme iron dependent enzymes also achieve substrate oxidation without input of exogenous electrons . for myo - inositol oxygenase ( miox ) , bollinger and krebs and co - workers have provided strong spectroscopic and kinetic evidence that a ferric - superoxo species is formed reversibly in the confines of an enzyme active site and that it is capable of abstracting a hydrogen atom from a ch bond that is activated by a geminal hydroxyl group . dft studies on isopenicillin n - synthase ( ipns ) concluded that the formation of a ferric superoxo form of the enzyme is favorable only because of charge donation from a thiolate ligand from substrate ; similar calculations on phenylalanine hydroxylase , another facial triad , mononuclear nonheme iron dependent enzyme , demonstrated that oxygen binding to the ferrous enzyme was highly unfavorable.(17 ) in the latter computations , two water molecules occupied the sites that in hepd are occupied by alkoxide and phosphonate ligands of the 2-hep substrate . although the oxygen ligands of 2-hep may not provide the same charge donation as the thiolate ligand of the -(l--aminoadipoyl)-l - cysteinyl - d - valine ( acv ) substrate of ipns , substrate coordination in hepd , as well as in hppe,(7 ) may also enhance oxygen binding and activation . with respect to the steps after hydrogen atom abstraction , this study with substrate analogs strongly supports a hydroperoxylation pathway for hepd . this transformation is most readily explained as a criegee - like rearrangement ( scheme 6 ) in which a phosphorus atom migrates . acetylphosphate is shown to be a potent inhibitor of hepd explaining why only a single turnover occurs , with the inhibition likely due to the resemblance of acetylphosphate to hmp - formyl ester . the proposed phosphorus migration has precedent in baeyervilliger oxidation of dialkyl acylphosphonates in organic solvents that have been shown to proceed through a criegee intermediate.(18 ) the hydroperoxylation mechanism can also explain the observed conversion of hmp into phosphate and formate with formylphosphate a likely intermediate ( scheme 6 ) . unlike acetyl phosphate , which has been shown to be stable at neutral ph for months,(19 ) formylphosphate is unstable and readily hydrolyzes nonenzymatically to phosphate and formate as observed by benkovic and co - workers for the reaction catalyzed by purt gar formyltransferase.(20 ) hepd is regenerated in its fe(ii ) state in this model and , unlike with 1-hep , the enzyme is not inactivated due to the rapid hydrolysis of formylphosphate , accounting for the catalytic turnover of hmp to phosphate and formate . elucidating the details of the mechanism by which the alkylhydroperoxides are formed from a ferric hydroperoxide and after initial hydroperoxylation , criegee - like rearrangement would result in trifluoroacetylphosphate , which is more reactive toward hydrolysis than acetylphosphate and is not detected directly . why hepd is inactivated during this process resulting in a single turnover is not clear as trifluoroacetate is not an inhibitor . it is possible that trifluoroacetyl phosphate remains bound to the iron center after its formation in the active site and that hydrolysis only occurs after protein denaturation associated with product analysis . the results of the detailed investigation of the conversion of ( 2r)-hpp to opp are also consistent with initial hydroperoxylation followed by a criegee rearrangement . previously , we did not observe hydrogen peroxide production,(4 ) which appeared to be incompatible with the hydroperoxylation mechanism because stoichiometric conversion of ( 2r)-hpp to opp should produce 1 equivalent of h2o2 . half an equivalent of h2o2 has been shown to inactivate approximately 1 equiv of fe(ii)-hepd,(4 ) and thus 0.5 equivalents of h2o2 should remain upon oxidation of ( 2r)-hpp and should have been detected . however , as noted in the supporting information of our previous study , a caveat in this model was that the conversion of ( 2r)-hpp to opp could not be quantitated at the time . in this study , we were able to analyze the products by p nmr spectroscopy after solving previous problems with peak broadening . the results show that conversion of ( 2r)-hpp to opp is not quantitative and that ( 2r)-hpp oxidation partitions between formation of opp and hmp along with acetate ( scheme 7 ) . the latter reaction is similar to the physiological reaction , conversion of 2-hep to hmp and formate . presumably , an intermediate in this process is o - acetyl - hydroxymethylphosphonate ( viii ) that is hydrolyzed to acetate and hmp . the partitioning also explains why h2o2 was not observed in the previous study since only 0.6 equivalents of opp ( and hence h2o2 ) are formed with respect to hepd . this amount is sufficient to completely oxidize the enzyme , but very little h2o2 would remain after hepd oxidation . the partitioning between carbon migration and peroxide elimination suggests that adding an additional methyl group to c2 results in less favorable orbital overlap for a criegee rearrangement such that peroxide elimination competes . the observed conversion of ( 2r)-hpp to opp is reminiscent of the conversion by hppe of ( 2r)-hpp , the enantiomer of the physiological substrate , to opp . thus , when a hydrogen atom is available for abstraction at c2 , both hepd and hppe carry out the same transformation with ( 2r)-hpp . on the other hand , unlike hppe , hepd can not be coerced to abstract a hydrogen atom from c1 of ( 2s)-hpp . we note that the observed oxidation of ( 2r)-hpp and not ( 2s)-hpp by hepd is consistent with the active site geometry and bidentate binding mode of 2-hep observed in the cocrystal structure of cd - hepd with 2-hep.(4 ) unfortunately , attempts to obtain direct evidence for the hydroperoxylation / criegee rearrangement mechanism with 2-hep as substrate have so far been inconclusive . ofhmp , the penultimate intermediate in the hydroperoxylation pathway with 2-hep , was synthesized and presented as a substrate for hepd but enzymatic hydrolysis was not observed . this result may be rationalized by inability of the product to bind to the reactive form of the enzyme , which may only be accessed during turnover . alternative direct support for the hydroperoxylation followed by a criegee rearrangement may be obtained with 2-hep stereospecifically labeled at c1 . net inversion of configuration is predicted by this mechanism ( retention in the criegee rearrangement step and inversion for subsequent hydrolysis at c1 ) , whereas net retention of configuration is most likely for the hydroxylation mechanism ; experiments to examine these predictions are ongoing . in summary , the studies presented here with substrate analogues provide evidence for a hydroperoxylation mechanism for hepd that is followed by a criegee - like rearrangement . as such , the enzyme has similarities with flavin dependent bayer - villigerases , except that the substrate for hepd is at a lower oxidation level than the carbonyl - containing substrates of these enzymes . the corollary of the hydroperoxylation mechanism of hepd is that the oo bond of molecular oxygen is not broken prior to substrate activation , as has been proposed for the aforementioned nonheme iron enzymes miox and ipns , for a diiron(ii / iii ) superoxide model complex,(25 ) and for the copper - dependent enzymes dopamine monooxygenase , peptidyl glycine -hydroxylating monooxygenase , and galactose oxidase . we note that seto and co - workers in pioneering studies had proposed that phosphonoacetaldehyde was converted to hmp via a baeyervilliger like oxidation to ofhmp followed by nonenzymatic hydrolysis.(31 ) although the molecular details turn out to be different , the overall biosynthetic logic is remarkably similar . hepd was overexpressed in e. coli as an n - terminal his6-fusion protein and purified via affinity chromatography as previously described.(4 ) protein concentrations were determined by the method of bradford using bovine serum albumin as the standard . in addition , a theoretical extinction coefficient of 79 995 m cm was calculated using expert protein analysis system ( expasy , http://www.expasy.org ) and concentrations determined using this value were within 10% of the concentrations determined by the bradford assay . thus , while small deviations from the reaction stoichiometries reported herein are possible , they would not change the interpretation of these studies . all activity assays were performed in hepes buffer ( 25 mm , ph = 7.5 ) unless otherwise noted . lc - ms was performed on an agilent 1200 series quad pump system equipped with a diode array detector and a g1956b mass spectrometer with a multimode - electrospray / atmospheric pressure chemical ionization ( mm - es+apci ) source . the column used for separation was a synergi 4 fusion - rp 80a column ( 150 4.6 mm , 4 m , phenomenex torrance , ca ) using a flow rate of 0.5 ml / min . gc - ms was performed on an agilent 6890n gas chromatograph equipped with an electron impact ( ei ) ionization source . nmr spectra were recorded on a varian unity 500 or varian unity inova 600 spectrometer . proton and carbon chemical shifts are reported in values relative to an external standard of 0.1% tetramethylsilane in cdcl3 ( 0.00 ppm ) . phosphorus shifts are reported in values relative to an external standard of 85% phosphoric acid ( 0.00 ppm ) . fluorine shifts are reported in values relative to an external standard of cfcl3 ( 0.00 ppm ) . fast atom bombardment ( fab ) mass spectrometry for characterization of synthetic compounds was performed by the university of illinois mass spectrometry center using a waters 70-se-4f mass spectrometer . 2-hep and hmp were synthesized as previously reported.(4 ) the following substrate analogues were synthesized according to literature procedures : 2-oh - hep,(14 ) ( 2r)- and ( 2s)-hpp,(12 ) 3-hydroxypropylphosphonate,(32 ) 2-hydroxybutylphosphonate,(33 ) 3-fluoro-2-hydroxypropylphosphonate,(34 ) 2-fluoroethylphosphonate,(35 ) phosphonoacetaldehyde,(36 ) and 1-hydroxyethylphosphonate.(37 ) ethylphosphonate and aminoethylphosphonate were purchased from sigma - aldrich . n-(3-dimethylaminopropyl)-n-ethylcarbodiimide ( edc ) was purchased from chem - impex ( wood dale , il ) . all other chemicals were purchased from sigma aldrich at the highest purity and used without further purification . a round - bottom flask equipped with a magnetic stir bar and reflux condenser was charged with dibenzyl hmp(38 ) ( 500 mg , 1.7 mmol ) , which was dissolved in formic acid ( 3 ml ) and heated under reflux while stirring overnight . the solution was allowed to cool to 25 c , and et2o ( 20 ml ) was added . a solution of aqueous naoh ( 1 m ) was added dropwise until a second layer formed . the layers were separated and water was removed from the aqueous ( bottom ) layer under reduced pressure to afford product as a white solid . ofhmp was relatively stable in buffered hepes solution ( 25 mm , ph = 7.5 ) . h nmr ( 500 mhz , d2o ) 4.09 ( d , j = 8.5 hz , 2h , ch2 ) , 7.99 ( s , 1h cho ) ; c nmr ( 125 mhz , d2o ) 59.4 ( d , j = 156.9 hz ) , 163.5 ; p nmr ( 202 mhz , d2o ) 13.7 . under a nitrogen atmosphere a round - bottom flask equipped with a magnetic stir bar and reflux condenser was charged with commercially available diethyl 1-hydroxy-2,2,2-trifluoroethylphosphonate ( 1 g , 4.2 mmol , 1 equiv ) , which was dissolved in dry dcm ( 10 ml ) . to the solution was added bromotrimethylsilane ( 2 ml , 15.1 mmol , 3.6 equiv ) and the orange solution was heated under reflux while stirring for 2 h. the solution was allowed to cool to 25 c and solvent was removed under reduced pressure . the resulting residue was taken up in 1:1 h2o : etoh ( 10 ml ) and solvent was removed under reduced pressure to afford product ( 663 mg , 3.7 mmol , 88% ) as a tan oil . h nmr ( 500 mhz , d2o ) 4.09 ( m ) ; c nmr ( 125 mhz , d2o ) 27.76 ( d , j = 8.5 ) , 66.01 ( m ) ; p nmr ( 202 mhz , d2o ) 11.13 ( bs ) ; f nmr ( 215 mhz , d2o ) 17.34 ( at ) ; hrms ( fab ) calcd for ( c2h4f3o4p+h ) 180.9878 m / z found 180.9880 m / z . enzymatic assays were quenched by adding h2so4 ( 30 mm ) and the protein was pelleted via microcentrifugation ( 1 min at 16.1 g ) . the supernatant was analyzed via lc - ms ( positive mode ) using aqueous 0.1% formic acid as an isocratic mobile phase and scanning masses of 50500 m / z . hepd was separated from the assay via centrifugal filtration ( millipore micron ym-30 , 30 kda nominal molecular weight limit ) . d2o ( 20% v / v ) was added to the flow - through and the p nmr spectrum was recorded on a varian ui600 ( frequency for phosphorus : 242 mhz ) . following a modified literature procedure,(39 ) to a 100 l sample was added propionic acid as an internal standard ( 10 l of a 1 mm stock solution in 1:1 pyridine : hcl ) , followed by edc ( 10 l of a 0.29 m stock solution ) , and subsequently nph ( 10 l of a 0.12 m stock solution in 250 mm aqueous hcl ) . the orange solution was heated to 60 c in a water bath for 15 min and precipitated protein was pelleted via microcentrifugation ( 1 min at 16.1 g ) . the supernatant ( 20 l ) was analyzed via lc - ms using an isocratic mobile phase ( 50:50 meoh : h2o + 0.1% formic acid ) for separation . authentic standards of sodium c - formate , sodium acetate , and propionic acid were also derivatized and analyzed by monitoring the absorbance at 400 nm and by mm - es+apci ( negative mode ) and found to have retention times of 5.81 , 5.93 , and 6.93 min , respectively . the observed m / z values for each derivatized compound corresponded to the monodeprotonated hydrazide and were : 181 ( c - formate ) , 194 ( acetate ) , and 208 ( propionic acid ) . solutions of varying concentrations of each of these acids were made in hepes buffer ( 25 mm , ph = 7.5 ) , derivatized , and analyzed via lc - ms as described previously using single ion monitoring ( sim ) for the derivatized acid of interest . the area of the peak corresponding to derivatized acid was divided by the area of the derivatized propionic acid peak to afford a response factor , which was plotted against the acid concentration to afford a linear relationship . this equation was used to quantify the organic acid concentration in the enzymatic assays . in a typical assay hepd ( 10 m ) and hmp ( 2 mm ) were added to oxygenated buffer ( 25 mm hepes , ph 7.5 ) and incubated at 25 c for 2 h. the sample was then analyzed via p nmr spectroscopy ( proton coupled and decoupled ) . the carbon - containing product was identified by running an assay similar to the one described above except using h2hmp as the substrate and subsequent derivatization and quantification of organic acids as described above . in a typical assay , o - formyl-2-hydroxymethylphosphonate ( ofhmp , 2 mm ) was incubated with hepd ( 10 m ) for 2 h and samples were analyzed via p nmr spectroscopy . a control sample without hepd was also analyzed in a similar fashion . to determine which enantiomer of hpp is oxidized , hepd ( 500 m ) and either ( 2s)- or ( 2r)-hpp ( 1.5 mm ) were incubated in oxygenated hepes buffer ( 25 mm , ph 7.5 ) for 2 h at 25 c . the masses for protonated 2-hpp ( 141 ) and opp ( 139 ) were extracted from the total ion chromatogram ( tic ) to afford a single peak in each extracted ion chromatogram ( eic ) with retention times of 6 and 7 min , respectively , that were identical to authentic standards of 2-hpp and opp . to determine the stoichiometry of the oxidation of ( 2r)-hpp to opp via lc - ms , first a linear relationship between opp concentration and area of the peak in the eic was established by analyzing varying concentrations of synthetic opp in buffer ( 50 , 100 , 250 , 500 , and 1000 m ) . similar assays as previously described were then carried out with varying concentrations of hepd ( 100 , 250 , and 500 m ) and ( 2r)-hpp ( 3-fold excess over hepd ) , which were analyzed via lc - ms . to determine the stoichiometry of the oxidation of ( 2r)-hpp to opp by nmr spectroscopy , ( 2r)-hpp ( 1.5 mm ) was incubated with hepd ( 500 m ) for 2 h. edta ( 50 mm ) then dithionite ( 10 mm ) and d2o ( 20% v / v ) were added and the p nmr spectrum was recorded . the edta and dithionite were necessary to abrogate the line broadening effect of a high concentration of paramagnetic fe(iii ) in the sample . additionally , ( 2r)-hpp ( 1.5 mm ) was incubated with hepd ( 500 m ) for 2 h and the sample was analyzed for organic acids as described above . to identify the carbon - containing product of 1-hydroxyethylphosphonate , hepd ( 100 m ) was incubated in oxygenated buffer with 1-hep ( 300 m ) for 2 h at 25 c . the organic acids were then derivatized and analyzed via lc - ms as described above . to identify the product of 1-hep - cf3 , hepd ( 100 m ) was incubated in oxygenated hepes buffer with 1-hep - cf3 ( 300 m ) for 2 h at 25 c , then d2o ( 20% v / v ) was added and the f nmr spectrum recorded . standards of 1-hep - cf3 and sodium trifluoroacetate showed peaks at 72.5 ppm and 76.0 ppm , respectively . to identify the phosphorus containing product , 1-hydroxy ethylphosphonates ( 1.5 mm ) were incubated with hepd ( 500 m ) for 2 h. edta ( 50 mm ) then dithionite ( 10 mm ) and d2o ( 20% v / v ) were added and the p nmr spectrum was recorded . standards of 1-hep and 1-hep - cf3 under similar conditions had chemical shifts of 19.2 ppm and 7.3 ppm , respectively . phosphate , phosphite , and acetyl phosphate under these conditions had chemical shifts of 2.3 ppm , 4 ppm , and 2.2 ppm respectively . hepd ( 2 m ) and 2-oh - hep ( 2 mm ) were added to oxygenated buffer , incubated at 25 c for 2 h , and then analyzed via lcms as described above . the masses for protonated hmp and oh - hmp ( 115 and 117 , respectively ) were extracted to afford a single peak in each eic with a retention time of 5.4 min . additionally , formate was analyzed via gc - ms as previously reported(4 ) to determine the isotopic composition .

hydroxyethylphosphonate dioxygenase ( hepd ) catalyzes the o2-dependent cleavage of the carboncarbon bond of 2-hydroxyethylphosphonate ( 2-hep ) to afford hydroxymethylphosphonate ( hmp ) and formate without input of electrons or use of any organic cofactors . two mechanisms have been proposed to account for this reaction . one involves initial hydroxylation of substrate to an acetal intermediate and its subsequent attack onto an fe(iv)-oxo species . the second mechanism features initial hydroperoxylation of substrate followed by a criegee rearrangement . to distinguish between the two mechanisms , substrate analogues were synthesized and presented to the enzyme . hydroxymethylphosphonate was converted into phosphate and formate , and 1-hydroxyethylphosphonate was converted to acetylphosphate , which is an inhibitor of the enzyme . these results provide strong support for a criegee rearrangement with a phosphorus - based migrating group and require that the oo bond of molecular oxygen is not cleaved prior to substrate activation . ( 2r)-hydroxypropylphosphonate partitioned between conversion to 2-oxopropylphosphonate and hydroxymethylphosphonate , with the latter in turn converted to phosphate and formate . collectively , these results support a mechanism that proceeds by hydroperoxylation followed by a criegee rearrangement .

Introduction Results Discussion Experimental Procedures

PMC3463953

increasing daily physical activity ( pa ) is a key strategy for preventing or minimizing numerous chronic diseases [ 1 , 2 ] . daily pa consists of all occupational , domestic , and leisure - time activities . while participation in leisure - time activities has remained relatively stable over the years , technological advancements have drastically reduced occupational pa . people spent more time at sedentary occupations or in work - related activities such as passive commuting ( e.g. , driving / riding in a motorized vehicle ) , which has a negative impact on total pa . given the benefits of regular pa and the current high prevalence of physical inactivity , people need strategies to increase their level of pa . in 1996 , the american college of sports medicine proposed pa guidelines which stated that 30 minutes of moderate - intensity exercise on most days provide some health benefits , notably , a decreased risk of chronic diseases . moderate intensity pa is defined as activity performed at 36 met and corresponds with the equivalent of walking 3 - 4 mph for most healthy adults . because inactive people show measurable health benefits with only small increases in activity levels , these people should be targeted in health - promotion programs . the physical activity level ( pal ) of an individual is widely used as an indirect measure of physical activity energy expenditure . it can be measured or estimated from the average of 24-hour total energy expenditure ( tee ) and the basal metabolic rate ( bmr ) ( i.e. , pal = tee / bmr ) . the expression of energy requirements of adults as pals provides a convenient way of controlling for age , sex , weight , and body composition . a meta - analysis of studies that involved a total of 411 men and women from 18 to 64 years of age with a predominantly sedentary western lifestyle showed a mean value for pal of 1.60 ( range 1.55 to 1.65 ) for both men and women . a pal value of 1.75 and higher includes the regular practice of pa at work or in leisure time with an intensity and duration that will reduce the risk of becoming overweight and developing a variety of noncommunicable chronic diseases . research has revealed an association between a person 's physical activity level and his / her lifestyle . according to the 1981 fao / who / uno expert consultation , an active or moderate active lifestyle corresponds with a pal value between 1.70 and 1.99 . according to this expert panel , the daily performance of one hour ( either continuous or in several bouts during the day ) of moderate to vigorous exercise , such as jogging / running , cycling , aerobic dancing , or various sports activities , can raise a person 's average pal from 1.55 ( corresponding to the sedentary category ) to 1.75 ( the moderately active category ) . physical activity awareness defined as the agreement between self - rated and actual activity level according to current guidelines has rarely been studied as a determinant of healthy behavior change . people are only expected to consider changing their behavior when they become aware that they personally engage in too little pa and are potentially putting their health at risk . one way of raising awareness of individual physical ( in)activity levels compared with perceived ( in)activity levels is to monitor their physical ( in)activity , for instance , by using objective measures such as a pedometer or a multisensor activity monitor . several studies have promoted pedometers as effective tools to increase walking [ 10 , 11 ] . pedometers measure walking through step counts and have been proven to be both reliable and valid [ 13 , 14 ] . most of the research on pedometers has focused on their use as a measurement device for assessing pa and not as a motivational tool . recently , studies examining the effectiveness of pedometers in promoting pa have shown that pa programs consisting of a pedometer , a daily goal defined in steps , and a step diary will increase physical activity levels by 2500 steps on average , compared with a sedentary control group [ 1618 ] . it therefore has been concluded that pedometers can motivate individuals by monitoring the step count and operate as a tracking device and goal setting tool . although pedometers are easy - to - use motivational instruments , their major drawbacks are that they can not reflect intensity of movement and therefore result in inaccurate energy expenditure estimations . pittsburgh , pa , usa ) is a multisensory device that is worn on the upper arm and receives information from a dual - axial accelerometer to measure motion and multiple sensors to measure skin temperature , heat flux , and galvanic skin response . the swa also works with a watch - like display device that gives the user real - time feedback on calories burned , steps taken , and time spent in various intensity zones . time spent in moderate , vigorous , and very vigorous activities corresponds with met values greater than or equal to 3 and less than 6 , greater than or equal to 6 , and greater than or less than 9 , respectively . such a sophisticated accelerometer - based device has advantages over pedometers when energy - related output variables are a desired outcome . when people are given information on the energy expenditure ( kilocalories ) and the time spent ( minutes ) of physical activities above a predetermined intensity level as well as the number of steps , they will have a very detailed description of their own physical ( in)activity pattern . to our knowledge , no study has so far investigated whether specific feedback on various dimensions of the physical ( in)activity behavior leads to a greater increase of the individual 's physical activity level compared with more basic feedback on their number of steps . when people are being measured , they become more self - conscious about their own behavior . it is well documented that promoting pa using simple , attainable behavioral strategies such as self - monitoring , goal setting , and physical ( in)activity behavior feedback is effective for interventions . self - monitoring increases awareness of energy expenditure , enhances self - efficacy , allows for individuals to monitor progress and change over time , and thus can be an effective tool for behavioral change . goal setting has shown some promise in promoting dietary and pa behavior change among adults . cullen et al . developed a four - step goal - setting process which consists of ( 1 ) recognizing a need for change , ( 2 ) establishing a goal , ( 3 ) adopting a goal - directed activity and self - monitoring it , and ( 4 ) self - rewarding goal attainment . adding feedback and rewards to the goal - setting process feedback can be described as knowledge of personal status about one 's selected goal and should be provided regularly to adapt or adjust to the required behavior . the simple retargeting of information about the physical ( in)activity behavior to the individual can have immediate impact on their exercise behavior . this pilot study examined whether giving activity feedback to obese , sedentary adults with type 2 diabetes would improve their adherence to a home - based walking program . since the authors could not significantly contribute the increase in pa to the feedback condition , they concluded that further studies with larger sample sizes and greater control of experimental conditions were needed to determine the utility of objective activity feedback . the sensewear display is a recent innovation which allows progress monitoring throughout the day and indicates in real time when goals have been met . this technology can help motivate people towards predetermined activity goals allowing them to have a clearer picture of their physical ( in)activity pattern . setting specific goals is an important aspect of coaching as the effectiveness of coaching revolves around the nature of the goals established by the individual and the processes by which they are achieved . coaching is a behavioral strategy recently emerging in the literature and works as an effective strategy in achieving lifestyle change . the social interactions provided by a coach may provide useful forms of modeling and social support that can enhance the initial adoption of health behaviors such as pa . understanding the factors that can motivate health - enhancing pa has considerable value given the role of this lifestyle behavior in promoting quality of life . one theoretical perspective that appears useful for understanding motivation and adherence to exercise is the self determination theory . sdt particularly describes the processes through which a person acquires the motivation for initiating new health - related behaviors and maintaining them over time . the theory focuses on the degree to which people 's motivation toward engagement in activities , such as pa , is more or less self - determined ( autonomous ) or controlled by external or internal pressures . people are autonomously motivated when they engage in an activity or cease an activity for reasons that are freely chosen . in a pa context , people are autonomously motivated if they choose to initiate pa for enjoyment and/or because they think pa is important and will help them attain valued goals and/or because of personal commitment to improving their health or quality of life . research has shown that autonomous motivation leads to greater long - term persistence , for example , maintained change toward healthier behaviors . sdt proposes that humans have three basic psychological needs of autonomy , competence , and relatedness that must be satisfied in order for growth and well - being to be achieved . autonomy relates to the desire to be the regulator of one 's actions , and that behavior is volitional . people perceive themselves to be competent when they feel capable of attaining health outcomes , such as meeting a pa goal . the construct of perceived competence is very similar to the self - efficacy concept [ 32 , 33 ] , which has been found to be one of the strongest predictors of pa in adults . relatedness refers to experiencing care and concern from important individuals and feeling connected and understood by others . sdt argues that developing a sense of autonomy and competence is critical to self - regulate and sustain health - promoting behavior . equally important is relatedness , as people are more likely to adopt behaviors promoted by those whom they trust . over the years , the number of social factors that have been found to affect people 's needs and , in turn , motivation has been growing . how other people behave has tremendous impact on the needs being satisfied and , in turn , on motivation being optimal or not . one of the key interpersonal dimensions studied is autonomy support which can be defined as the active support of the person 's capacity to be self - initiating and autonomous . autonomous supportive environments can be established by acknowledging the feelings and perspectives of the individual , by providing sufficient information , by minimizing pressure and control , and by offering freedom of choice . silva et al . implemented an sdt - based intervention for weight management , facilitating exercise adherence by enhancing the more autonomous forms of behavioral regulation . patrick and canevello used a computerized intervention based on sdt to better understand the psychological mechanisms in regard to pa frequency , intensity , and duration in sedentary young adults . these studies highlight the positive influence that autonomy support can have on facilitating health behavior change as well as associated physical and psychological health benefits . well - designed randomized controlled trials ( rcts ) could help in understanding what types of interventions promote change in a particular behavior . research shows that individually tailored interventions are more likely to be read , saved , remembered , and discussed , and that goal setting in combination with self - monitoring is more successful [ 27 , 43 ] . we hypothesize that the use of the feedback generated by a sensewear display or a pedometer will increase awareness of employees own physical ( in)activity pattern and feelings of competence and results in an increased pal in comparison with a minimal intervention group . for motivational purposes , a simple , low - cost step counter may suffice , and these are more feasible from a cost - efficiency perspective than sophisticated accelerometer - based devices . because the swa display gives continuous feedback about people 's ( in)activity behavior and specific activity goals can be set , research is needed to compare the individual 's adherence with a healthy lifestyle when feedback is given by a pedometer or a multisensor activity monitor . this study has the additional purpose to compare the effectiveness of feedback given by a pedometer and a multisensor device . we hypothesize that activity monitors that display specific feedback on several parameters of an individual 's activity pattern , namely , steps , calories , and minutes spent on pa will motivate people to increase their activity level more than monitors who only track step counts . this theory states that the extent that the environment allows one to experience feelings of competence , autonomy , and relatedness will influence the person 's motivation toward a given task . the behavior of a coach has a strong impact on people 's needs being satisfied and , in turn , on their motivation being optimal or not . it can be speculated that if the relation with a coach leads to relatedness satisfaction , the message and values transmitted by the coach may be more readily internalized by the participant . furthermore , to the extent that the coach also supports the client 's autonomy , such internalization will be autonomous in nature , thereby leading to positive and long - lasting pa changes in the participant . our intervention study wants to determine the effectiveness of continuous self - monitoring and real - time feedback from the sensewear armband alone and in combination with a weekly meeting with a personal coach . we hypothesize that the weekly meeting with a coach will help create an autonomous supportive environment and stimulate the feeling of relatedness . we performed a 12-month randomized controlled trial in which we randomly assigned sedentary male ( n = 103 ) and female ( n = 124 ) employees to one of four intervention groups : a minimal intervention group received no feedback during the intervention period ( mig ; n = 56 ) ; a pedometer group received information on their daily step count and received a step diary to write down their daily steps ( pg ; n = 57 ) ; a display group got personalized feedback by means of the swa display and received a diary to write done their additional activities ( dg ; n = 57 ) ; a coaching group also received the display and diary and additionally had weekly meetings with a coach to discuss the progress they had made ( coachg ; n = 57 ) . during those meetings , the coach attempted to create an autonomy - supportive environment by encouraging choice and initiation ( how would you increase your activities this week ? ) , providing participants with different options ( maybe you could go for a walk during your lunch break or travel to work by bike ? ) , giving informational positive feedback , and acknowledging that the feeling of competence grows from feedback inherent to task ( well done , this week you participated at least more than one hour a day in pa ) . outcome measures were assessed during a baseline assessment and again at 3 , 6 , and 12 months . the primary outcome of the study is the level of physical activity ( pal ) . secondary outcome measures include step count , minutes of physical ( in)activity ( sedentary , light , moderate , vigorous , and very vigorous intensity pa ) , daily energy expenditure in pa , percentage of participants losing fat percentage , and stages of motivational readiness for pa . all participants were recruited from working places in flanders ( belgium ) through flyers , emails , pharmacists , and word of mouth . employees ( male and female ) aged between 19 and 67 years who mentioned not being physically active during the last year , who are interested in knowing how many calories they burn in daily life , and who are motivated wearing the sensewear armband during 6 weeks were recruited . all study participants progressed through the study , beginning with an initial contact in which participants are invited to go to an information session . at this session , potential participants received an explanation regarding the purpose of the study and study expectations . this discussion included the participant responsibilities and potential health benefits and risks related to study participation . all interested participants had to sign an informed consent approved by the medical ethics committee of the ku leuven and were scheduled to take part in a baseline measurement . during baseline measurement , participants completed standardized measurements including height , weight , resting blood pressure , resting heart rate , waist and hip circumference , and measurement of fat percentage . participants received the armband with oral and written instructions for proper wear and were given the opportunity to wear and adjust the armband . they were asked to wear the armband for 7 days to assess baseline physical activity level ( bpal ) . participants were asked to fill in the flemish physical activity computerized questionnaire ( fpacq ; ) about their self - reported physical ( in)activity pattern and were asked to complete some other questionnaires regarding their medical history , sports and activity history , and job status . previous research showed that both saturday and sunday and at least 3 weekdays needed to be monitored to obtain reliable measures of habitual physical ( in)activity . only participants with a bpal value lower than 1.71 , wearing the armband for more than 95% of the time and had at least 3 days of monitoring from monday through friday and both saturday and sunday measured , were eligible to continue . after successfully completing the baseline measurement and signing the informed consent , eligible participants were randomized into one of the four groups . during the randomization visit , all participants received an evaluation of their baseline measurement . the feedback report also gave information about the benefits of an active lifestyle , the current pa recommendations , how to get active in daily life , and tips for starting pa . our population includes participants with a bpal 1.71 met ( act ) , individuals that dropped out from the intervention trial ( do ) , and the four intervention arms ( mig , pg , dg , and coachg ) . the randomization process achieves a reasonable balance across the four intervention arms at baseline . after exclusion of the active group and the drop - out group , post hoc tests reveal a difference in bpal between the pg and dg of 0.08 met for the total group and 0.11 met for the male group . no difference in bpal exists between the pg and dg for the female group . followup data collection visits occurred at 3 , 6 , and 12 months after randomization and was collected between october 2010 and july 2012 . participants were also asked to fill in the fpacq to assess detailed information on several dimensions of pa and sedentary behavior over a usual week . after each followup measurement , participants received feedback about their physical ( in)activity level and a comparison was made with previous measurements . secondary outcome measures include step count , minutes of physical ( in)activity ( sedentary , light , moderate , vigorous , and very vigorous intensity pa ) , daily energy expenditure in pa , percent of participants losing fat percentage , and stages of motivational readiness for pa . during each assessment , several measures were obtained including height , weight , hip , and waist circumference , resting blood pressure , resting heart rate , and fat percentage . resting heart rate and resting blood pressure height and weight were assessed without shoes using a portable stadiometer of martin ( gpm anthropological instruments , zurich , switzerland ) and a digital scale ( seca , hamburg , germany ) . bmi is calculated as body weight / height . the same measuring tape with tension rod the waist was measured at the smallest circumference of the natural waist , just above the belly button . hip circumference was assessed with the measuring tape being placed just above both iliac crests . bioelectrical impedance analysis ( bia ) was used to obtain a complete analysis of the body composition ( percent distribution of body fat ) . the armband is a pa monitor that is worn on the upper left arm halfway between the acromion and olecranon processes . this device measures physical ( in)activity using accelerometry technology augmented by two heat sensors ( a thermistor - based skin surface sensor and a proprietary heat flux sensor ) and a galvanic skin response sensor . these four internal sensors turn on the monitor ( i.e. , with skin contact ) and estimate armband compliance , daily energy expenditure , step count , sleep efficiency , and the intensity , duration , and frequency of pa bouts . collected pa data all armband data were analyzed by computer - based software ( version 6.1 ) at 1-minute intervals using demographic information ( i.e. , gender , age , height , and weight at prior assessment ) and proprietary algorithms . throughout the study , the dg and coachg group participants used the self - monitoring device to aid behavior change via real - time lifestyle feedback targeting pa . baseline pa occurred over a one - year period ( july 2010july 2011 ) which makes it possible to account for seasonal variation in pa in a predominantly sedentary population . independent researchers have validated the sensewear system , especially with respect to comparing energy expenditure measurements from the armband to estimates from gold - standard laboratory equipment [ 4750 ] . an extensive review of the literature shows different validation studies where several sensewear models ( sensewear , sensewear pro , sensewear pro2 , and sensewear pro3 ) and software algorithms are used . the sensewear armband has been validated as a measure of daily pa with findings suggesting that both minute - by - minutes as well as average energy expenditure can be a valid measurement of free - living activity [ 51 , 52 ] . two recent studies [ 53 , 54 ] discussed the validation of the swa at higher intensity levels and found a significant underestimation of the armband compared to indirect calorimetry for vigorous and very vigorous exercise intensities . differences between the four study arms in pa outcomes all participants ' data will be analyzed according to their group assignment at randomization , regardless of adherence to the intervention . the pa outcomes being studied are daily energy expenditure ( dee ) , active energy expenditure ( aee ) , minutes of pa , physical activity level ( pal ) , and steps . a primary comparison is between the pa outcomes of groups who receive feedback ( step group , display group and coaching group ) and the group receiving no feedback ( minimal intervention group ) . we hypothesize that getting feedback on the pa pattern will alter the pal compared to having no information . secondly , a comparison will be made on the difference in pa variables between participants who received feedback by means of the swa display ( display group ) and participants who are only given information on their daily step count using a simple pedometer ( step group ) . since the swa display gives more detailed information on different pa variables ( minutes of pa , dee , and steps ) in contrast to the pedometer which only gives information on the daily step count , it is expected that people from the display group will have more benefits of the intervention compared to those belonging to the step group . finally , the additional effect of a coach having weekly meetings with the participants will be evaluated . we hypothesize that the power of the interaction of a coach will result in an increase of the pa outcomes compared to subjects only receiving information by means of a technological instrument ( display group and step group ) . differences in pa outcome measures will be examined using analysis of covariance ( ancova ) models of 12-month change scores . self - reported pa , awareness , and cognitions will be analyzed as secondary outcomes . all analyses take into account specified covariates , including age , gender , bmi , education , percentage of armband wearing time , and baseline values of pa outcome measures . based on our results from a pilot study ( n = 52 ; palpre = 1.41 0.11 mets ) , an a priori power analysis was performed to determine the sample size necessary to detect a significant increase in energy expenditure in the intervention groups . sample size calculations showed that 57 subjects in each group would give 80% power to detect a difference in pal between baseline and posttest at a significance level of p < 0.05 . all intervention groups receive a feedback report on the physical activity level , amount of steps taken , and minutes of physical ( in)activity . in a personal contact with a coach , the report is discussed . the intervention study was executed by one coach to maximize the reliability of the intervention trial . during the 4-week intervention period , this group ( 25 male , 31 female ) wears the sensewear armband but gets no further feedback . the pedometer group ( 26 male , 31 female ) receives feedback on the daily step count by means of a pedometer ( yamax digiwalker , sw200 ) and wears the sensewear armband for measuring the physical activity level . the yamax sw digiwalker is a waist - mounted device and is the most widely used pedometer in research studies . previous studies comparing 10 or more pedometer models identified it as one of the most accurate and reliable electronic pedometers available . every day participants of the pedometer group write down their amount of steps in a step diary and also mention the reason for walking 10,000 steps a day . after an intervention period of 4 weeks , the pedometer is returned and participants are asked to wear the sensewear armband for one more week without knowing the amount of steps taken every day . the display group ( 26 male , 31 female ) receives a sensewear display which allows participants to concurrently track total calories burned , minutes spent performing pa , and number of steps taken throughout the day . participants wear the sensewear armband and matching sensewear display for a period of 4 weeks . every day participants of the display group write down their amount of calories burned , their amount of steps taken , and their minutes of pa a day in an activity diary . they are asked to mention the reason for achieving or not succeeding in their personal goals . every participant has individualized target goals for total calories , number of steps , and minutes spent performing pa . the individualized energy expenditure goal corresponds with an increase of the individual physical activity level with 0.10 met . individuals are asked to walk 10,000 steps a day and to be physically active for 60 minutes a day . after an intervention period of 4 weeks , the sensewear display is returned and participants are asked to wear the sensewear armband for one more week without knowing the amount of calories burned , amount of steps taken , or amount of minutes spent in pa every day . the coaching group ( 26 male , 31 female ) receives the same intervention as the display group ( see above ) but additionally has a weekly meeting with a coach to discuss the progress they have made . the session format is as follows : check - in of sensewear data , discussion of results , evaluation of the individual goal setting , summary of current session , and preview of the next session . this intervention study will evaluate the effectiveness of four approaches to enhance pa in daily life over a one - year period in sedentary flemish employees . the focus of this study is to evaluate the efficacy of providing real - time feedback on physical ( in)activity patterns . secondly our intervention study will investigate whether feedback on several parameters ( including energy expenditure , steps , and minutes of physical ( in)activity performed ) will increase the physical activity level in comparison with a standard 10,000 step program . our final aim is to evaluate whether a weekly meeting with a personal coach will help create an autonomous supportive environment and stimulate the feeling of relatedness thereby influencing the pa behavior . it should be noted that the participants in this study are volunteers and , as such , may be different ( e.g. , psychologically or motivationally ) from other sedentary individuals .

purpose . the sensewear armband ( swa ) is a multisensor activity monitor that can be used in daily life to assess an individual 's physical activity level ( pal ) . the primary goal of this study was to analyze the impact of different types of feedback on the pal of flemish employees . methods / design . we recruited 320 sedentary employees ( age , 41.0 10.7 years ; bmi , 26.2 4.2 kg / m2 ) to participate in the 12-month study . participants were randomized into one of four intervention groups after being measured for 7 days and nights by means of the swa : ( 1 ) a minimal intervention group received no feedback ( mig , n = 56 ) ; ( 2 ) a pedometer group was provided only information on their daily step count ( pg , n = 57 ) ; ( 3 ) a display group received feedback on calories burned , steps taken , and minutes of physical activity by means of the swa display ( dg , n = 57 ) ; ( 4 ) a coaching group also received the display and had weekly meetings with a personal coach ( coachg , n = 57 ) . we hypothesize that participants receiving feedback ( sg , dg , and coachg ) will have a greater increase in physical activity outcome variables compared to participants of the minimal intervention group .

1. Introduction 2. Methods/Design 3. Intervention 4. Discussion

PMC4555192

a large amount of scientific research is directed towards anterior cruciate ligament ( acl ) injuries . however , recent epidemiological studies have demonstrated a continued high incidence of acl injuries despite the implementation of intervention programs . as these injuries remain a significant problem , research directed at rehabilitation is needed . an injured athlete is under pressure to return to competition as soon as possible and there is significant interest among sports medicine professionals in identifying an adequate indicator of safe return to activity [ 12 , 17 , 29 , 30 ] . between 43 and 92 % of athletes return to their sports within 612 months following acl reconstruction . another important fact to note is the unacceptably high re - injury rate of reconstructed acls . in addition to clinical examinations such as lysholm s score and the cincinnati knee ligament rating scale , functional performance tests are commonly used to predict athlete s abilities to participate in sports . some clinicians use isolated functional tests , such as hop tests [ 5 , 15 , 26 , 27 ] , but these tests have been criticized for not being suitable for the evaluation of sufficient functional capability in acl - reconstructed patients . muscle strength tests were used to identify differences between injured and non - injured legs . the results showed that the patients reported poor results with respect to knee function during activities and a high fear of re - injury despite sufficient muscle strength capacity in both legs [ 2 , 4 , 25 ] . this observation demonstrated that muscle strength tests are not sensitive enough to distinguish between the functional differences between injured and non - injured legs and time since - surgery criteria next to single functional test , there are a few studies that examined test batteries in acl - reconstructed patients as a measure of function [ 9 , 10 , 22 ] , but they did not provide information necessary for determining readiness for return to sport . most of these tests in a clinical setting were applied as an outcome measure based on scores obtained from the acl - reconstructed patient . in order to classify whether the test result can be considered as a functionally average outcome , there is no study that provides data of functional measurements in a healthy population as a reference . furthermore , most of these studies were conducted at least 1 year postoperatively ; investigations performed during the early rehabilitation period ( 36 months ) are missing . evaluations of progress over time , which are necessary to guarantee a confident postoperative return to training and competition within an appropriate time frame , are still a major concern . there is a need to identify complex , persistent functional deficits related to the initial acl reconstruction . to our knowledge , there is only one test battery that combines various subtests to broadly address sport - specific requirements . the large - scale setting design and lab - based materials in this study may limit a regular application . to support decision - making , we therefore developed and evaluated a standardized test protocol combining various subtests ( power , speed , agility , coordination , unilateral cutting and side - to - side movements ) that require no sophisticated equipment and can be used multiple times within the rehabilitation period . we classified test results into five different performance categories and established potential limb asymmetries in a healthy population . we hypothesized that the test battery represents the basis for a clinical setting by objectively determine the right time point for a safe return to sport post acl reconstruction . we further hypothesized that healthy individuals did not exhibit relevant limb asymmetries independent of the test requirements , age and gender . test results from an acl - reconstructed patient should be at least classified as a functionally good outcome to support a safe return to sports and to decrease the likelihood of re - injury by returning to sport too quickly . for reliability testing , data from twenty eight ( thirteen female , fifteen male ) healthy subjects ( age 24.1 2.5 years ) were obtained for each test . exclusion criteria included previous knee , hip or ankle injuries as well as current pain somewhere of the musculoskeletal system . before testing , each subject completed a 15-min warm - up at a submaximal level on a bike cycle , followed by 5 min of individual dynamic stretching and jumping . the order of the test stations were similar to the subsequent recommendations of the present test battery , starting with tl - st and ol - st on a mft challenge disc , followed by all jumping test with the myotester ( tl - cmj , ol - cmj , tl - py ) , the ol - sy and finally the tl - qft . after a rest of 3 min , the test was performed until three successful and valid trials were made . to ensure adequate recovery between tests , each subject got 5 min of rest between the test stations . test procedure and equipment were the same as used for the evaluation of normative data . the test leader of each station was the same between test and re - test . there was a 5-day interval between the tests , and subjects were asked to avoid strenuous physical activity 3 days before testing . over the 2-year period of data collection , the participants were not engaged in competitive sports and had no experience with activities similar to the exercises used in the test battery . prior to testing , the procedure , including possible risks and benefits , was explained to the subjects . they ( or , if under 18 years of age , their legal guardians ) were asked to read and sign the informed consent document . we only included subjects who performed all tests in the specified order . in total , data from 434 subjects were included in the study . for each subject , age , height , weight , body mass index ( bmi ) and the dominant leg based on the study by coren et al . who used similar survey items to determine a person s dominant leg , we asked the following question : which leg do you use to push a ball as strongly as possible ? based to the result , each participant had to perform the following two tests , in which the first leg used was defined as the dominant leg.climb a stairwalkerparticipants were prodded into action by us giving them a slight pushtable 1descriptive characteristics of healthy subjects in all age groupsage ( years)height ( m)weight ( kg)bmi ( index)dominant leg ( % ) children ( 1014 years ) male ( n = 57)10.9 ( 0.7)1.47 ( 0.06)39.8 ( 8.8)18.4 ( 3.4)98-right female ( n = 50)11.7 ( 0.7)1.52 ( 0.08)43.3 ( 9.5)18.8 ( 4.0)96-rightyouth ( 1519 years ) male ( n = 57)16.2 ( 0.8)1.77 ( 0.07)67.8 ( 10.8)21.6 ( 3.1)96-right female ( n = 70)16.1 ( 1.1)1.67 ( 0.06)59.8 ( 9.8)21.4 ( 3.5)97-rightyoung adults ( 2029 years ) male ( n = 51)24.8 ( 2.6)1.79 ( 0.06)74.8 ( 6.3)23.3 ( 1.7)85-right female ( n = 51)23.6 ( 2.5)1.65 ( 0.06)59.5 ( 7.1)20.9 ( 1.9)69-rightadults ( 3050 years ) male ( n = 51)38.2 ( 7.3)1.79 ( 0.05)77.3 ( 7.2)24.1 ( 1.8)86-right female ( n = 47)37.5 ( 6.1)1.69 ( 0.06)61.3 ( 7.0)21.5 ( 2.1)75-rightall values are expressed as means ( sd ) participants were prodded into action by us giving them a slight push descriptive characteristics of healthy subjects in all age groups all values are expressed as means ( sd ) before testing , the subjects completed a warm - up consisting of 10 min of stationary cycling ( female 1.5 w / kg , male 2.0 w / kg ) . the test battery included seven different tests conducted in a systematic order . for each test , two practice trials , followed by three approved trials , were performed . table 2 provides an overview of the test descriptions and variables measured . for the one - leg tests , all procedures were reviewed and approved ( id - number 022014 ) by the board of ethical questions in science of the university of innsbruck.table 2description of the test batterytestdescriptionstability test to assess postural control , tests were performed on an mft challenge disc ( tst trendsport , grosshflein , austria ) connected to a pc . while balancing on the disc , coordi software provides instant feedback about the position of the disc . to avoid the influence of different shoe types , all trials were performed without shoes . subjects were instructed to stand in the centre with their arms at their sides variable level of stability [ index ] two - leg stability test ( tl - st ) subjects had to stand with both legs on the disc while maintaining their balance for 30 s. data collection was immediately stopped in the case of a loss of balance one - leg stability test ( ol - st ) based on the two - leg test results , the test was performed with one leg . the subject was not allowed to stabilize the raised leg against the plate or standing legjumping test all jump tests were performed using myotest ( myotest s.a . , the subjects carried a belt around their hips , and the myotester was placed above the greater trochanter of the hip . before jumping , the subjects had to stand in an upright and still position variables jump height ( cm ) , power ( w / kg ) , ground contact time ( ms ) , reactivity ( mm / ms ) two - leg countermovement jump ( tl - cmj ) a sound signal from the myotester announced the start of the jump . from an upright position , the subjects quickly bent their knees and then immediately jumped upward , attempting to maximize their height . during this hop , arms were placed on the hips one - leg countermovement jump ( ol - cmj ) this was similar to the two - leg test , but this test was performed with one leg plyometric jump ( tl - pj ) the subject had to perform three consecutive two - leg jumps , focusing on a maximum jump height and a fast ground contact time . arms could be used to assist with the jump speedy jump ( ol - sy ) the speedy basic jump set ( tst trendsport , grosshflein , austria ) was used to create the jump coordination path ( right panel ) . the subjects performed one - footed jumps through the course of red ( forward backward forward jumps ) and blue ( sideway jumps ) hurdles , completing 16 jumps . this had to be performed as quickly as possible by jumping on one leg without a rest between the hurdles . twisting of the hip was not allowed , and the test was immediately stopped when the raised leg touched the ground or the subject had direct contact with the speedy basic jump hurdles . time was measured using two stopwatches beginning as soon as the subject started to jump and ending when he / she reached the finish line with one leg . the mean value was recorded for each jump variables time ( s ) quick feet test ( tl - qft ) again , the speedy basic jump set ( tst trendsport , grosshflein , austria ) was used for the quick feet test as displayed in the picture . the subject had to step in and out with one foot after the other until 15 repetitions were completed . arms could be used to maintain balance , and stepping on the speedy pole was not allowed variables time ( s ) description of the test battery to assess the test retest reliability , measurements from the first testing day were correlated with measurements from the same tester s second testing day . test retest reliability was determined using the intraclass correlation coefficient ( icc 1/1 ) in the one - way random effects model . this correlation value reflects the percentage of variance between the day - to - day measurements . to enable an objective evaluation , half of the standard deviation was both added to the mean value ( good , very good ) and subtracted from it ( weak , very weak ) . in total , seven categories were generated for each test based on gender and age . in addition , we calculated the lower limb symmetry index ( lsi ) for the one - leg stability test and countermovement jump to determine whether there was a side - to - side difference between the dominant- and non - dominant legs . the lsi is defined as the ratio of the dominant leg score and the contralateral leg score expressed as a percentage ( dominant / non - dominant 100 = lsi ) [ 8 , 24 ] . for the one - leg tests , we also defined specificity , expressed as the number of subjects classified as having a normal lsi divided by the total number of subjects . according to previous studies , for reliability testing , data from twenty eight ( thirteen female , fifteen male ) healthy subjects ( age 24.1 2.5 years ) were obtained for each test . exclusion criteria included previous knee , hip or ankle injuries as well as current pain somewhere of the musculoskeletal system . before testing , each subject completed a 15-min warm - up at a submaximal level on a bike cycle , followed by 5 min of individual dynamic stretching and jumping . the order of the test stations were similar to the subsequent recommendations of the present test battery , starting with tl - st and ol - st on a mft challenge disc , followed by all jumping test with the myotester ( tl - cmj , ol - cmj , tl - py ) , the ol - sy and finally the tl - qft . after a rest of 3 min , the test was performed until three successful and valid trials were made . to ensure adequate recovery between tests , each subject got 5 min of rest between the test stations . test procedure and equipment were the same as used for the evaluation of normative data . the test leader of each station was the same between test and re - test . there was a 5-day interval between the tests , and subjects were asked to avoid strenuous physical activity 3 days before testing . over the 2-year period of data collection , more than 450 subjects were randomly selected to participate in this study . the participants were not engaged in competitive sports and had no experience with activities similar to the exercises used in the test battery . prior to testing , the procedure , including possible risks and benefits , was explained to the subjects . they ( or , if under 18 years of age , their legal guardians ) were asked to read and sign the informed consent document . exclusion criteria included previous knee , hip or ankle injuries . additionally , none of the subjects exhibited evidence of acute pain . we only included subjects who performed all tests in the specified order . in total , for each subject , age , height , weight , body mass index ( bmi ) and the dominant leg were recorded ( table 1 ) . based on the study by coren et al . who used similar survey items to determine a person s dominant leg , we asked the following question : which leg do you use to push a ball as strongly as possible ? based to the result , each participant had to perform the following two tests , in which the first leg used was defined as the dominant leg.climb a stairwalkerparticipants were prodded into action by us giving them a slight pushtable 1descriptive characteristics of healthy subjects in all age groupsage ( years)height ( m)weight ( kg)bmi ( index)dominant leg ( % ) children ( 1014 years ) male ( n = 57)10.9 ( 0.7)1.47 ( 0.06)39.8 ( 8.8)18.4 ( 3.4)98-right female ( n = 50)11.7 ( 0.7)1.52 ( 0.08)43.3 ( 9.5)18.8 ( 4.0)96-rightyouth ( 1519 years ) male ( n = 57)16.2 ( 0.8)1.77 ( 0.07)67.8 ( 10.8)21.6 ( 3.1)96-right female ( n = 70)16.1 ( 1.1)1.67 ( 0.06)59.8 ( 9.8)21.4 ( 3.5)97-rightyoung adults ( 2029 years ) male ( n = 51)24.8 ( 2.6)1.79 ( 0.06)74.8 ( 6.3)23.3 ( 1.7)85-right female ( n = 51)23.6 ( 2.5)1.65 ( 0.06)59.5 ( 7.1)20.9 ( 1.9)69-rightadults ( 3050 years ) male ( n = 51)38.2 ( 7.3)1.79 ( 0.05)77.3 ( 7.2)24.1 ( 1.8)86-right female ( n = 47)37.5 ( 6.1)1.69 ( 0.06)61.3 ( 7.0)21.5 ( 2.1)75-rightall values are expressed as means ( sd ) participants were prodded into action by us giving them a slight push descriptive characteristics of healthy subjects in all age groups all values are expressed as means ( sd ) before testing , the subjects completed a warm - up consisting of 10 min of stationary cycling ( female 1.5 w / kg , male 2.0 w / kg ) . the test battery included seven different tests conducted in a systematic order . for each test , two practice trials , followed by three approved trials , were performed . table 2 provides an overview of the test descriptions and variables measured . for the one - leg tests , all procedures were reviewed and approved ( id - number 022014 ) by the board of ethical questions in science of the university of innsbruck.table 2description of the test batterytestdescriptionstability test to assess postural control , tests were performed on an mft challenge disc ( tst trendsport , grosshflein , austria ) connected to a pc . while balancing on the disc , coordi software provides instant feedback about the position of the disc . to avoid the influence of different shoe types , subjects were instructed to stand in the centre with their arms at their sides variable level of stability [ index ] two - leg stability test ( tl - st ) subjects had to stand with both legs on the disc while maintaining their balance for 30 s. data collection was immediately stopped in the case of a loss of balance one - leg stability test ( ol - st ) based on the two - leg test results , the test was performed with one leg . the subject was not allowed to stabilize the raised leg against the plate or standing legjumping test all jump tests were performed using myotest ( myotest s.a . , sion , switzerland ) equipment . the subjects carried a belt around their hips , and the myotester was placed above the greater trochanter of the hip . before jumping , the subjects had to stand in an upright and still position variables jump height ( cm ) , power ( w / kg ) , ground contact time ( ms ) , reactivity ( mm / ms ) two - leg countermovement jump ( tl - cmj ) a sound signal from the myotester announced the start of the jump . from an upright position , the subjects quickly bent their knees and then immediately jumped upward , attempting to maximize their height . during this hop , arms were placed on the hips one - leg countermovement jump ( ol - cmj ) this was similar to the two - leg test , but this test was performed with one leg plyometric jump ( tl - pj ) the subject had to perform three consecutive two - leg jumps , focusing on a maximum jump height and a fast ground contact time . arms could be used to assist with the jump speedy jump ( ol - sy ) the speedy basic jump set ( tst trendsport , grosshflein , austria ) was used to create the jump coordination path ( right panel ) . the subjects performed one - footed jumps through the course of red ( forward backward forward jumps ) and blue ( sideway jumps ) hurdles , completing 16 jumps . this had to be performed as quickly as possible by jumping on one leg without a rest between the hurdles . twisting of the hip was not allowed , and the test was immediately stopped when the raised leg touched the ground or the subject had direct contact with the speedy basic jump hurdles . time was measured using two stopwatches beginning as soon as the subject started to jump and ending when he / she reached the finish line with one leg . the mean value was recorded for each jump variables time ( s ) quick feet test ( tl - qft ) again , the speedy basic jump set ( tst trendsport , grosshflein , austria ) was used for the quick feet test as displayed in the picture . the subject had to step in and out with one foot after the other until 15 repetitions were completed . arms could be used to maintain balance , and stepping on the speedy pole was not allowed variables time ( s ) description of the test battery to assess the test retest reliability , measurements from the first testing day were correlated with measurements from the same tester s second testing day . test retest reliability was determined using the intraclass correlation coefficient ( icc 1/1 ) in the one - way random effects model . this correlation value reflects the percentage of variance between the day - to - day measurements . to enable an objective evaluation , normative data from healthy subjects were established for each test . to generate these data , the mean values in each category were calculated and were defined as average . to establish further categories , half of the standard deviation was both added to the mean value ( good , very good ) and subtracted from it ( weak , very weak ) . in total , seven categories were generated for each test based on gender and age . in addition , we calculated the lower limb symmetry index ( lsi ) for the one - leg stability test and countermovement jump to determine whether there was a side - to - side difference between the dominant- and non - dominant legs . the lsi is defined as the ratio of the dominant leg score and the contralateral leg score expressed as a percentage ( dominant / non - dominant 100 = lsi ) [ 8 , 24 ] . for the one - leg tests , we also defined specificity , expressed as the number of subjects classified as having a normal lsi divided by the total number of subjects . according to previous studies , to assess the test retest reliability , measurements from the first testing day were correlated with measurements from the same tester s second testing day . test retest reliability was determined using the intraclass correlation coefficient ( icc 1/1 ) in the one - way random effects model . this correlation value reflects the percentage of variance between the day - to - day measurements . to enable an objective evaluation , normative data from healthy subjects were established for each test . to generate these data , the mean values in each category were calculated and were defined as average . to establish further categories , half of the standard deviation was both added to the mean value ( good , very good ) and subtracted from it ( weak , very weak ) . in total , seven categories were generated for each test based on gender and age . in addition , we calculated the lower limb symmetry index ( lsi ) for the one - leg stability test and countermovement jump to determine whether there was a side - to - side difference between the dominant- and non - dominant legs . the lsi is defined as the ratio of the dominant leg score and the contralateral leg score expressed as a percentage ( dominant / non - dominant 100 = lsi ) [ 8 , 24 ] . for the one - leg tests , we also defined specificity , expressed as the number of subjects classified as having a normal lsi divided by the total number of subjects . according to previous studies , table 1 presents the descriptive characteristics and the number of participants in all age groups . the right leg was found to be the dominant leg for most of the participants . the icc indicated a high reproducibility for the tl - cmj and a moderate reproducibility for the tl - st . table 3 presents the icc values for all the functional tests.table 3icc values of the functional testsfunctional testunitlegtestretesticctl - stlevel2.60 ( 0.47)2.49 ( 0.36)0.688ol - stleveldoml2.51 ( 0.50)2.27 ( 0.49)0.763n - doml2.40 ( 0.45)2.31 ( 0.46)0.819tl - cmjheight ( m)38.1 ( 0.7)37.6 ( 0.6)0.921power ( w / kg)45.7 ( 7.8)47.2 ( 7.7)0.889ol - cmjheight ( m)doml23.6 ( 4.5)23.9 ( 4.1)0.897power ( w / kg)doml36.2 ( 8.1)36.0 ( 7.8)0.822height ( m)n - doml20.2 ( 5.2)21.1 ( 4.9)0.778power ( w / kg)n - doml32.8 ( 7.8)33.5 ( 8.5)0.814tl - pyindex2.0 ( 0.4)1.9 ( 0.5)0.838ol - stime ( s)doml6.3 ( 0.8)6.1 ( 0.7)0.792n - doml6.4 ( 0.9)6.3 ( 0.9)0.825tl - qtime ( s)8.7 ( 1.3)8.4 ( 1.0)0.803values are expressed as means ( sd ) icc values of the functional tests values are expressed as means ( sd ) for the data analyses , the subjects were categorized into the following 4 different groups according to their age : children ( 1014 years ) , youth ( 1519 years ) , young adults ( 2029 years ) and adults ( 3050 years ) . the establishment of the functional test values allowed for the creation of five normative categories . for each test , normative data were defined according to age group and gender . as an example , table 4 represents the five categories of normative data for the one - leg stability test . for all one - leg tests , all categories were colour - coordinated to facilitate classification as very good ( dark green ) , good ( light green ) , average ( yellow ) , weak ( orange ) and very weak ( red ) . results for the ol - st revealed a score of 98 % in male children , indicating a better performance ( less time ) for the non - dominant leg . in the female young adult and male adult groups , no differences could be found between the dominant and non - dominant legs ( lsi 100 % ) . only female adults showed better performance in the dominant leg compared with the non - dominant leg , with an lsi of 107 % . similar results , with small performance differences between the two legs , were reported for the ol - st , where the lsi ranged from 101 to 104 % . in contrast , high values of the lsi were found for the ol - cmj , indicating a better performance of the dominant leg ( better jump height ) . female children ( 118 % ) and male adults ( 124 % ) , specifically , exhibited large side - to - side differences.table 4examples of normative values for the one - leg stability test for the five categoriesnormdatanormdatafemale ; non - dominant legfemale ; dominant leglevel ( index)descriptionlevel ( index)description1.74very good1.76very good1.752.05good1.772.00good2.062.90average2.012.72average3.003.30weak2.732.96weak3.31very weak2.97very weaknormdatanormdatamale ; non - dominant legfemale ; dominant leglevel ( index)descriptionlevel ( index)description1.98very good2.00very good1.992.31good2.012.30good2.323.09average2.313.19average3.303.62weak3.203.49weak3.63very weak3.50very weakfig . 1limb symmetry index of all one - leg tests examples of normative values for the one - leg stability test for the five categories limb symmetry index of all one - leg tests table 5 shows the specificity of the three one - leg tests . the one - leg countermovement jump had the lowest specificity in all age groups , ranging from 40 % ( male adults ) to 63 % ( male children ) . independent of gender and age , the one - leg stability test showed a mean specificity ranging from 66 % ( male adults ) to 78 % ( male children ) . the test reached a specificity of greater than 90 % in female and male children , as well as in female and male young adults.table 5specificity results for all one - leg testsage categorygenderspecificity ( % ) ol - stol - cmjol - sychildrenfemale714193male786395youthfemale685388male675990young adultsfemale765192male744793adultsfemale714586male664087 specificity results for all one - leg tests the icc indicated a high reproducibility for the tl - cmj and a moderate reproducibility for the tl - st . table 3 presents the icc values for all the functional tests.table 3icc values of the functional testsfunctional testunitlegtestretesticctl - stlevel2.60 ( 0.47)2.49 ( 0.36)0.688ol - stleveldoml2.51 ( 0.50)2.27 ( 0.49)0.763n - doml2.40 ( 0.45)2.31 ( 0.46)0.819tl - cmjheight ( m)38.1 ( 0.7)37.6 ( 0.6)0.921power ( w / kg)45.7 ( 7.8)47.2 ( 7.7)0.889ol - cmjheight ( m)doml23.6 ( 4.5)23.9 ( 4.1)0.897power ( w / kg)doml36.2 ( 8.1)36.0 ( 7.8)0.822height ( m)n - doml20.2 ( 5.2)21.1 ( 4.9)0.778power ( w / kg)n - doml32.8 ( 7.8)33.5 ( 8.5)0.814tl - pyindex2.0 ( 0.4)1.9 ( 0.5)0.838ol - stime ( s)doml6.3 ( 0.8)6.1 ( 0.7)0.792n - doml6.4 ( 0.9)6.3 ( 0.9)0.825tl - qtime ( s)8.7 ( 1.3)8.4 ( 1.0)0.803values are expressed as means ( sd ) icc values of the functional tests values are expressed as means ( sd ) for the data analyses , the subjects were categorized into the following 4 different groups according to their age : children ( 1014 years ) , youth ( 1519 years ) , young adults ( 2029 years ) and adults ( 3050 years ) . the establishment of the functional test values allowed for the creation of five normative categories . for each test , normative data were defined according to age group and gender . as an example , table 4 represents the five categories of normative data for the one - leg stability test . for all one - leg tests , all categories were colour - coordinated to facilitate classification as very good ( dark green ) , good ( light green ) , average ( yellow ) , weak ( orange ) and very weak ( red ) . results for the ol - st revealed a score of 98 % in male children , indicating a better performance ( less time ) for the non - dominant leg . in the female young adult and male adult groups , no differences could be found between the dominant and non - dominant legs ( lsi 100 % ) . only female adults showed better performance in the dominant leg compared with the non - dominant leg , with an lsi of 107 % . similar results , with small performance differences between the two legs , were reported for the ol - st , where the lsi ranged from 101 to 104 % . in contrast , high values of the lsi were found for the ol - cmj , indicating a better performance of the dominant leg ( better jump height ) . female children ( 118 % ) and male adults ( 124 % ) , specifically , exhibited large side - to - side differences.table 4examples of normative values for the one - leg stability test for the five categoriesnormdatanormdatafemale ; non - dominant legfemale ; dominant leglevel ( index)descriptionlevel ( index)description1.74very good1.76very good1.752.05good1.772.00good2.062.90average2.012.72average3.003.30weak2.732.96weak3.31very weak2.97very weaknormdatanormdatamale ; non - dominant legfemale ; dominant leglevel ( index)descriptionlevel ( index)description1.98very good2.00very good1.992.31good2.012.30good2.323.09average2.313.19average3.303.62weak3.203.49weak3.63very weak3.50very weakfig . 1limb symmetry index of all one - leg tests examples of normative values for the one - leg stability test for the five categories limb symmetry index of all one - leg tests table 5 shows the specificity of the three one - leg tests . the one - leg countermovement jump had the lowest specificity in all age groups , ranging from 40 % ( male adults ) to 63 % ( male children ) . independent of gender and age , the one - leg stability test showed a mean specificity ranging from 66 % ( male adults ) to 78 % ( male children ) . the test reached a specificity of greater than 90 % in female and male children , as well as in female and male young adults.table 5specificity results for all one - leg testsage categorygenderspecificity ( % ) ol - stol - cmjol - sychildrenfemale714193male786395youthfemale685388male675990young adultsfemale765192male744793adultsfemale714586male664087 specificity results for all one - leg tests the most important finding of the present study was the evidence of limb asymmetries of more than 10 % in healthy individuals during hop performance . in one - leg hop testing , the lsi has been suggested as an indicator of normal or abnormal side - to - side differences . the european board of sports rehabilitation recommends hop performance differences of 10 % between the injured and non - injured legs for competitive sports ; however , to date , evidence - based data that can be used to distinguish between normal and abnormal performances are limited . in the present study , one maximum one - leg hop test ( ol - cmj ) and one endurance hop test ( ol - sy ) were used to increase the opportunity to detect differences in hop performances between legs . the present study demonstrated an lsi of up to 124 % for the ol - cmj in healthy male adults . there are conflicting results regarding the functional differences between dominant and non - dominant legs . kimura et al . reported no equivalent muscular output between the right and left legs in vertical jumps . in contrast , a study by abrams et al . demonstrated an lsi of > 90 % in healthy subjects . the subjects were allowed to use their hands , while subjects in the present study had to keep their hands on their hips . if subjects are restricted from using additional centrifugal mass , the examination of the isolated jump power is dominant , which may explain the higher side - to - side differences . to further investigate the lsi for the one - leg tests , we calculated its specificity , which indicated the probability that the one - leg tests would reveal a normal lsi for healthy subjects . specificities for the ol - cmj ranged from 41 to 63 % and were lower than those derived from the one - leg stability test ( 6678 % ) and the one - leg speedy test ( 8695 % ) . these findings are similar to those in the study of barber et al . , where the specificity ranged from 48 to 90 % , and many of the healthy subjects performed the vertical jump outside of the normal lsi . according to the literature , jump performance seems to be an important outcome factor for a safe return to sport following injury . . stated that patients with good hop performances were more likely to return to sport compared with those with poor performances . we also generated normative data for the two - leg plyometric jump to assess the bounce quality and intermuscular coordination of both legs . the plyometric jump demands a consecutive output of instantaneous force production , which is often required in sports . a new test , this test required knee joint stability in multiple planes and directions . in addition , a dynamic balance ability and coordination of the whole body is required to maintain balance . interestingly , when the hop test demanded an increased endurance and more complex task , the difference in the lsi between the dominant and non - dominant legs decreased to 101104 % . in addition , the specificity of this test showed more acceptable values , between 86 and 95 % , and was higher than that of the other one - leg tests . the importance of quick movements in a fatigued state has been emphasized in relation to the prevention of acl injuries . therefore , we administered the quick feet test as the last test in the battery . in addition to speed , the test also requires a high degree of concentration to avoid mistakes . furthermore , neuromuscular control and good coordination have also been documented to decrease the likelihood of knee injuries . despite the importance of coordination and balance in knee injury prevention , the results of one - leg stability tests have not yet been correlated with return to sport . the stability test on the mft challenge disc requires balance and coordination in both two- and one - legged performance . the comparison between the dominant and non - dominant legs revealed an lsi of 98 % , indicating a poor performance in the assumed dominant leg . the selection of the dominant leg in this study was based on the subjective dominance of fundamental movements . however , the definition of the dominant leg is not clear . according to kimura and asaeda as well as miyaguchi and demura , the ball kicking movement showed marked right - leg dominance . in the present study , the right leg was declared as the dominant leg for most of the participants . however , with regard to return to sport , it needs to be considered that both legs are used equally in some sports ( e.g. running , alpine skiing , swimming ) and that other sports require a specific leg ( e.g. jumping events in track and field , ball games ) . in practice , functional movement tests within a test battery were utilized as an outcome measure or a measurement of function . myer et al . tested functional deficits in athletes following acl rupture compared with a healthy control group . the low number of participants in the acl group ( 18 ) may limit the results . stated that none of the clinicians utilized functional performance tests as a measure of readiness to return to sport . the present study collected information from more than 400 healthy subjects that allows for the creation of normative data . these data serve as a basis for the classification of the potential of the knee during the rehabilitation process and , therefore , evidence for use in decision - making regarding an unrestricted return to activity . to represent age- and population - based acl injuries , we tested active subjects to generate normative data because most acl ruptures appear in athletic individuals who want to become active again . the present test battery can now be used in the day - by - day clinical work to draw conclusions regarding its ability to assist in decision - making regarding returning to sport . based on the normative data , the test results of an acl - reconstructed patient within the rehabilitation period can now be objectively evaluated . only an at least functionally good outcome in each subtest should be reached to minimize the risk of a re - ruptures of the acl . however , there is often a large discrepancy between clinical outcomes and the rate of successful return to high - level sport . therefore , more demanding criteria and functional tests need to be established to determine sport - specific return - to - sport outcomes in high - level competitive athletes . overall , it must be considered that the test battery facilitates the attainment of information regarding the actual functional status of the knee ; however , in addition to physical parameters , the psychological status of an athlete who experienced an acl injury is of great importance for guaranteeing a successful return to sport . thus , in addition to the functional test battery , psychological outcome measures must be added to ensure that participants in sports are physically and psychologically capable of returning to sport . the present study features normative data from seven functional performance tests and , thus , aids in the return - to - sport timeline by identifying functional deficits of the knee in clinical work . at this point , it needs to be considered that limb asymmetries of more than 10 % were evident in healthy subjects within the one - leg hop testing . this aspect will be discussed in the second part of the study , where the clinical utility of the test battery will be explained . it can be concluded that all tests are simple to administer , and the application of quick on - field tests that do not require sophisticated and expensive devices simplifies testing during the rehabilitation period .

purposereturn to activity remains the most common concern following an injury . to facilitate the decision regarding a patient s return to sport , we developed a standardized and easy - to - use test battery to enable an objective evaluation of knee function.methodsthe test battery consisted of seven functional tests : the two - leg stability test , one - leg stability test ( ol - st ) , two - leg countermovement jump ( cmj ) , one - leg cmj ( ol - cmj ) , plyometric jumps , speedy test and quick feet test . for each test , the reliability was determined based on the intraclass correlation coefficient . for all one - leg tests , the limb symmetry index ( lsi ) was calculated . resultsall tests showed a moderate - to - high reliability . normative data from 434 participants were included in the analysis . the subjects were categorized according to age as follows : children ( 1014 years ) , youth ( 1519 years ) , young adults ( 2029 years ) and adults ( 3050 years ) . the establishment of the functional test values allowed the classification into five normative categories . the lsi for the ol - st ( 98 % ) indicated a better performance of the non - dominant leg . in contrast , high lsi values were found for the ol - cmj ( 124 % ) , indicating a better performance of the dominant leg.conclusioneach test was found to be reliable and simple to perform . the better performance of the non - dominant leg in stability tasks must be considered when interpreting side - to - side differences . the established norm data from healthy individuals of each test battery represents an important basis for a clinical setting . test results from an acl - reconstructed patient should be at least classified as a functionally average outcome to support a safe return to sports.level of evidenceiv .

Level of evidence Introduction Materials and methods Reliability Normative data Statistical analysis Reliability Normative data Results Reliability Normative data Discussion Conclusion Conflict of interest

PMC4885601

mu - opioid receptor ( mor ) agonists , such as morphine , are the gold standard in the treatment of moderate to severe pain , however , their therapeutic use is often limited due to serious side effects including tolerance , dependence , respiratory depression , and constipation . numerous studies have shown promise for ameliorating mor - related side effects through simultaneous action at -opioid receptors ( dor ) . dor agonists have been shown to reduce the development of mor agonist tolerance and dependence , presumably due to the ability of dor agonists to potentiate mor mediated analgesia . dor agonists have also been shown to block -opioid receptor ( kor ) mediated antinociception which has possible implications in reversing kor related addictive behaviors . strong evidence has also accumulated for the potential of dor antagonists as agents to mitigate mor - mediated side effects . recently , there have been multiple reports on the utility and application of mixed - efficacy compounds that simultaneously produce mor activation while acting as dor antagonists . while the mechanism remains unclear , this profile has been shown to elicit the desired analgesia of a mor agonist but with reduced risk of tolerance and dependence in a number of preclinical models of analgesia . we have previously described a series of peptidomimetics in which key opioid pharmacophore elements were transferred from a peptide scaffold to a tetrahydroquinoline ( thq ) core , a smaller , more drug - like scaffold . expanding upon this initial report , we modified the thq core in an effort to increase metabolic stability in two ways : through n - acetylation of the thq nitrogen and through replacement the thq core with a tetrahydronaphthalene ( thn ) core . through these modifications , we found that n - acetylation of the thq core of the mixed - efficacy mor agonist / dor antagonist peptidomimetics improves dor affinity , which results in an overall better balance of affinities at mor and dor and provides selectivity over the -opioid receptor ( kor ) . although the optimal balance of mor affinity and dor affinity is unknown , we reasoned that similar high affinity profiles at both mor and dor was a reasonable goal , as this would ensure that both mor and dor character would be similarly represented . in the same report , we proposed that an additional polar contact between the carbonyl of the n - acetyl moiety and tyr of dor in transmembrane 3 ( tm3 ) helix is responsible for this increase in dor affinity . analogues in the present structure activity relationship ( sar ) campaign were therefore designed ( 1 ) to include a carbonyl moiety in order to maintain the high dor affinity seen in the initial analogue series and ( 2 ) to incorporate various aliphatic , cyclic , aromatic , and heteroatom - containing functionalities ( figure 1 ) to probe the effect of such modifications on the binding affinity and efficacy profiles not only at dor but also at mor and kor . peptidomimetic scaffold and n - substitutions ; * previously published in ref ( 8) , * * previously published in ref ( 9 ) . analogues 4a and 4b have been previously reported and are included here to serve as standards of comparison for the additional benzyl pendant n - substituted series . the majority of the remaining peptidomimetics ( 4c4j ) reported herein were prepared in four steps starting from the dihydroquinolinone 1 , which was prepared as previously reported ( scheme 1 ) . compound 1 was subjected to acylation using either an acid anhydride or acyl chloride to form intermediates 2c32j . it should be noted that the low yield for the synthesis of 2d was due to the use of pyridine , which was originally included to help catalyze the reaction . however , we found that running the reactions in neat anhydride at temperatures between 90 and 120 c led to better product conversion , and for the remainder of syntheses , we excluded pyridine in this reaction step . next , intermediates 2c2j were treated with ( r)-t - butanesulfinamide and ti(oet)4 to yield imines in situ , which were reduced with nabh4 to form the desired r - stereochemistry of intermediates 3c3j . the ellman auxiliary was cleaved using concentrated hydrochloric acid , forming hydrochloride salts , which were then coupled to di - boc protected 2,6-dimethyl - l - tyrosine ( di - boc - dmt ) and subsequently deprotected using trifluoroacetic acid ( tfa ) to yield analogues 4c4j . as we have previously shown , the ellman chemistry employed here produces the desired ( r)-stereocenter with high enantiomeric excess , as evidenced in the current syntheses by the absence of nmr - detectable disateromer after coupling to diboc - dmt . ( a ) neat acid anhydride ( excess ) , 100 c , 24 h ( for compunds 2c , 2d ) ; ( b ) acid chloride ( eq ) , dcm , ( for 2e j ) ; ( c ) ( r)-t - butanesulfinamide ( 23 equiv ) , thf , ti(oet)4 ( 46 equiv ) , 0 c , then reflux at 75 c ; ( d ) nabh4 ( 6 equiv ) , thf , 50 c to rt , then meoh , rt ; ( e ) hcl ( 6 equiv ) , dioxane , rt ; ( f ) diboc - dmt ( 1.05 equiv ) , pybop ( 1 equiv ) , 6cl - hobt ( 1 equiv ) , dipea ( 10 equiv ) , dmf , rt ; ( g ) 1:1 tfa : dcm ( excess ) . note : not all crude final product was purified ; as such a yield was not calculated . the synthesis of the remaining analogues 4k4 m began by protecting the nitrogen in 1 with a tert - butyloxycarbonyl ( boc ) group forming 5 ( scheme 2 ) , as previously described . intermediate 5 was treated with ( r)-t - butanesulfinamide and ti(oet)4 to yield an imine in situ , which was reduced with nabh4 to give the desired r - stereochemistry of intermediate 6 . to ensure both the cleavage of the ellman auxiliary and the removal of the boc group , 6 was treated first with tfa then with conc hcl in dioxane to ultimately yield the hydrochloride salt 7 . intermediate 7 was coupled to diboc - dmt , forming 8 . from this intermediate , 8 , the syntheses of 4k , 4l , and 4 m diverged . the synthesis of 4k was completed by first performing a finkelstein reaction on 2-chloroacetamide to form 2-iodoacetamide , which was cannulated into a solution containing 8 and n , n - diisopropylethylamine ( dipea ) to give 9k . intermediate 9k was treated with tfa to remove the boc groups , yielding 4k . to complete the syntheses of 4l and 4 m , ethyl 2-bromoacetate was added to intermediate 8 in the presence of potassium carbonate ( k2co3 ) to give 9l . intermediate 9l was then treated with tfa to remove boc groups and afford compound 4l . compound 4 m was completed by first saponification of the ester of 9l with lithium hydroxide , which was subsequently treated with tfa to remove the boc groups and yield compound 4 m . ( a ) boc2o ( 1.22 equiv ) , dmap ( 0.1 equiv ) , dipea ( 1.22 equiv ) , dcm , 60 c ; ( b ) ( r)-t - butanesulfinamide ( 23 equiv ) , thf , ti(oet)4 ( 46 equiv ) , 0 c , then reflux at 75 c ; ( c)nabh4 ( 6 equiv ) , thf , 50 c to rt , then meoh , rt ; ( d ) 1:1 tfa : dcm ( excess ) ; ( e ) hcl ( 6 equiv ) , dioxane , rt ; ( f ) diboc - dmt ( 1.05 equiv ) , pybop ( 1 equiv ) , 6cl - hobt ( 1 equiv ) , dipea ( 10 equiv ) , dmf , rt ; ( g ) nal ( 2 equiv ) , 2-chloroacetamide ( 2 equiv ) , then dipea ( 1.5 equiv ) ; ( h ) ethyl 2-bromoacetate ( 10 equiv ) , k2co3 ( 2 equiv ) , ch3cn , 24 h ; ( i ) lioh ( excess ) , etoh , 60 c , 2.5 h ; ( j ) 3 n hcl ( excess ) , rt . therefore , only 1.6 mg of 95% pure product was isolated , which was sufficient for opioid receptor binding and efficacy assays . analogues 15a and 15b have been previously reported and are included here to serve as standards of comparison for the additional 2-methylnaphthyl pendant n - substituted series . synthesis of the n - acylated peptidomimetics containing the 2-methylnaphthyl pendant began by forming intermediate 10 , which was synthesized as previously described . intermediate 10 was subjected to suzuki cross - coupling to incorporate the 2-methylnaphthyl moiety and yield 11 . intermediate 11 was treated with tfa to remove the boc group , forming 12 . intermediate 12 was treated with propionic anhydride to yield 13c , butyric anhydride to yield 13d , or methylchloroformate to yield 13e . intermediates 13c13e were treated with ( r)-t - butanesulfinamide and ti(oet)4 to form a chiral imine in situ , which was reduced with nabh4 to form the desired r - stereochemistry for intermediate 14c14e . the ellman auxiliary was cleaved using concentrated hcl forming primary amines , which were then coupled to diboc - dmt and deprotected to yield compound 15c15e ( scheme 3 ) . ( f ) 2-naphthalenylboronic acid , pd(dppf)cl2 , k2co3 , 3:1 acetone : h2o ; ( g ) 1:1 tfa : dcm ; ( h ) neat acid anhydride , reflux , 24 h ( for 15c and 15d ) ; ( i ) mecoocl , dcm , rt , 16 h ( for 15e ) ; ( j ) ( r)-t - butanesulfinamide , thf , ti(oet)4 , 0 c , then reflux at 75 c ; ( k ) nabh4 , thf , 50 c to rt , 3 h , then meoh , rt ; ( l ) hcl , dioxane , rt , 3 h ; ( m ) diboc - dmt , pybop , 6cl - hobt , dipea , dmf , rt . * not all of the crude compound was purified , so yield was not calculated or appears low . analogues 4c4 m and 15c15e were evaluated in in vitro mor , dor , and kor binding and efficacy assays , as previously described ( tables 1 and 2 ) . binding affinities ( ki ( nm ) ) were obtained by competitive displacement of radiolabeled [ h]diprenorphine in membrane preparations from c6 cells stably expressing mor ( c6-mor ) or dor ( c6-dor ) or cho cells stably expressing kor ( cho - kor ) , as previously described ( table 1 ) . efficacy data were obtained using agonist - induced stimulation of [ s]gtps binding to g protein in the same cell preparations as previously mentioned . efficacy data are represented as percent maximal stimulation relative to standard agonist ( damgo at mor , dpdpe at dor , and u69593 at kor ) , and potency ( ec50 ( nm ) ) values are calculated as the concentration of drug required to produce half - maximal stimulation ( table 1 ) . briefly , all analogues in both the benzyl and 2-methylnaphthyl series maintained high affinity binding and efficacy at mor . additionally , as predicted , all analogues across both series maintained high binding affinity at dor , relative to the unsubstituted analogues 4a and 15a , excluding 4 m , which displayed reduced affinity across all three receptors . interestingly , most of the n - substituted analogues in the benzyl pendant series produced increased dor efficacy ( table 1 ) , creating varying degrees of mor agonist / dor agonist ligands in the in vitro assays , while all analogues in the n - substituted 2-methylnaphthyl series displayed minimal or no efficacy at dor , maintaining the mor agonist / dor antagonist profile ( table 2 ) . lastly , while all analogues maintained selectivity for mor and dor over kor , kor affinity for many of the n - substituted analogues in the benzyl series increased relative to 4a ( table 1 ) . binding affinities ( ki ( nm ) ) were obtained by competitive displacement of radiolabeled [ h]diprenorphine in membrane preparations . efficacy is represented as percent maximal stimulation relative to standard agonist damgo ( mor ) , dpdpe ( dor ) , or u69,593 ( kor ) at 10 m . potency ( ec50 ( nm ) ) is calculated from the percent maximal stimulation as the concentration of drug required to produce half the maximal stimulation all values are expressed as the mean with sem in parentheses for n = 3 independent assays in duplicate , unless otherwise noted . dns : does not stimulate . published in ref ( 8) . published in ref ( 9 ) . binding affinities ( ki ( nm ) ) were obtained by competitive displacement of radiolabeled [ h]diprenorphine in membrane preparations . efficacy is represented as percent maximal stimulation relative to standard agonist damgo ( mor ) , dpdpe ( dor ) , or u69,593 ( kor ) at 10 m . potency ( ec50 ( nm ) ) is calculated from the percent maximal stimulation as the concentration of drug required to produce half the maximal stimulation all values are expressed as the mean with sem in parentheses for n = 3 independent assays in duplicate , unless otherwise noted . dns : does not stimulate . published in ref ( 8) . published in ref ( 9 ) . although the benzyl pendant n - substituted series displayed varying degrees of stimulation at dor , all analogues still exhibited both high affinity and efficacy at mor ; consequently , we elected to test select analogues ( 4e4k ) for in vivo efficacy in the wwtw assay using a cumulative dosing procedure . additionally , because all new compounds in the 2-methylnaphthyl pendant series displayed the desired mor agonist / dor antagonist profile , compounds 15c15e were also tested for in vivo efficacy in the same assay . briefly , withdrawal latencies were determined by placing a mouse into a cylindrical plastic restrainer and immersing 23 cm of the tail tip into a water bath maintained at 50 c . the latency to withdrawal or to rapidly flick the tail back and forth was recorded with a maximum cutoff time of 20 s to prevent tissue damage . acute antinociceptive effects were determined using a cumulative dosing procedure , as previously described . each animal received an intraperitoneal ( ip ) injection of saline , and then 30 min later , the baseline withdrawal latencies were recorded . following baseline determinations , three increasing doses ( 1 , 2.2 , and 6.8 mg / kg ) of a peptidomimetic ( dissolved in either deionized water or 1:1:8 solution of ethanol : alkumuls : deionized water , as was the case for 15c15e ) were given ip at 30 min intervals to provide final doses of 1 , 3.2 , and 10 mg / kg . thirty minutes after each injection , the tail withdrawal latency was measured as described above . to determine the duration of antinociceptive action , the tail - withdrawal test was performed at varying times following administration of a peptidomimetic ( 10 mg / kg , ip ) . of all compounds tested in vivo , only two of the novel n - substituted analogues tested displayed in vivo efficacy after ip administration ( figure 2a ) . analogue 4f produced partial antinociception with an 11 s latency to tail flick at the 10 mg / kg dosage following ip administration , while 4h produced full antinociception , reaching the 20 s latency to tail flick cutoff time point at the same dosage . because 4h displayed full antinociception , we performed a time course assay to determine the duration of antinociceptive action . as can be seen in figure 2b , 4h has a similar duration of action to that of 4a , producing maximal antinociception for approximately 1 h following a 10 mg / kg ip dose . ( a ) cumulative antinociceptive dose response curves of select benzyl pendant analogues ( n = 3 for all analogues ) in the mouse wwtw assay following ip administration . plotted as average sem . * * * * , p < 0.0001 for 4a and 4h for the 10 mg / kg dose when compared to baseline , p ( b ) time course of antinociception of 4a and 4h ( n = 3 ) in the mouse wwtw assay following ip administration of 10 mg / kg . plotted as average sem . all new analogues across both series sets , excluding 4 m , maintained high binding affinity at mor and exhibited an increased dor binding affinity when compared to the unsubstituted parent compounds 4a and 15a , presumably due to the carbonyl moiety incorporated into each of the n - substitutions , as had been previously observed for 15b . the only exception to this trend , 4 m , contains a carboxylic acid moiety and displayed significantly decreased affinity across all three opioid receptors . the poor binding affinity of 4 m could be potentially due to electrostatic repulsion between its carboxylic acid moiety and a conserved negatively charged residue in tm5 ( asp in dor , asp in kor , and glu in mor ) , which may also be complemented by acidic glu in kor . on the basis of these initial in vitro results and previously reported computational modeling , it appears that a carbonyl - containing moiety is beneficial in maintaining higher binding affinity at dor while a negatively charged species is not well tolerated . an additional trend seen in the benzyl series is that increasing the n - acyl chain length or overall bulk increases dor stimulation , with the exception of 4j , which contains a benzoyl moiety and produces no stimulation at dor . as seen in table 1 , the unsubstituted compound 4a shows very weak stimulation at dor ( 16% maximal stimulation ) while the n - acetyl analogue 4b increased maximal stimulation at dor to 26% and the n - propionyl ( 4c ) and n - butyryl ( 4d ) analogues further increased dor stimulation to between 40 and 60% . analogues 4e4 g contain branched or cyclic n - substitutions , and in the case of 4f ( containing a cyclopropanecarbonyl moiety ) and 4 g ( containing a cyclobutanecarbonyl moiety ) , dor stimulation increased to 69% and 58% , respectively . the computational model of 4 g in the active conformation of dor ( figure 3a ) demonstrates the presence of a hydrophobic cavity in the ligand binding pocket formed by residues from extracellular loop 2 ( leu and phe ) and tm5 ( thr and val ) , which is preserved in mor and kor . it can be seen that the cyclobutanecarbonyl moiety extends into this cavity forming favorable hydrophobic interactions . these interactions may contribute to the improved binding affinity of compounds 4c4f toward the active receptor conformation and to their increased efficacy at dor . as also seen in figure 3a , compound 4j has a benzoyl moiety which appears to be too bulky to fit this cavity in the active receptor conformation although it can be easily accommodated in the slightly enlarged ligand pocket of the inactive receptor state . ( a ) structure of 4 g ( teal ) and 4j ( magenta ) bound in the active conformation of dor with the area of the binding pocket occupied by the acyl chain circled ( black ) . ( b ) structure of 4k ( green ) bound in the active conformation of kor with a potential polar contact between e and the terminal amide of the ligand which could be responsible for the increased kor affinity and efficacy . similarly , hydrophobic interactions with the nonpolar cavity may also contribute to the improved binding affinity toward kor of compounds 4c , 4f , 4 g , and 4j with long or bulky nonpolar n - substitutions ( ki values ranging from 8 to 56 nm , as compared to ki = 68 nm of 4a ) , which act as partial agonists . however , this can not explain the observed activity of 4k , which has a polar amide group as an n - substitution . compound 4k not only displays increased affinity at kor ( 4.3 vs 68 nm for 4a ) but also exhibits significant kor stimulation ( 49% ) . the docking of 4k into the model of the active conformation of kor ( figure 3b ) suggests that formation of a hydrogen bond between n - substituted amide of 4k and glu from tm6 of kor may be the reason for the improved binding and agonist properties of this compound . while analogues 4c and 4e g all represent a subset of analogues with a mor agonist / dor agonist in vitro profile , 4 g displayed balanced binding affinities ( ki = 0.23 nm at mor and 0.15 nm at dor ) , low nanomolar potency ( ec50 = 2.1 nm at mor and 5.1 nm at dor ) , and high efficacy at both mor and dor ( 94% and 58% , respectively ) , along with weak binding and no efficacy at kor . while this profile was not our initial focus , several reports have shown that coadministration of a dor agonist with a mor agonist can increase both the potency and efficacy of the mor agonist . these findings suggest ligands producing a mixed - efficacy mor agonist / dor agonist profile could allow for the effective management of pain with a decrease in side effects seen with the administration of only a mor agonist . indeed , this has been observed with mmp-2200 , a mor agonist / dor agonist peptide ligand that produces in vivo activity in mice with reduced side effects . additionally , we have previously shown that 15b , with a 2-methylnaphthyl pendant and an acetyl n - substitution produces in vivo antinociception for greater than 3 h in the wwtw assay . thus , we hypothesized that compounds containing a 2-methylnaphthyl pendant with carbonyl - containing n - substitutions could produce similar , or longer lasting , in vivo activity when compared to 15b . we synthesized a modest set of 2-methylnaphthyl analogues and found that as the acyl chain length increases , the balanced - affinity mor agonist / dor antagonist profile seen with 15b remains essentially constant , while kor affinity slightly decreases , indicating that increased chain length helps provide mor and dor selectivity over kor ( table 2 ) . the high affinity binding and lack of stimulation at dor corroborates our previous findings that additional hydrophobic bulk in the pendant region of our peptidomimetic series increases affinity to dor in the inactive conformation while being incompatible with the smaller dor active state binding site . this trend suggests that the differences in dor efficacy between most of the compounds in the benzyl series ( 1670% ) and the 2-methylnaphthyl series ( 015% ) is due to differences in bulk between these two pendant moieties . while the in vitro profile of the 2-methylnaphthyl analogues is very promising in that all analogues are high affinity , potent mor agonists and high affinity dor antagonists , none of the novel analogues in this series ( 15c15e ) produced antinociception in the wwtw assay . finally , as we have seen with other peptidomimetic series , in vivo activity is not predictable from in vitro profiles and physiochemical parameters . this fact remains true for this series of n - substituted analogues where 4h is the only analogue to emerge that produces full antinociception in the wwtw assay at a 10 mg / kg dose . 4h is a mixed - efficacy peptidomimetic with nearly balanced affinities at mor and dor ( ki = 0.19 and 0.51 nm , respectively ) and produces potent ( ec50 = 0.78 ) , full stimulation at mor ( 95% ) , and moderately potent ( ec50 = 14 nm ) stimulation at dor ( 40% ) and selectivity for mor and dor over kor . further , 4h produces in vivo activity for 1 h , a similar duration of action to 4a . planned pharmacokinetic and metabolic stability testing of key members of our series are expected to shed light on the as yet unexplained differences in in vivo activity . in conclusion , we have expanded our opioid peptidomimetic library by incorporating various n - substitutions on our previously published , unsubstituted peptidomimetics 4a and 15a after observing that n - acetylation of the thq nitrogen in 4a and 15a to give 4b and 15b increases dor affinity . all of the n - substitutions in the present study contain a carbonyl moiety , which appears to be beneficial in maintaining high dor affinity , an important factor in the development of bifunctional mor / dor ligands . through this synthetic campaign , we have also discovered that an increased acyl chain length or additional bulk increases dor stimulation within the benzyl pendant series while increasing acyl chain length on the 2-methylnaphthyl series did not increase dor stimulation , suggesting that pendant moieties play an important role in anchoring the ligand to dor in the inactive conformation . analogue 4h , which displays high efficacy at mor and moderate efficacy at dor and produces full in vivo antinociception in the wwtw assay at a 10 mg / kg dose , may serve as a lead in the development of bifunctional mor / dor agonists . all reagents and solvents were obtained from commercial sources and used without additional purification . suzuki couplings were performed on a discover s - class ( cem ) microwave in a closed vessel with maximum power input of 300 w and temperature set at 110 c for 1060 min under the standard method from their synergy software . flash column chromatography was carried out using p60 silica gel ( 230400 mesh ) either manually or with a biotage isolera instrument . before flash column chromatograph was performed , crude mixtures were analyzed using thin - layer chromatography using 2:3 ea : hex , 1:1 ea : hex , and/or 3:2 ea : hex . the rf values of products and impurities were calculated then copied into either the linear gradient or stepwise gradient wizard on the biotage isolera instrument . the tlc wizard then determined the optimal purification technique which was used to purify crude mixtures . purification of final compounds was performed using a waters semipreparative hplc with a vydac protein and peptide c18 reverse phase column , using a linear gradient of 0% solvent b ( 0.1% tfa in acetonitrile ) in solvent a ( 0.1% tfa in water ) to 100% solvent b in solvent a at a rate 1% per minute and monitoring uv absorbance at 230 nm . purity of synthesized compounds was determined on a waters alliance 2690 analytical hplc instrument and a vydac protein and peptide c18 reverse phase column , using a linear gradient of 0% solvent b in solvent a to 45% , 70% , or 90% solvent b in solvent a in 45 , 70 , or 90 min , respectively , and uv absorbance at 230 nm ( gradient a ) . purities of the final compounds used for testing were 95% , unless otherwise stated , as determined by hplc . h nmr and c nmr data were obtained on either a 400 or 500 mhz varian spectrometer using cdcl3 or cd3od solvents . the identity of each compound was verified by mass spectrometry using an agilent 6130 lc ms mass spectrometer in positive mode . to a flame - dried round - bottom flask under ar the reaction vessel was placed back under vacuum for 5 min then excess acid anhydride ( propionic or butyric ) was added via syringe and the solution stirred for 5 min . the round - bottom flask was flooded with ar , equipped with a condenser , and placed in oil bath at 90120 c . the reaction stirred at reflux for 1220 h under ar and was monitored by tlc . once the reaction was complete , the solvent was removed under reduced pressure , yielding a crude yellow oil which was purified using silica gel chromatograph to obtain the pure product . to a flame - dried round - bottom flask with stir bar under to a round - bottom flask already containing dried , desiccated n - substituted dihydroquinolinone intermediate ( 1.0 equiv ) was added ( r)-2-methylpropane-2-sulfinamide ( 3.0 equiv ) , then the round - bottom flask was placed under vacuum for 10 min . meanwhile , a reflux condenser was flame - dried under vacuum and then flooded with ar . next , anhyd thf ( 20 ml ) was added to the reaction vessel containing starting reagents via syringe . the reaction solution allowed to stir under vacuum for 5 min and then was flooded with ar . the round - bottom flask was placed in ice bath and allowed to equilibrate . once addition was complete , the reaction vessel was taken out of the ice bath and placed in an oil bath at 7075 c , equipped with condenser , and stirred for 1648 h under ar . once sufficient conversion to the tert - butanesulfinyl imine was observed , the reaction vessel was taken out of the oil bath and cooled to ambient temperature . meanwhile , an additional round - bottom flask containing a stir bar was flame - dried under vacuum , then flooded with ar , then nabh4 was added quickly , and then reaction vessel was placed back under vacuum for 5 min . minimal anhyd thf was added ( 5 ml ) , and the vessel allowed to stir under vacuum for 5 min , then was flooded with ar . the round - bottom flask was placed in dry ice / xylenes bath and allowed to equilibrate . contents from the round - bottom flask containing the imine intermediate were transferred to round - bottom flask containing nabh4 via cannula . once contents completely added , the reaction was taken out of dry ice / xylenes bath and allowed to warm to room temperature . the reaction stirred at ambient temperature for 23 h. once the reaction was complete , meoh was added to quench . the residue was resuspended in dcm , solid remained , and the remaining solid was removed by filtration through a cotton plug and the mother liquor was concentrated and purified using silica gel chromatography to yield pure sulfinamide . to a round - bottom flask already containing sulfinamide intermediate was added 1520 ml of dioxane followed by conc hcl ( 6.0 equiv ) . the reaction stirred at rt for up to 3 h. solvent was removed under reduced pressure to yield slightly yellow , clear residue . if a white solid precipitated ( the hcl salt of the amine ) : solid was removed via filtration as product without any further purification necessary . if a white solid did not precipitate , but residue remains as film on flask : residue washed with fresh et2o ( 3 5 ml ) and dried without any further purification necessary . the ( r)-amine intermediate ( 1.0 equiv ) and diboc - dmt ( 1.01.05 equiv ) and the coupling reagents pybop ( 1.0 equiv ) or hatu ( 1.0 equiv ) , hobt - cl ( 1.0 equiv ) , were dissolved in dmf ( 1015 ml ) followed by the addition of the and dipea ( 10.0 equiv ) . after concentration under reduced pressure , the crude residue was dissolved in a 1:1 mixture of dcm and tfa ( 10 ml ) and stirred for 1 h. the mixture was concentrated and purified by semipreparative hplc , then lyophilized to yield the final compound . 2c was synthesized according to a modified general procedure a using 1 ( 110 mg , 0.47 mmol , 1.0 equiv ) and excess propionic anhydride . after reaction was complete , reaction mixture was cooled to rt , then transferred to a separatory funnel , and ea and nahco3 were added and the layers separated . the aqueous layer was extracted with ea , and the combined organic layers washed with brine then dried over mgso4 and purified using silica gel chromatography to yield the title compound 2c as a clear , colorless oil ( 120 mg , 85% ) . h nmr ( 500 mhz , cdcl3 ) 7.86 ( d , j = 0.9 , 1h ) , 7.39 ( br s , 1h ) , 7.35 ( dd , j = 8.2 , 2.2 , 1h ) , 7.29 ( t , j = 7.4 , 2h ) , 7.237.16 ( m , 3h ) , 4.20 ( t , j = 6.2 , 2h ) , 3.98 ( s , 2h ) , 2.75 ( t , j = 6.2 , 2h ) , 2.58 ( q , j = 7.4 , 2h ) , 1.20 ( t , j = 7.4 , 3h ) . c nmr ( 126 mhz , cdcl3 ) 194.27 , 173.03 , 142.21 , 140.13 , 138.78 , 134.63 , 128.90 , 128.71 , 127.62 , 126.49 , 126.11 , 124.44 , 43.88 , 41.29 , 39.62 , 27.98 , 9.91 . 2d was synthesized according to a modified general procedure a using 1 ( 0.18 g , 0.78 mmol , 1.0 equiv ) and excess butyric anhydride ( 5 ml ) and pyridine ( 4 ml ) . after reaction was complete ( 24 h ) , the reaction was quenched with h2o . dcm was added , and the aqueous layer was extracted with dcm . combined organic extracts were washed with 2 m naoh and dried with mgso4 . solvents were filtered and removed under reduced pressure , and the crude residue was purified via silica gel chromatography to yield the title compound 2d as an oil ( 0.029 g , 12% ) . h nmr ( 400 mhz , cdcl3 ) 7.87 ( t , j = 1.3 , 1h ) , 7.397.34 ( m , 2h ) , 7.337.25 ( m , 2h ) , 7.257.16 ( m , 3h ) , 4.21 ( t , j = 6.2 , 2h ) , 3.99 ( s , 2h ) , 2.76 ( t , j = 6.2 , 2h ) , 2.54 ( t , j = 8.0 , 2h ) , 1.791.66 ( m , 2h ) , 0.95 ( t , j = 7.4 , 3h ) . c nmr ( 101 mhz , cdcl3 ) 194.37 , 172.35 , 142.31 , 140.18 , 138.87 , 134.71 , 128.99 , 128.80 , 127.77 , 126.58 , 126.18 , 124.55 , 43.97 , 41.38 , 39.82 , 36.57 , 19.25 , 13.94 . 2e was synthesized according to general procedure b starting from 1 ( 100 mg , 0.42 mmol , 1.0 equiv ) to yield the title compound 2e as a clear , colorless oil ( 105 mg , 80.8% ) . h nmr ( 500 mhz , cdcl3 ) 7.87 ( d , j = 2.0 hz , 1h ) , 7.377.25 ( m , 4h ) , 7.257.17 ( m , 3h ) , 4.21 ( t , j = 6.3 hz , 2h ) , 3.99 ( s , 2h ) , 3.14 ( hept , j = 6.7 hz , 1h ) , 2.75 ( t , j = 6.2 hz , 2h ) , 1.18 ( d , j = 6.7 hz , 6h ) . c nmr ( 126 mhz , cdcl3 ) 194.33 , 176.97 , 142.29 , 140.05 , 138.84 , 134.59 , 128.90 , 128.87 , 128.68 , 127.64 , 126.46 , 126.15 , 124.22 , 43.90 , 41.25 , 39.79 , 31.13 , 19.88 . 2f was synthesized following a modified general procedure b starting with 1 ( 0.15 g , 0.65 mmol , 1.0 equiv ) and also using et3n ( 0.18 ml , 1.29 mmol , 2.0 equiv ) . the reaction stirred for 3 h , after which time satd nahco3 was added . solvents were filtered and removed under reduced pressure , and crude residue was purified via silica gel chromatography to yield the title compound 2f as a colorless oil ( 0.15 g , 78% ) . h nmr ( 500 mhz , cdcl3 ) 7.88 ( d , j = 2.2 , 1h ) , 7.46 ( d , j = 8.3 , 1h ) , 7.35 ( dd , j = 8.4 , 2.2 , 1h ) , 7.317.26 ( m , 2h ) , 7.227.17 ( m , 3h ) , 4.26 ( t , j = 6.3 , 2h ) , 3.98 ( s , 2h ) , 2.76 ( t , j = 6.3 , 2h ) , 2.061.96 ( m , 1h ) , 1.211.15 ( m , 2h ) , 0.910.82 ( m , 2h ) . c nmr ( 126 mhz , cdcl3 ) 194.41 , 173.00 , 142.45 , 140.13 , 138.53 , 134.49 , 128.89 , 128.71 , 127.74 , 126.49 , 125.85 , 124.09 , 43.53 , 41.28 , 39.70 , 13.74 , 9.77 . 2 g was synthesized following general procdure b starting with 1 ( 0.15 g , 0.62 mmol , 1.0 equiv ) . the reaction stirred for 3 h , after which time satd nahco3 was added . solvents were filtered and removed under reduced pressure , and crude residue was purified via silica gel chromatography to yield the title compound 2 g as a white solid ( 0.20 g , 99% ) . h nmr ( 500 mhz , cdcl3 ) 7.85 ( d , j = 1.3 , 1h ) , 7.487.37 ( br s , 1h ) , 7.35 ( dd , j = 8.2 , 2.2 , 1h ) , 7.29 ( t , j = 7.5 , 2h ) , 7.247.13 ( m , 3h ) , 4.12 ( t , j = 6.2 , 2h ) , 3.98 ( s , 2h ) , 3.52 ( p , j = 8.4 , 1h ) , 2.72 ( t , j = 6.2 , 2h ) , 2.43 ( dq , j = 11.8 , 9.2 , 2h ) , 2.13 ( q , j = 9.9 , 2h ) , 2.021.88 ( m , 2h ) . c nmr ( 126 mhz , cdcl3 ) 194.09 , 174.02 , 142.14 , 140.14 , 138.52 , 134.63 , 128.85 , 128.64 , 127.49 , 126.41 , 125.75 , 123.84 , 43.78 , 41.24 , 39.57 , 37.95 , 25.72 , 17.83 . 2h was synthesized following general procedure b starting from the 1 ( 150 mg , 0.63 mmol , 1.0 equiv ) to yield the title compound 2h as a clear , colorless oil ( 111 mg , 59.4% ) . h nmr ( 500 mhz , cdcl3 ) 7.86 ( d , j = 2.1 hz , 1h ) , 7.71 ( d , j = 8.6 hz , 1h ) , 7.34 ( dd , j = 8.6 , 2.2 hz , 1h ) , 7.29 ( t , j = 7.5 hz , 2h ) , 7.237.16 ( m , 3h ) , 4.18 ( t , j = 6.3 hz , 2h ) , 3.97 ( s , 2h ) , 3.84 ( s , 3h ) , 2.76 ( t , j = 6.3 hz , 2h ) . c nmr ( 126 mhz , cdcl3 ) 193.79 , 154.23 , 141.74 , 140.24 , 137.32 , 134.79 , 128.74 , 128.51 , 127.13 , 126.25 , 124.79 , 123.58 , 53.30 , 44.40 , 41.09 , 38.81 . 2i was synthesized following general procedure b starting from the 1 ( 150 mg , 0.63 mmol , 1.0 equiv ) to yield the title compound 2i as a white solid ( 176 mg , 89.8% ) . h nmr ( 500 mhz , cdcl3 ) 7.87 ( d , j = 2.2 hz , 1h ) , 7.37 ( dd , j = 8.4 , 2.2 hz , 1h ) , 7.28 ( t , j = 7.6 hz , 2h ) , 7.237.15 ( m , 3h ) , 4.26 ( s , 2h ) , 4.17 ( t , j = 6.3 hz , 2h ) , 3.98 ( s , 2h ) , 3.45 ( s , 3h ) , 2.78 ( t , j = 6.2 hz , 2h ) . c nmr ( 126 mhz , cdcl3 ) 193.62 , 168.07 , 141.25 , 139.91 , 139.02 , 134.72 , 128.77 , 128.58 , 128.55 , 127.56 , 126.37 , 125.78 , 123.66 , 76.74 , 71.84 , 59.24 , 43.95 , 41.17 , 39.41 . 2j was synthesized following general procedure b starting from the 1 ( 100 mg , 0.42 mmol , 1.0 equiv ) to yield the title compound 2j as a clear , slightly yellow oil ( 139 mg , 96.5% ) . h nmr ( 500 mhz , cdcl3 ) 7.78 ( d , j = 2.1 hz , 1h ) , 7.427.34 ( m , 3h ) , 7.317.25 ( m , 2h ) , 7.227.17 ( m , 2h ) , 7.147.10 ( m , 1h ) , 7.087.04 ( m , 2h ) , 7.01 ( dd , j = 8.5 , 2.2 hz , 1h ) , 6.81 ( d , j = 8.4 hz , 1h ) , 4.21 ( t , j = 6.3 hz , 2h ) , 3.85 ( s , 2h ) , 2.77 ( t , j = 6.3 hz , 2h ) . c nmr ( 126 mhz , cdcl3 ) 193.73 , 170.04 , 142.60 , 139.99 , 138.06 , 135.06 , 134.31 , 130.99 , 128.78 , 128.58 , 128.47 , 128.39 , 127.38 , 126.36 , 124.82 , 124.63 , 45.30 , 41.10 , 39.50 . 3c was synthesized following general procedure c using 2c ( 120 mg , 0.40 mmol , 1.0 equiv ) , ( r)-2-methylpropane-2-sulfinamide ( 150 mg , 1.2 mmol , 3.0 equiv ) , and ti(oet)4 ( 0.63 ml , 2.4 mmol , 6.0 equiv ) to form the ( r)-tert - butanesulfinyl imine intermediate in situ . once sufficient ketone was converted into imine intermediate ( after 17 h ) , the reaction mixture was transferred via cannula to a round - bottom flask containing nabh4 ( 90 mg , 2.4 mmol , 6.0 equiv ) and 20 ml of thf in a xylenes dry ice bath ; after addition , the solution was stirred at room temperature for 3 h before being quenched with meoh . once resultant solid was removed , crude residue was purified using silica gel chromatography to yield the title compound 3c as a clear , colorless oil ( 120 mg , 78% ) . h nmr ( 500 mhz , cdcl3 ) 7.69 ( d , j = 8.2 hz , 1h ) , 7.297.21 ( m , 2h ) , 7.207.15 ( qd , j = 6.5 , 5.4 , 1.6 hz , 4h ) , 7.06 ( dd , j = 8.5 , 2.3 hz , 1h ) , 4.52 ( q , j = 3.4 hz , 1h ) , 3.973.87 ( m , 3h ) , 3.57 ( tdd , j = 12.9 , 4.2 , 1.7 hz , 1h ) , 3.32 ( bs , 1h ) , 2.202.13 ( m , 1h ) , 1.991.90 ( m , 1h ) , 1.50 ( s , 9h ) , 1.19 ( s , 9h ) . c nmr ( 126 mhz , cdcl3 ) 153.43 , 140.77 , 136.51 , 136.42 , 128.88 , 128.71 , 128.55 , 128.47 , 128.34 , 125.97 , 123.89 , 80.97 , 55.52 , 50.36 , 41.08 , 40.00 , 29.41 , 28.22 , 22.48 . 3d was synthesized following general procedure c using 2d ( 0.029 g , 0.094 mmol , 1.0 equiv ) to yield the title compound 3d as a colorless oil ( 0.010 g , 26% ) . h nmr ( 400 mhz , cdcl3 ) 7.337.27 ( m , 2h ) , 7.247.18 ( m , 3h ) , 7.10 ( dd , j = 8.3 , 2.1 , 1h ) , 4.53 ( t , j = 4.6 , 1h ) , 3.95 ( s , 2h ) , 3.943.85 ( m , 1h ) , 3.803.68 ( m , 1h ) , 3.34 ( br s , 1h ) , 2.512.41 ( m , 2h ) , 2.272.15 ( m , 1h ) , 2.081.97 ( m , 1h ) , 1.741.63 ( m , 2h ) , 1.20 ( s , 9h ) , 0.92 ( t , j = 7.4 , 3h ) . 3e was synthesized following general procedure c using 2e ( 105 mg , 0.34 mmol , 1.0 equiv ) to yield the title compound 3e as a clear , colorless oil ( 70 mg , 50% ) . h nmr ( 500 mhz , cdcl3 ) 7.347.26 ( m , 3h ) , 7.247.19 ( m , 3h ) , 7.10 ( dd , j = 8.2 , 2.2 hz , 1h ) , 4.53 ( q , j = 3.9 hz , 1h ) , 3.96 ( s , 2h ) , 3.943.89 ( m , 1h ) , 3.753.67 ( m , 1h ) , 3.30 ( bs , 1h ) , 3.12 ( p , j = 6.9 hz , 1h ) , 2.242.15 ( m , 1h ) , 2.081.98 ( m , 1h ) , 1.231.16 ( s , 9h ) , 1.13 ( dd , j = 9.0 , 6.7 hz , 6h ) . c nmr ( 126 mhz , cdcl3 ) 177.35 , 140.53 , 138.70 , 136.67 , 128.92 , 128.67 , 128.58 , 128.54 , 126.28 , 124.60 , 55.76 , 51.04 , 41.37 , 40.00 , 31.09 , 30.96 , 22.55 , 20.03 , 20.01 . 3f was synthesized following general procedure c using 2f ( 0.16 g , 0.51 mmol , 1.0 equiv ) to yield the title compound 3f as a colorless oil ( 0.12 g , 55% ) . h nmr ( 400 mhz , cdcl3 ) 7.36 ( d , j = 8.2 , 1h ) , 7.327.26 ( m , 3h ) , 7.237.18 ( m , 3h ) , 7.09 ( dd , j = 8.3 , 2.0 , 1h ) , 4.55 ( q , j = 4.3 , 1h ) , 4.013.93 ( m , 3h ) , 3.75 ( ddd , j = 12.9 , 9.3 , 5.6 , 1h ) , 3.33 ( d , j = 3.6 , 1h ) , 2.23 ( dq , j = 14.9 , 5.1 , 1h ) , 2.101.89 ( m , 3h ) , 1.20 ( s , 9h ) , 1.141.08 ( m , 2h ) , 0.78 ( dd , j = 7.9 , 2.5 , 2h ) . c nmr ( 101 mhz , cdcl3 ) 173.36 , 140.65 , 138.53 , 136.83 , 131.83 , 128.98 , 128.71 , 128.58 , 128.51 , 126.36 , 124.97 , 55.85 , 51.18 , 41.47 , 39.85 , 30.74 , 22.61 , 13.65 , 9.28 . 3 g was synthesized following general procedure c using 2 g ( 0.20 g , 0.62 mmol , 1.0 equiv ) to yield the title compound 3 g as a colorless oil ( 0.16 g , 60% ) . h nmr ( 500 mhz , cdcl3 ) 7.317.25 ( m , 2h ) , 7.237.17 ( m , 2h ) , 7.09 ( dd , j = 8.3 , 2.1 , 1h ) , 4.51 ( q , j = 4.3 , 1h ) , 3.95 ( s , 2h ) , 3.863.77 ( m , 1h ) , 3.743.63 ( m , 1h ) , 3.503.39(m , 2h ) , 2.442.30 ( m 2h ) , 2.19 ( dq , j = 14.3 , 4.9 , 1h ) , 2.121.97 ( m , 3h ) , 1.971.82 ( m , 2h ) , 1.21 ( s , 9h ) . c nmr ( 126 mhz , cdcl3 ) 174.44 , 140.63 , 138.39 , 136.58 , 128.89 , 128.73 , 128.56 , 128.50 , 126.23 , 124.20 , 55.77 , 50.96 , 41.37 , 39.90 , 38.22 , 30.72 , 25.80 , 22.60 , 17.84 . 3h was synthesized following general procedure c using 2h ( 111 mg , 0.376 mmol , 1.0 equiv ) to yield the title compound 3h as a clear , colorless oil ( 124 mg , 82.1% ) . h nmr ( 500 mhz , cdcl3 ) 7.73 ( d , j = 8.7 hz , 1h ) , 7.27 ( t , j = 7.8 hz , 2h ) , 7.237.16 ( m , 4h ) , 7.117.06 ( m , 1h ) , 4.54 ( q , j = 3.6 hz , 1h ) , 4.01 ( dt , j = 13.0 , 4.6 hz , 1h ) , 3.93 ( s , 2h ) , 3.78 ( d , j = 1.0 hz , 3h ) , 3.64 ( ddd , j = 12.9 , 11.1 , 3.8 hz , 1h ) , 3.26 ( d , j = 2.7 hz , 1h ) , 2.242.14 ( m , 1h ) , 2.031.94 ( m , 1h ) , 1.20 ( d , j = 1.0 hz , 9h ) . c nmr ( 126 mhz , cdcl3 ) 154.94 , 140.75 , 137.11 , 136.11 , 129.11 , 128.79 , 128.77 , 128.57 , 128.44 , 126.09 , 126.08 , 123.68 , 55.59 , 52.94 , 50.24 , 41.15 , 40.23 , 29.43 , 22.51 . 3i was synthesized following general procedure c using 2i ( 176 mg , 0.60 mmol , 1.0 equiv ) to yield the title compound 3i as a clear , colorless oil ( 148 mg , 62.7% ) . h nmr ( 500 mhz , cdcl3 ) 7.307.24 ( m , 3h ) , 7.18 ( d , j = 7.6 hz , 3h ) , 7.09 ( dd , j = 8.3 , 2.0 hz , 1h ) , 4.52 ( q , j = 4.3 hz , 1h ) , 4.17 ( s , 2h ) , 3.93 ( s , 2h ) , 3.79 ( d , j = 40.2 hz , 11h ) , 3.44 ( d , j = 3.6 hz , 1h ) , 3.39 ( s , 3h ) , 2.20 ( dq , j = 14.5 , 4.9 hz , 1h ) , 2.04 ( qd , j = 9.2 , 8.7 , 5.0 hz , 1h ) , 1.20 ( s , 35h ) , 1.17 ( s , 10h ) . c nmr ( 126 mhz , cdcl3 ) 168.45 , 140.40 , 135.69 , 128.97 , 128.82 , 128.68 , 128.50 , 128.46 , 128.21 , 126.20 , 126.14 , 123.95 , 71.69 , 59.16 , 55.72 , 55.22 , 50.80 , 41.38 , 41.29 , 39.90 , 30.47 , 29.60 , 22.52 , 22.21 , 22.07 . 3j was synthesized following general procedure c using 2j ( 139 mg , 0.41 mmol , 1.0 equiv ) to yield the title compound 3j as an off - white solid ( 127 mg , 69.8% ) . h nmr ( 500 mhz , cdcl3 ) 7.31 ( td , j = 8.0 , 1.4 hz , 3h ) , 7.277.15 ( m , 5h ) , 7.147.09 ( m , 1h ) , 7.097.04 ( m , 2h ) , 6.78 ( t , j = 9.0 hz , 2h ) , 4.54 ( q , j = 3.9 hz , 1h ) , 3.93 ( dt , j = 12.5 , 5.1 hz , 1h ) , 3.82 ( s , 2h ) , 3.783.68 ( m , 1h ) , 3.32 ( d , j = 3.1 hz , 1h ) , 2.20 ( dq , j = 14.0 , 4.7 hz , 1h ) , 2.02 ( ddt , j = 14.5 , 9.9 , 5.0 hz , 1h ) , 1.13 ( d , j = 1.5 hz , 11h ) . c nmr ( 126 mhz , cdcl3 ) 170.22 , 140.50 , 138.06 , 136.69 , 135.96 , 130.33 , 129.87 , 128.79 , 128.68 , 128.46 , 128.34 , 128.32 , 128.23 , 126.15 , 125.31 , 60.32 , 55.72 , 50.61 , 41.47 , 41.19 , 30.24 , 22.53 , 20.99 , 14.15 . 4c was synthesized following general procedure d using 3c ( 120 mg , 0.31 mmol , 1.0 equiv ) and conc hcl ( 0.1 ml , 1.0 mmol , 6.0 equiv ) . after removing solvent , after washing the solid 3 with fresh et2o , the remaining et2o was decanted off , yielding a white solid amine hydrochloride salt ( 27 mg ) . the ( r)-amine intermediate ( 27 mg , 0.082 mmol , 1.0 equiv ) and diboc - dmt ( 33 mg , 0.082 mmol , 1.0 equiv ) and the coupling reagents hatu ( 31 mg , 0.082 mmol , 1.0 equiv ) and hobt - cl ( 14 mg , 0.082 mmol , 1.0 equiv ) were dissolved in dmf ( 1015 ml ) , followed by the addition of the and dipea ( 14 ml , 0.82 mmol , 10.0 equiv ) . after concentration under reduced pressure , the crude residue was dissolved in a 1:1 mixture of dcm and tfa ( 10 ml ) and stirred for 1 h. the mixture was concentrated and purified by semipreparative hplc to yield the title compound as a white solid . note that not all crude product was purified , so a yield was not calculated . h nmr ( 500 mhz , cd3od ) 8.15 ( d , j = 8.2 , 1h ) , 7.42 ( br s , 1h ) , 7.277.19 ( m , 2h ) , 7.187.11 ( m , 4h ) , 7.05 ( dd , j = 8.4 , 2.1 , 1h ) , 6.51 ( s , 2h ) , 4.964.91 ( m , 1h ) , 3.91 ( s , 2h ) , 3.86 ( dd , j = 11.5 , 5.0 , 1h ) , 3.78 ( br s , 1h ) , 3.26 ( dd , j = 13.6 , 11.5 , 1h ) , 3.183.10 ( m , 1h ) , 3.03 ( dd , j = 13.7 , 5.1 , 1h ) , 2.582.39 ( m , 2h ) , 2.27 ( s , 6h ) , 1.901.80 ( m , 1h ) , 1.481.39 ( m , 1h ) , 1.11 ( t , j = 7.4 , 3h ) . 4d was synthesized following general procedure d using 3d ( 10 mg , 0.024 mmol , 1.0 equiv ) with the coupling reagent pybop to yield the tfa salt of the title compound , 4d , as a white solid . h nmr ( 500 mhz , cd3od ) 7.39 ( br s , 1h ) , 7.277.20 ( m , 2h ) , 7.187.11 ( m , 4h ) , 7.06 ( dd , j = 8.4 , 2.1 , 1h ) , 6.50 ( s , 2h ) , 4.93 ( t , j = 6.5 , 1h ) , 3.91 ( s , 2h ) , 3.883.75 ( m , 2h ) , 3.25 ( dd , j = 13.6 , 11.5 , 1h ) , 3.153.11 ( m , 1h ) , 3.02 ( dd , j = 13.7 , 5.1 , 1h ) , 2.542.39 ( m , 2h ) , 2.27 ( s , 6h ) , 1.911.81 ( m , 1h ) , 1.691.58 ( m , 2h ) , 1.441.40 ( m , 1h ) , 0.93 ( t , j = 7.2 , 3h ) . 4e was synthesized following general procedure d using 3e ( 70 mg , 0.17 mmol , 1.0 equiv ) with the coupling reagent pybop to yield the tfa salt of the title compound , 4e , as a white solid ( 42 mg , 40.7% ) . h nmr ( 500 mhz , cd3od ) 8.18 ( d , j = 8.2 hz , 0h ) , 7.19 ( dt , j = 35.4 , 7.7 hz , 6h ) , 7.04 ( d , j = 8.3 hz , 3h ) , 6.51 ( s , 2h ) , 3.92 ( d , j = 14.2 hz , 3h ) , 3.26 ( t , j = 12.6 hz , 1h ) , 3.12 ( ddt , j = 41.3 , 13.4 , 5.3 hz , 4h ) , 2.27 ( s , 7h ) , 1.86 ( dq , j = 12.6 , 6.2 hz , 1h ) , 1.37 ( dd , j = 12.8 , 6.4 hz , 1h ) , 1.12 ( d , j = 6.6 hz , 5h ) , 1.05 ( d , j = 6.9 hz , 3h ) . c nmr ( 126 mhz , cd3od ) 179.32 , 169.29 , 162.72 , 157.37 , 142.26 , 140.05 , 137.53 , 129.77 , 129.45 , 129.20 , 127.13 , 125.65 , 123.29 , 116.40 , 53.46 , 46.89 , 42.12 , 32.38 , 31.92 , 31.83 , 20.44 , 20.17 , 19.90 . esi - ms 500.3 [ m + h ] and 522.3 [ m + na ] . 4f was synthesized following general procedure d using 3f ( 120 mg , 0.28 mmol , 1.0 equiv ) with the coupling reagent pybop to yield the tfa salt of the title compound , 4f , as a white solid . note that not all crude product was purified , so a yield was not calculated . h nmr ( 500 mhz , cd3od ) 7.39 ( d , j = 8.3 , 1h ) , 7.267.20 ( m , 2h ) , 7.197.12 ( m , 4h ) , 7.07 ( dd , j = 8.4 , 2.1 , 1h ) , 6.50 ( s , 2h ) , 4.96 ( t , j = 6.3 , 1h ) , 3.92 ( s , 2h ) , 3.913.80 ( m , 2h ) , 3.293.20 ( m , 2h ) , 3.072.99 ( m , 1h ) , 2.28 ( s , 6h ) , 1.981.92 ( m , 1h ) , 1.891.81 ( m , 1h ) , 1.471.38 ( m , 1h ) , 1.071.01 ( m , 1h ) , 0.990.92 ( m , 1h ) , 0.910.79 ( m , 2h ) . 4 g was synthesized following general procedure d using 3 g ( 160 mg , 0.37 mmol , 1.0 equiv ) with the coupling reagent pybop to yield the tfa salt of the title compound , 4 g , as a white solid . note that not all crude product was purified , so a yield was not calculated . h nmr ( 500 mhz , cd3od ) 7.39 ( br s , 1h ) , 7.267.21 ( m , 2h ) , 7.187.11 ( m , 4h ) , 7.05 ( dd , j = 8.3 , 2.1 , 1h ) , 6.50 ( s , 2h ) , 4.984.89 ( m , 1h ) , 3.91 ( s , 2h ) , 3.85 ( dd , j = 11.5 , 5.1 , 1h ) , 3.68 ( br s , 1h ) , 3.50 ( p , j = 8.5 , 1h ) , 3.25 ( dd , j = 13.6 , 11.6 , 1h ) , 3.123.01 ( m , 2h ) , 2.351.94 ( m , 11h ) , 1.891.77 ( m , 2h ) , 1.431.32 ( m , 1h ) . 4h was synthesized following general procedure d using 3h ( 124 mg , 0.31 mmol , 1.0 equiv ) and the coupling reagent pybop to yield the tfa salt of the title compound , 4h , as a white solid ( 50 mg , 69.5% ) . h nmr ( 400 mhz , cd3od ) 7.65 ( d , j = 8.6 hz , 1h ) , 7.267.20 ( m , 2h ) , 7.177.12 ( m , 3h ) , 7.07 ( d , j = 2.2 hz , 1h ) , 7.03 ( dd , j = 8.8 , 1.9 hz , 1h ) , 6.50 ( s , 2h ) , 4.97 ( t , j = 5.2 hz , 1h ) , 3.87 ( s , 2h ) , 3.863.81 ( m , 1h ) , 3.75 ( d , j = 1.4 hz , 4h ) , 3.25 ( ddd , j = 13.1 , 11.5 , 1.4 hz , 1h ) , 3.16 ( tdd , j = 6.7 , 3.8 , 1.4 hz , 12h ) , 3.052.94 ( m , 2h ) , 2.27 ( d , j = 1.4 hz , 7h ) , 1.901.82 ( m , 12h ) , 1.77 ( tt , j = 9.7 , 4.8 hz , 1h ) , 1.541.45 ( m , 1h ) . esi - ms 488.3 [ m + h ] and 510.3 [ m + na ] . 4i was synthesized following general procedure d using 3i ( 148 mg , 0.36 mmol , 1.0 equiv ) and the coupling reagent pybop to yield the tfa salt of the title compound , 4i , as a white solid . note that not all crude product was purified , so a yield was not calculated . h nmr ( 400 mhz , cd3od ) 7.23 ( t , j = 7.8 hz , 2h ) , 7.14 ( dd , j = 9.3 , 2.6 hz , 4h ) , 7.07 ( dd , j = 8.4 , 1.9 hz , 1h ) , 6.51 ( s , 2h ) , 4.96 ( t , j = 5.7 hz , 1h ) , 4.25 ( dd , j = 14.6 , 1.1 hz , 1h ) , 4.14 ( d , j = 14.5 hz , 1h ) , 3.90 ( s , 2h ) , 3.84 ( dd , j = 11.5 , 5.0 hz , 1h ) , 3.40 ( s , 3h ) , 3.25 ( t , j = 12.6 hz , 1h ) , 3.15 ( tdd , j = 6.7 , 3.7 , 1.2 hz , 7h ) , 3.02 ( dd , j = 13.7 , 5.0 hz , 1h ) , 2.27 ( s , 6h ) , 1.881.81 ( m , 8h ) , 1.48 ( s , 1h ) . esi - ms 502.3 [ m + h ] and 524.3 [ m + na ] . 4j was synthesized following general procedure d using 3j ( 182 mg , 0.41 mmol , 1.0 equiv ) and the coupling reagent pybop to yield the tfa salt of the title compound , 4j , as a white solid . note that not all crude product was purified , so a yield was not calculated . h nmr ( 400 mhz , cd3od ) 7.477.42 ( m , 1h ) , 7.37 ( d , j = 6.8 hz , 4h ) , 7.257.20 ( m , 2h ) , 7.15 ( d , j = 7.6 hz , 1h ) , 7.11 ( d , j = 7.6 hz , 2h ) , 6.876.76 ( m , 2h ) , 6.49 ( s , 2h ) , 5.04 ( t , j = 6.0 hz , 1h ) , 3.923.81 ( m , 4h ) , 3.25 ( d , j = 12.3 hz , 1h ) , 3.06 ( dd , j = 13.8 , 5.1 hz , 1h ) , 2.28 ( s , 6h ) , 1.94 ( t , j = 10.4 hz , 1h ) , 1.48 ( dd , j = 13.0 , 6.3 hz , 1h ) . c nmr ( 126 mhz , cd3od ) 172.42 , 169.14 , 157.49 , 142.26 , 140.04 , 139.67 , 137.75 , 137.19 , 131.74 , 130.37 , 129.71 , 129.50 , 129.45 , 129.39 , 128.99 , 127.15 , 126.27 , 123.18 , 116.45 , 53.53 , 49.00 , 46.85 , 42.06 , 31.98 , 31.42 , 20.44 . esi - ms 534.3 [ m + h ] and 556.2 [ m + na ] . after removal of solvent under reduced pressure , the crude product was purified by semipreparative hplc and lyophilized to yield the tfa salt of title compound , 9k , as a white powder ( 2 mg , 18% ) . h nmr ( 500 mhz , cd3od ) 8.33 ( d , j = 7.8 hz , 1h ) , 7.19 ( t , j = 7.4 hz , 2h ) , 7.147.07 ( m , 3h ) , 6.92 ( d , j = 8.5 hz , 1h ) , 6.86 ( s , 1h ) , 6.46 ( s , 2h ) , 6.38 ( d , j = 8.5 hz , 1h ) , 3.91 ( d , j = 17.8 hz , 1h ) , 3.80 ( dd , j = 11.8 , 5.1 hz , 1h ) , 3.76 ( s , 2h ) , 3.663.59 ( m , 1h ) , 3.25 ( t , j = 12.4 hz , 1h ) , 3.00 ( dd , j = 13.6 , 5.1 hz , 1h ) , 2.86 ( d , j = 12.1 hz , 1h ) , 2.38 ( t , j = 12.1 hz , 1h ) , 1.81 ( t , j = 13.1 hz , 1h ) , 1.52 ( d , j = 13.4 hz , 1h ) , 1.29 ( s , 1h ) . c nmr ( 126 mhz , cd3od ) 175.88 , 157.43 , 144.84 , 140.04 , 131.51 , 131.21 , 130.86 , 129.63 , 129.29 , 126.85 , 123.21 , 121.80 , 116.48 , 112.92 , 66.62 , 55.53 , 53.45 , 49.00 , 47.37 , 47.27 , 46.62 , 41.77 , 31.89 , 28.93 , 20.46 . esi - ms = 487.3.3 [ m + h ] and 509.3 [ m + na ] . 4l was synthesized by treating crude 9l with 3 n hcl to remove the remaining boc - groups and yield the crude product which was purified by semipreparative hplc and lyophilized to yield the title compound as the tfa salt of 4l , as a white powder ( 44 mg , 41.1% ) . h nmr ( 500 mhz , cd3od ) 8.17 ( d , j = 7.6 hz , 1h ) , 7.227.17 ( m , 2h ) , 7.147.07 ( m , 3h ) , 6.926.85 ( m , 2h ) , 6.43 ( s , 2h ) , 6.39 ( d , j = 8.4 hz , 1h ) , 4.87 ( s , 6h ) , 4.24 ( s , 0h ) , 4.234.15 ( m , 2h ) , 3.81 ( q , j = 5.3 hz , 1h ) , 3.76 ( d , j = 8.6 hz , 2h ) , 3.31 ( s , 2h ) , 3.25 ( dd , j = 13.5 , 11.8 hz , 1h ) , 3.00 ( dd , j = 13.6 , 5.0 hz , 1h ) , 2.85 ( dt , j = 11.8 , 4.1 hz , 1h ) , 2.42 ( td , j = 12.3 , 3.2 hz , 1h ) , 2.27 ( s , 6h ) , 1.75 ( tt , j = 12.8 , 4.1 hz , 1h ) , 1.57 ( dq , j = 13.3 , 3.5 hz , 1h ) , 1.29 ( td , j = 7.1 , 1.0 hz , 3h ) . c nmr ( 126 mhz , cd3od ) 173.03 , 168.05 , 157.42 , 144.44 , 143.28 , 140.00 , 131.60 , 131.03 , 130.84 , 129.64 , 129.29 , 126.84 , 123.20 , 121.53 , 116.47 , 112.40 , 62.35 , 53.49 , 53.20 , 49.51 , 49.34 , 49.17 , 49.00 , 48.83 , 48.66 , 48.49 , 47.56 , 46.18 , 41.75 , 31.87 , 28.78 , 20.48 , 14.51 . esi - ms 516.3 [ m + h ] and 538.3 [ m + na ] . 4 m was formed by treating the crude residue of 9 m with 1:1 tfa : dcm to form crude product which was purified by semipreparative hplc and lyophilized to yield the tfa salt of the title compound , 4 m ( 1.6 mg , 2.2% ) . note that purification was difficult and not all crude product was purified , so a yield was not calculated . esi - ms 488.3 [ m + h ] and 510.3 [ m + na ] . no h or c nmr data acquired . instead , formation of final product 4 m verified by mass spectrometry . 6 was synthesized following general procedure c using 5 ( 1.1 g , 4.8 mmol , 1.0 equiv ) , ( r)-2-methylpropane-2-sulfinamide ( 175 mg , 1.44 mmol , 3.0 equiv ) , and ti(oet)4 ( 0.604 ml , 2.88 mmol , 6.0 equiv ) to form the ( r)-tert - butanesulfinyl imine intermediate in situ . once sufficient ketone was converted into imine intermediate ( after 48 h ) , the reaction mixture was transferred via cannula to a round - bottom flask containing nabh4 ( 109 mg , 2.44 mmol , 6.0 equiv ) and 20 ml of thf in a xylenes dry ice bath ; after addition , the solution was stirred at room temperature for 3 h before being quenched with meoh . once resultant solid was removed , crude residue was purified using silica gel chromatography to yield the title compound 6 as a clear , colorless oil ( 175 mg , 82.2% ) . h nmr ( 500 mhz , cdcl3 ) 7.69 ( d , j = 8.2 hz , 1h ) , 7.297.21 ( m , 2h ) , 7.207.15 ( qd , j = 6.5 , 5.4 , 1.6 hz , 4h ) , 7.06 ( dd , j = 8.5 , 2.3 hz , 1h ) , 4.52 ( q , j = 3.4 hz , 1h ) , 3.973.87 ( m , 3h ) , 3.57 ( tdd , j = 12.9 , 4.2 , 1.7 hz , 1h ) , 3.32 ( bs , 1h ) , 2.202.13 ( m , 1h ) , 1.991.90 ( m , 1h ) , 1.50 ( s , 9h ) , 1.19 ( s , 9h ) . c nmr ( 126 mhz , cdcl3 ) 153.43 , 140.77 , 136.51 , 136.42 , 128.88 , 128.71 , 128.55 , 128.47 , 128.34 , 125.97 , 123.89 , 80.97 , 55.52 , 50.36 , 41.08 , 40.00 , 29.41 , 28.22 , 22.48 . 7 was synthesized by first treating 6 ( 400 mg , 0.904 mml , 1.0 equiv ) with excess 1:1 tfa : dcm . next , the ellman auxiliary was cleaved using conc hcl ( 0.133 ml , 5.43 mmol , 6.0 equiv ) . after removing solvent , after washing the solid 3 with fresh et2o , the remaining et2o was decanted off , yielding the title 7 compound as tan solid ( 110 mg , 44.4% from 7 ) . h nmr ( 500 mhz , cd3od ) 7.68 ( d , j = 1.8 hz , 1h ) , 7.44 ( dd , j = 8.3 , 1.8 hz , 1h ) , 7.37 ( d , j = 8.3 hz , 1h ) , 7.317.24 ( m , 4h ) , 7.227.17 ( m , 1h ) , 4.77 ( t , j = 6.0 hz , 1h ) , 4.06 ( s , 2h ) , 3.73 ( ddd , j = 12.4 , 9.0 , 3.1 hz , 1h ) , 3.65 ( ddd , j = 13.0 , 7.8 , 3.3 hz , 1h ) , 2.61 ( dddd , j14.8 , 9.0 , 5.7 , 3.3 hz , 1h ) , 2.38 ( dddd , j = 14.8 , 7.8 , 6.4 , 3.1 hz , 1h ) . 8 was synthesized following a modified version of general procedure d using 7 ( 110 mg , 0.40 mmol , 1.0 equiv ) . after coupling to diboc - dmt , residue was not deprotected with tfa . instead , the crude residue was purified by semipreparative hplc and lyophilized to yield the title compound 8 ( 287 mg , quant ) . esi - ms 630.3 [ m + h ] and esi - ms 652.3 [ m + na ] . first acetone was degassed for 30 min , then flooded with ar for an additional 30 min . to a flame - dried round - bottom flask equipped with a stir bar with ar atmosphere was added sodium iodide ( 25 mg , 0.16 mmol , 2.0 equiv ) and 2-chloroacetamide ( 15 mg , 0.16 mmol , 2.0 equiv ) , which was then placed back under vacuum . next , 10 ml of degassed acetone was added to the reaction vessel via cannula and reaction stirred for 10 min . meanwhile , to another flame - dried round - bottom flask equipped with a stirbar was added 8 , degassed acetone , followed by dipea ( 0.021 ml , 0.12 mmol , 1.5 equiv ) , and the solution stirred for 10 min . next , the contents of the round - bottom flask containing the sodium iodide and 2- chloroacetamide was transferred to the flask containing 8 via cannula . the reaction stirred stirred at room temp for 24 h. the next day , the solvent was removed under reduced pressure to yield the crude product ( 13 mg ) that was taken ahead to next step ( formation of 4k ) without further purification , isolation , or characterization . esi - ms 687.3 [ m + h ] and esi - ms 709.3 [ m + na ] . no h or c nmr data acquired . to synthesize 91 , 8 ( 214 mg , 0.34 mmol , 1.0 equiv ) and k2co3 ( 94 mg , 0.68 mmol , 2.0 equiv ) were placed in a round - bottom flask and the atmosphere was evacuated then the reaction vessel was flooded with ar . next , bromo ethyl acetate ( 0.377 ml , 3.4 mmol , 10 equiv ) was added to the reaction vessel , which was equipped with a condenser and placed in an 80 c oil bath . after 6 h , after 24 h , the solvent was removed under reduced pressure and crude residue was resuspended in dcm and di h2o and the layers separated . the organic layer was washed with 5% citric acid solution ( 1 20 ml ) and then with brine ( 1 20 ml ) and dried over mgso4 to yield the crude residue of 91 . esi - ms 716.3 [ m + h ] and esi - ms 738.3 [ m + na ] . the residue was carried forward ( to synthesize 4l and 4 m ) without further purification , isolation , or characterization . 9 m was synthesized by treating crude 9l ( 107 mg , 0.15 mmol , 1.0 equiv ) with lioh ( excess ) in etoh at 60 c for 2.5 h. reaction was monitored for loss of starting material by analytical hplc ( gradient a ) : starting material retention time = 72.4 ) . the residue was carried forward ( 4 m ) without further purification , isolation , or characterization . 11 was synthesized by first degassing a solution of 3:1 acetone : di h2o for 1 h , then ar was bubbled through solution for 1 h to ensure removal and displacement of ambient oxygen . intermediate 10 ( 1.0 equiv ) , 2-naphthylboronic acid ( 2.0 equiv ) , k2co3 ( 3.0 equiv ) , and pd(dppf)cl2 ( 0.1 equiv ) were added to a microwave tube , and the tube was placed under vacuum for 15 min , then flooded with ar . roughly 12 ml of the 3:1 acetone : di h2o solution was added to a tube via syringe , then the tube was placed in microwave for 3060 min with a maximum power of 300 w and a maximum temperature of 100 c with the powermax option enabled . once the microwave reaction was complete , reaction mixture was filtered through a pad of celite to remove palladium and solvents were removed under reduced pressure to yield a crude brown residue which was purified using silica gel chromatography to obtain the title compound 11 as an off - white solid . h nmr ( 500 mhz , cdcl3 ) 7.90 ( d , j = 2.2 hz , 1h ) , 7.817.73 ( m , 3h ) , 7.69 ( d , j = 8.7 hz , 1h ) , 7.63 ( s , 1h ) , 7.497.40 ( m , 2h ) , 7.35 ( dt , j = 8.6 , 1.8 hz , 1h ) , 7.30 ( dt , j = 8.4 , 1.5 hz , 1h ) , 4.164.10 ( m , 4h ) , 2.782.71 ( m , 2h ) , 1.54 ( d , j = 1.3 hz , 9h ) . c nmr ( 126 mhz , cdcl3 ) 194.26 , 152.75 , 142.47 , 137.89 , 136.72 , 134.71 , 133.58 , 132.13 , 128.24 , 127.61 , 127.54 , 127.35 , 127.31 , 127.25 , 127.08 , 126.78 , 126.05 , 125.46 , 124.82 , 123.87 , 82.12 , 44.28 , 41.33 , 38.99 , 28.29 . 12 was synthesized by deprotecting 11 with a 1:1 mixture of tfa : dcm . after stirring at rt for 1 h , solvent was removed under reduced pressure to yield the crude final product , which was then purified using silica gel chromatography to yield the title compound 12 as a yellow solid . note that not all crude product was purified , so a yield was not calculated . h nmr ( 500 mhz , cdcl3 ) 7.847.72 ( m , 4h ) , 7.62 ( s , 1h ) , 7.497.38 ( m , 2h ) , 7.337.24 ( m , 1h ) , 7.16 ( ddd , j = 8.3 , 4.8 , 1.9 hz , 1h ) , 6.65 ( ddd , j = 15.4 , 8.3 , 2.4 hz , 1h ) , 4.03 ( s , 1h ) , 3.593.53 ( m , 2h ) , 2.772.66 ( m , 2h ) , 2.25 ( s , 1h ) . c nmr ( 126 mhz , cdcl3 ) 193.66 , 138.64 , 136.34 , 136.11 , 133.56 , 132.05 , 130.95 , 128.11 , 127.58 , 127.51 , 127.40 , 127.36 , 127.20 , 126.87 , 125.95 , 125.31 , 119.67 , 119.42 , 116.41 , 116.22 , 42.57 , 42.41 , 41.14 , 38.08 . 13c was synthesized according to general procedure a using 12 ( 150 mg , 0.52 mmol , 1.0 equiv ) and excess propionic anhydride . once complete , excess anhydride was removed and the crude residue was purified using silica gel chromatography to yield the title compound 13c as a clear , pale - orange oil ( 156 mg , 87.2% ) . h nmr ( 500 mhz , cdcl3 ) 7.91 ( s , 1h ) , 7.817.73 ( m , 3h ) , 7.63 ( s , 1h ) , 7.477.32 ( m , 4h ) , 7.28 ( dd , j = 8.4 , 1.9 hz , 1h ) , 4.224.15 ( m , 2h ) , 4.12 ( s , 2h ) , 2.74 ( td , j = 6.3 , 1.9 hz , 2h ) , 2.612.52 ( m , 2h ) , 1.21 ( t , 3h ) . c nmr ( 126 mhz , cdcl3 ) 194.08 , 172.83 , 142.08 , 138.43 , 137.45 , 134.74 , 134.52 , 133.42 , 132.01 , 128.23 , 127.51 , 127.49 , 127.41 , 127.17 , 127.03 , 126.03 , 125.92 , 125.45 , 124.30 , 123.97 , 43.69 , 41.27 , 39.43 , 27.80 , 9.71 . 13d was synthesized according to general procedure a using 12 ( 155 mg , 0.54 mmol , 1.0 equiv ) and excess butyric anhydride . once complete , excess anhydride was removed and the crude residue was purified using silica gel chromatography to yield the title compound 13d as a clear , slightly dark - red oil ( 112 mg , 58.0% ) . h nmr ( 500 mhz , cdcl3 ) 7.94 ( s , 1h ) , 7.857.76 ( m , 3h ) , 7.66 ( s , 1h ) , 7.517.39 ( m , 4h ) , 7.32 ( d , j = 8.5 hz , 1h ) , 4.20 ( t , j = 6.3 hz , 2h ) , 4.15 ( s , 2h ) , 2.76 ( t , j = 5.9 hz , 2h ) , 2.602.49 ( m , 2h ) , 1.75 ( h , j = 7.7 hz , 2h ) , 0.97 ( t , j = 7.6 hz , 3h ) . c nmr ( 126 mhz , cdcl3 ) 194.00 , 171.99 , 142.10 , 138.38 , 137.42 , 134.69 , 134.47 , 133.42 , 132.01 , 128.21 , 127.49 , 127.47 , 127.39 , 127.16 , 127.02 , 126.00 , 125.90 , 125.42 , 124.32 , 124.00 , 43.71 , 41.25 , 39.50 , 36.28 , 18.94 , 13.66 . 13e was synthesized according to general procedure b using 12 ( 150 mg , 0.52 mmol , 1.0 equiv ) and methylchloroformate ( 0.200 ml , 2.3 mmol , 2.3 equiv ) . the reaction stirred overnight ; once complete , solvent was removed and the crude residue was purified using silica gel chromatography to yield the title compound 109 as a clear oil ( 65 mg , 36.1% ) . additionally , not all product was purified as the rf values for the starting material and product were similar and separation was difficult . h nmr ( 500 mhz , cdcl3 ) 7.92 ( d , j = 2.2 hz , 1h ) , 7.827.74 ( m , 3h ) , 7.72 ( d , j = 8.6 hz , 1h ) , 7.64 ( s , 1h ) , 7.487.41 ( m , 2h ) , 7.38 ( dd , j = 8.6 , 2.3 hz , 1h ) , 7.30 ( dd , j = 8.4 , 1.8 hz , 1h ) , 4.18 ( t , j = 6.3 hz , 2h ) , 4.13 ( s , 2h ) , 3.84 ( s , 3h ) , 2.77 ( t , j = 6.3 hz , 2h ) . c nmr ( 126 mhz , cdcl3 ) 193.81 , 154.23 , 141.81 , 137.73 , 137.18 , 134.88 , 133.49 , 132.05 , 128.21 , 127.55 , 127.47 , 127.26 , 127.23 , 127.03 , 126.01 , 125.42 , 124.81 , 123.63 , 53.32 , 44.39 , 41.26 , 38.81 . 14c was synthesized according to general procedure c using 13c ( 156 mg , 0.45 mmol , 1.0 equiv ) , ( r)-2-methylpropane-2-sulfinamide ( 165 mg , 1.36 mmol , 3.0 equiv ) , and ti(oet)4 ( 0.57 ml , 2.7 mmol , 6.0 equiv ) to form the ( r)-tert - butanesulfinyl imine intermediate in situ . once sufficient ketone was converted into imine intermediate ( after 48 h ) , the reaction mixture was transferred via cannula to a round - bottom flask containing nabh4 ( 103 mg , 2.7 mmol , 6.0 equiv ) and 20 ml of thf in a xylenes dry ice bath , after addition , the solution was stirred at room temperature for 3 h before being quenched with meoh . once resultant solid was removed , crude residue was purified using silica gel chromatography to yield the title compound 14c as a clear , colorless oil ( 148 mg , 72.5% ) . no h or c nmr data acquired . instead , product taken ahead to next reaction ( formation of 15c ) without any further isolation , purification , or characterization . 14d was synthesized according to general procedure c using 13d ( 112 mg , 0.31 mmol , 1.0 equiv ) , ( r)-2-methylpropane-2-sulfinamide ( 114 mg , 0.94 mmol , 3.0 equiv ) , and ti(oet)4 ( 0.39 ml , 1.9 mmol , 6.0 equiv ) to form the ( r)-tert - butanesulfinyl imine intermediate in situ . once sufficient ketone was converted into imine intermediate ( after 48 h ) , the reaction mixture was transferred via cannula to a round - bottom flask containing nabh4 ( 71 mg , 1.9 mmol , 6.0 equiv ) and 20 ml of thf in a xylenes a dry ice bath ; after addition , the solution was stirred at room temperature for 3 h before being quenched with meoh . once resultant solid was removed , crude residue was purified using silica gel chromatography to yield the title compound 14d as a clear , colorless oil ( 54 mg , 37.2% ) . h nmr ( 500 mhz , cdcl3 ) 7.78 ( q , j = 7.7 hz , 3h ) , 7.66 ( s , 1h ) , 7.44 ( pd , j = 7.1 , 3.4 hz , 2h ) , 7.33 ( dd , j = 6.4 , 2.1 hz , 2h ) , 7.14 ( dt , j = 8.2 , 2.0 hz , 1h ) , 4.52 ( t , j = 4.4 hz , 1h ) , 4.11 ( s , 2h ) , 3.89 ( dt , j = 12.2 , 5.5 hz , 1h ) , 3.74 ( ddd , j = 13.6 , 9.8 , 6.2 hz , 1h ) , 3.32 ( d , j = 3.3 hz , 1h ) , 2.522.39 ( m , 2h ) , 2.20 ( dh , j = 15.3 , 7.4 , 5.6 hz , 1h ) , 2.04 ( dq , j = 12.7 , 4.6 , 3.9 hz , 1h ) , 1.741.61 ( m , 2h ) , 1.17 ( d , j = 1.9 hz , 9h ) , 0.91 ( t , j = 7.5 hz , 3h ) . c nmr ( 126 mhz , cdcl3 ) 187.72 , 172.68 , 138.03 , 136.64 , 133.57 , 132.10 , 128.79 , 128.72 , 128.57 , 128.21 , 127.58 , 127.52 , 127.46 , 127.10 , 126.03 , 125.43 , 124.85 , 124.60 , 55.73 , 50.96 , 41.52 , 39.95 , 36.69 , 30.68 , 22.51 , 19.14 , 13.88 . 14e was synthesized according to general procedure c using 13e ( 65 mg , 0.19 mmol , 1.0 equiv ) , ( r)-2-methylpropane-2-sulfinamide ( 68 mg , 0.57 mmol , 3.0 equiv ) , and ti(oet)4 ( 0.24 ml , 1.1 mmol , 6.0 equiv ) to form the ( r)-tert - butanesulfinyl imine intermediate in situ . once sufficient ketone was converted into imine intermediate ( after 48 h ) , the reaction mixture was transferred via cannula to a round - bottom flask containing nabh4 ( 43 mg , 1.1 mmol , 6.0 equiv ) and 20 ml of thf in a xylenes dry ice bath , after addition , the solution was stirred at room temperature for 3 h before being quenched with meoh . once resultant solid was removed , crude residue was purified using silica gel chromatography to yield the title compound 14e as a clear , colorless oil ( 34 mg , 40.0% ) . h nmr ( 500 mhz , cdcl3 ) 7.817.71 ( m , 4h ) , 7.64 ( d , j = 5.2 hz , 1h ) , 7.487.39 ( m , 2h ) , 7.347.26 ( m , 2h ) , 7.13 ( ddd , j = 8.5 , 6.0 , 2.2 hz , 1h ) , 4.55 ( h , j = 3.7 hz , 1h ) , 4.09 ( s , 2h ) , 4.053.96 ( m , 1h ) , 3.78 ( s , 3h ) , 3.693.59 ( m , 1h ) , 3.33 ( q , j = 2.7 hz , 1h ) , 2.18 ( ddt , j = 12.8 , 10.4 , 4.3 hz , 1h ) , 2.061.93 ( m , 1h ) , 1.18 ( s , 9h ) . c nmr ( 126 mhz , cdcl3 ) 154.94 , 138.25 , 136.95 , 136.16 , 133.53 , 132.02 , 129.23 , 128.84 , 128.65 , 128.08 , 127.52 , 127.48 , 127.43 , 126.96 , 125.92 , 125.30 , 123.70 , 55.60 , 52.95 , 50.35 , 41.33 , 40.28 , 29.49 , 22.49 . 15c was synthesized following general procedure d using 14c ( 148 mg , 0.33 mmol , 1.0 equiv ) and conc hcl ( 6 drops ) . after removing solvent , after washing the solid 3 with fresh et2o , the remaining et2o was decanted off , yielding a white solid amine hydrochloride salt ( 126 mg ) . the synthesis was completed by following general procedure f with newly formed ( r ) amine intermediate ( 60 mg , 0.16 mmol , 1.0 equiv ) to yield crude product which was purified by semipreparative hplc and lyophilized to yield the title compound 15c as a tfa salt ( 15 mg , 14.7% ) . note that not all crude product was purified . h nmr ( 500 mhz , cdcl3 ) 7.76 ( ddd , j = 21.9 , 8.2 , 3.1 hz , 3h ) , 7.60 ( d , j = 3.2 hz , 1h ) , 7.477.37 ( m , 2h ) , 7.28 ( dt , j = 8.6 , 2.2 hz , 1h ) , 7.19 ( d , j = 3.5 hz , 1h ) , 7.157.09 ( m , 1h ) , 6.51 ( d , j = 3.4 hz , 2h ) , 4.93 ( p , j = 5.2 hz , 1h ) , 4.08 ( d , j = 3.4 hz , 2h ) , 3.86 ( dt , j = 11.2 , 4.3 hz , 1h ) , 3.78 ( s , 1h ) , 3.24 ( td , j = 12.6 , 11.2 , 3.5 hz , 1h ) , 3.15 ( s , 1h ) , 3.03 ( dt , j = 13.9 , 4.5 hz , 1h ) , 2.582.41 ( m , 2h ) , 2.26 ( d , j = 3.6 hz , 6h ) , 1.85 ( dtd , j = 13.5 , 8.7 , 4.7 hz , 1h ) , 1.45 ( s , 1h ) , 1.11 ( td , j = 7.1 , 3.2 hz , 3h ) . c nmr ( 126 mhz , cd3od ) 175.85 , 169.20 , 157.47 , 140.07 , 139.86 , 137.62 , 135.07 , 133.59 , 129.47 , 129.07 , 128.58 , 128.46 , 128.38 , 127.87 , 127.09 , 126.46 , 125.87 , 123.24 , 116.45 , 53.49 , 47.11 , 42.27 , 31.95 , 31.29 , 28.92 , 20.42 , 10.06 . esi - ms 536.3 [ m + h ] and 558.3 [ m + na ] . 15d was synthesized following general procedure d using 14d ( 34 mg , 0.076 mmol , 1.0 equiv ) and conc hcl ( 3 drops ) . after removing solvent , after washing the solid 3 with fresh et2o , the remaining et2o was decanted off , yielding a white solid amine hydrochloride salt ( 46 mg ) . the synthesis was completed by following general procedure f with newly formed ( r ) amine intermediate ( 46 mg , 0.12 mmol , 1.0 equiv ) to yield crude product which was purified by semipreparative hplc and lyophilized to yield the title compound 15d as a tfa salt ( 12 mg , 15.6% ) . note that not all crude product was purified . esi - ms 550.3 [ m + h ] and 572.3 [ m + na ] . 15e was synthesized following general procedure d using 14e ( 34 mg , 0.076 mmol , 1.0 equiv ) and conc hcl ( 3 drops ) . after removing solvent , after washing the solid 3 with fresh et2o , the remaining et2o was decanted off , yielding a white solid amine hydrochloride salt ( 19 mg ) . the synthesis was completed by following general procedure f with newly formed ( r ) amine intermediate ( 19 mg , 0.0.16 mmol , 1.0 equiv ) to yield crude product which was purified by semipreparative hplc and lyophilized to yield the title compound 15e as a tfa salt ( 13 mg , 40.6% ) . h nmr ( 500 mhz , cd3od ) 7.78 ( d , j = 7.9 hz , 1h ) , 7.73 ( dd , j = 8.3 , 2.9 hz , 2h ) , 7.67 ( d , j = 8.5 hz , 1h ) , 7.59 ( s , 1h ) , 7.42 ( dt , j = 8.9 , 6.2 hz , 2h ) , 7.27 ( dt , j = 8.4 , 1.7 hz , 1h ) , 7.12 ( s , 1h ) , 7.09 ( dt , j = 8.6 , 1.8 hz , 1h ) , 6.50 ( s , 2h ) , 4.96 ( t , j = 5.1 hz , 1h ) , 4.04 ( s , 2h ) , 3.873.80 ( m , 1h ) , 3.75 ( d , j = 1.5 hz , 4h ) , 3.283.19 ( m , 1h ) , 3.00 ( dt , j = 12.6 , 8.3 hz , 2h ) , 2.26 ( d , j = 1.5 hz , 7h ) , 1.78 ( ddt , j = 14.7 , 9.9 , 4.7 hz , 1h ) , 1.50 ( ddd , j = 13.4 , 8.6 , 4.8 hz , 1h ) . c nmr ( 126 mhz , cd3od ) 168.96 , 157.48 , 156.61 , 139.99 , 138.03 , 137.48 , 135.07 , 133.57 , 130.31 , 129.72 , 129.02 , 128.78 , 128.57 , 128.46 , 128.39 , 127.80 , 127.05 , 126.41 , 124.63 , 123.19 , 116.43 , 111.40 , 53.54 , 53.41 , 47.06 , 42.32 , 42.14 , 31.92 , 30.30 , 20.44 . esi - ms 538.3 [ m + h ] and 560.3 [ m + na ] . all tissue culture reagents were purchased from gibco life sciences ( grand island , ny , u.s . ) . c6-rat glioma cells stably transfected with a rat ( c6-mor ) or rat ( c6-dor ) opioid receptor and chinese hamster ovary ( cho ) cells stably expressing a human ( cho - kor ) opioid receptor were used for all in vitro assays . cells were grown to confluence at 37 c in 5% co2 in dulbecco s modified eagle medium ( dmem ) containing 10% fetal bovine serum and 5% penicillin / streptomycin . membranes were prepared by washing confluent cells three times with ice - cold phosphate buffered saline ( 0.9% nacl , 0.61 mm na2hpo4 , 0.38 mm kh2po4 , ph 7.4 ) . cells were detached from the plates by incubation in warm harvesting buffer ( 20 mm hepes , 150 mm nacl , 0.68 mm edta , ph 7.4 ) and pelleted by centrifugation at 1600 rpm for 3 min . the cell pellet was suspended in ice - cold 50 mm tris - hcl buffer , ph 7.4 , and homogenized with a tissue tearor ( biospec products , inc . , bartlesville , ok , u.s . ) for 20 s. the homogenate was centrifuged at 15000 rpm for 20 min at 4 c . the pellet was rehomogenized in 50 mm tris - hcl with a tissue tearor for 10 s , followed by recentrifugation . the final pellet was resuspended in 50 mm tris - hcl and frozen in aliquots at 80 c . protein concentration was determined via a bca protein assay ( thermo scientific pierce , waltham , ma , u.s . ) using bovine serum albumin as the standard . radiolabeled compounds were purchased from perkinelmer ( waltham , ma , u.s . ) . opioid ligand binding assays were performed by competitive displacement of 0.2 nm [ h]diprenorphine ( 250 ci , 1.85 tbq / mmol ) by the peptidomimetic from membrane preparations containing opioid receptors as described above . the assay mixture , containing membranes ( 20 g protein / tube ) in 50 mm tris - hcl buffer ( ph 7.4 ) , [ h]diprenorphine , and various concentrations of test peptidomimetic , was incubated at room temperature for 1 h to allow binding to reach equilibrium . the samples were rapidly filtered through whatman gf / c filters using a brandel harvester ( brandel , gaithersburg , md , u.s . ) and washed five times with 50 mm tris - hcl buffer . bound radioactivity on dried filters was determined by liquid scintillation counting , after saturation with ecolume liquid scintillation cocktail , in a wallac 1450 microbeta ( perkinelmer , waltham , ma , u.s . ) . the results presented are the mean standard error ( se\m ) from at least three separate assays performed in duplicate . ki ( nm ) values were calculated using nonlinear regression analysis to fit a logistic equation to the competition data using graphpad prism , version 6.0c , for mac os x ( graphpad software inc . , la jolla , ca ) . agonist stimulation of [ s]guanosine 5-o-[-thio]triphosphate ( [ 5s]gtps , 1250 ci , 46.2 tbq / mmol ) binding to g - protein was measured as described previously . briefly , membranes ( 1020 g of protein / tube ) were incubated 1 h at 25 c in gtps buffer ( 50 mm tris - hcl , 100 mm nacl , 5 mm mgcl2 , ph 7.4 ) containing 0.1 nm [ s]gtps , 30 m guanosine diphosphate ( gdp ) , and varying concentrations of test peptidomimetic . g - protein activation following receptor stimulation of [ s]gtps ( % stimulation ) with peptidomimetic was compared with 10 m of the standard compounds [ d - ala2,n - mephe4,gly - ol]enkephalin ( damgo ) at mor , d - pen2,5-enkephalin ( dpdpe ) at dor , or u69,593 at kor . the reaction was terminated by vacuum filtration of gf / c filters that were washed 10 times with gtps buffer . bound radioactivity was measured as described above . the results are presented as the mean standard error ( sem ) from at least three separate assays performed in duplicate ; potency ( ec50 ( nm ) ) and % stimulation were determined using nonlinear regression analysis with graphpad prism , as above . adult male c57bl/6 mice weighing between 20 and 30 g at 816 weeks old purchased from harlan ( indianapolis , in ) . mice were group - housed and had free access to food and water at all times . experiments were conducted in the housing room , which was maintained on a 12 h light / dark cycle ( with lights on at 0700 ) ; experiments were conducted during the light cycle . each mouse was used only once . studies were performed in accordance with the university of michigan committee on the use and care of animals and the guide for the care and use of laboratory animals ( national research council , 2011 publication ) . three - dimensional ( 3d ) models of opioid receptors in the inactive conformation were produced as previously described using x - ray structures of the mouse mor ( pdb i d : 4dkl ) , the human dor ( pdb i d : 4n6h ) , and the human kor ( pdb i d : 4djh ) as structural templates . the recently obtained crystal structure of mouse mor in the active conformation ( pdb i d : 5c1 m ) was used as a template for homology modeling of active conformations of dor and kor . structures of receptor loops in the active state were kept similar to those in the crystal structures of corresponding receptors in the inactive state . n - termini of dor ( residues 3345 ) and kor ( residues 4557 ) were modeled using the structure of mor n - terminus in the active conformation with a few adjustments that transformed the central bulge ( residues 5357 ) into the extended conformation of the polypeptide chain . such modifications increased the size of the binding pocket and allowed docking of relatively bulky tetra- and pentapeptides and peptidomimetics without hindrances with the n - terminus . structures of peptidomimetic ligands were generated using the 3d - builder application of quanta ( accelrys , inc . ) , followed by conformational search included in the program package . ligand conformations that demonstrated the best superposition of aromatic substituents of the thq core with the pharmacophore elements ( tyr and phe ) of receptor - bound conformations of cyclic tetrapeptides were selected and minimized with charmm implemented in quanta ( adopted - basis newton raphson method , 100 steps , = 10 ) . low energy conformations ( within 2 kcal / mol ) were manually positioned inside the receptor binding cavity to reproduce the binding modes of cyclic tetrapeptides . the docking pose of each ligand was subsequently refined using the solid docking module of quanta . the receptor ligand complexes were then minimized with charmm ( adopted - basis newton raphson method , 30 steps , = 10 ) . models of opioid ligand

n - acetylation of the tetrahydroquinoline ( thq ) core of a series of -opioid receptor ( mor ) agonist/-opioid receptor ( dor ) antagonist ligands increases dor affinity , resulting in ligands with balanced mor and dor affinities . we report a series of n - substituted thq analogues that incorporate various carbonyl - containing moieties to maintain dor affinity and define the steric and electronic requirements of the binding pocket across the opioid receptors . 4h produced in vivo antinociception ( ip ) for 1 h at 10 mg / kg .

Introduction Results Discussion and Conclusions Experimental Section

PMC4590978

compression therapy is used for the treatment of venous pathologies such as deep vein thrombosis and chronic - venous insufficiency . the application of the external pressure on the lower limbs acts to compress the veins thus reducing their diameter . in consequence the velocity increases , which in turn encourages the return of blood to the heart and reduces pooling . many studies have demonstrated an increase in mean deep venous velocity , reduced venous pooling , and an improved venous return in patients who wore graduated compression stockings [ 35 ] . the use of compression garments in sport is becoming increasingly popular due to claims that they can improve recovery from exercise , by exerting these hemodynamic effects . evidence for the efficacy of compression garments in recovery is solid with recent meta - analysis supporting the use of compression to alleviate symptoms associated with fatigue [ 6 , 7 ] . suggested mechanisms include enhanced venous return and blood flow in passive conditions [ 8 , 9 ] that may aid the removal of metabolic waste [ 10 , 11 ] , reduce edema [ 12 , 13 ] , and improve muscle oxygenation [ 14 , 15 ] . however in some cases there is little evidence to support some of the purported benefits and gaps in knowledge are still evident [ 16 , 17 ] . the heterogeneity of effects could be explained by the disparity in terms of compression garments in all studies [ 6 , 7 ] . for example , findings of french et al . indicate that recovery and regeneration appear unaffected by wearing compression garments for 12 h after a hard training session compared with passive recovery . however , it should be clearly stated that one limitation of this study is that compression garments used were reported to have an average compression pressure of 12 mmhg at the calf . indeed venous return and other hemodynamic benefits which may aid recovery were previously described to be significantly improved with a minimum compression pressure of 1520 mmhg and with a peak for a pressure of around 40 mmhg [ 2 , 18 ] . some other studies present such limitation [ 19 , 20 ] and recent meta - analysis suggest understanding the discrepancy of results well to focus on the characteristics of compression garments especially compression pressure [ 6 , 7 ] . therefore the aim of the present study was to assess the changes in tissue oxygen saturation ( sto2 ) in response to the application of the main commercially available calf compression sleeves . we focused on sto2 because it has been extensively documented as a hemodynamic benefit of compression garments during recovery . it also presents the advantage of being noninvasively measured using near - infrared spectroscopy ( nirs ) . eight healthy subjects ( no history of cardiopulmonary disease or medication ) were studied ( mean sd : age , 32.4 4.8 years ; height , 174.5 1.9 cm ; body mass , 66.1 2.2 kg ; calf circumference at larger part : 36.4 0.2 cm ) . they were informed about the procedures and risks associated with participation in the study and all provided their written informed consent prior to participation . the study protocol was approved by the local ethics committee and was in accordance with the declaration of helsinki of the world medical association with regard to research conduct . the subjects came to the laboratory ( temperature : 20.0 1.0c ; humidity : 50 1.0% ) to complete a session in seated position including 10 min of quiet rest followed by 3 min measuring calf sto2 without compression sleeves and then alternating of 3 min of quiet rest and 3 min measuring sto2 with calf compression sleeves . a total of 15 different commercially available compression sleeves were studied in a randomized order ( figure 1 ) . using nirs technique , the inspectra sto2 tissue oxygenation monitor , model 650 ( hutchinson , mn , usa ) , provides continuous noninvasive assessment of sto2 at a maximum depth of 15 mm . the measurement principles of this technology have been described and its accuracy and reproducibility have been previously established . the microcirculatory sto2 assessment is defined as the ratio [ hbo2]/([hb]+[hbo2 ] ) expressed as percent , with hbo2 and hb being oxy- and deoxygenated hemoglobin , respectively . sto2 was measured at the level of the right gastrocnemius muscle , 12 cm below the fibula head . a transparent film was placed between the skin and the probe to protect it from sweat . sto2 was measured during three minutes without compression and during three minutes with each calf compression sleeve . sto2 values were analyzed with sto2 researcher 's analysis software version 4 ( hutchinson , mn , usa ) . only the mean of the last minute of each 3 min period was considered . the pressure of each calf sleeve was measured at the same area as the sto2 measurement using a pneumatic measuring system ( picopress , microlabitalia , padua , italy ) . the pressure transducer consists of a flat plastic pressure probe ( 5 cm diameter ) that is filled with 2 ml of air for the pressure measurement . fluctuations of pressure on this probe are transformed into electronic signals that can be recorded continuously . data are presented as mean sd . a p value < 0.05 was considered as significant . the normality of distribution was tested using the kolmogorov - smirnov test . to assess the effects of the calf compression sleeves , sto2 was significantly increased with all compression sleeves ( p < 0.05 ) compared with no compression ( from + 6.9% for a to + 22.6% for o ) . sto2 was positively correlated with compression pressure ( p < 0.05 ; r = 0.84 ) . the present study aimed at investigating the changes in sto2 in response to the application of the main commercially available calf compression sleeves . ( 1 ) wearing all compression sleeves significantly increased sto2 and ( 2 ) the most effective increases of sto2 were correlated with the highest compression pressures . firstly , sto2 increased with calf compression sleeves ( from + 6.9% for a to + 22.6% for o ) . these results are in accordance with previous works that reported an increase in sto2 with calf compression sleeves before and after running or cycling exercises . this higher sto2 was attributed to the increased muscle flow rate [ 8 , 9 ] and changes in skin blood flow [ 26 , 27 ] . indeed , wearing compression on the lower limbs venous emptying may increase arteriovenous pressure gradient , increasing arterial flow rate , oxygen supply , and therefore sto2 . as previously described , arterial vessels dilate in response to a fall of the transmural vessel pressure gradient . the pressure applied by sleeves is transmitted into the tissue and thus reduces the transmural pressure gradient of the arterial vessels . finally , changes in skin blood flow must also be considered [ 26 , 27 ] . indeed , sto2 was recorded at a maximum depth of 15 mm , including cutaneous and muscular vessels . moreover , previous studies suggest that compression sleeves may affect cutaneous sto2 through temperature changes [ 14 , 27 ] and pressure - induced skin vasodilation [ 26 , 30 ] . this study also revealed that increase of calf sto2 with compression sleeves was positively correlated with compression pressure ( p < 0.05 ; r = 0.84 ) . this finding is in agreement with previous studies suggesting a dose - response of compression pressure on venous hemodynamics including the velocity of venous circulation , venous pump function , or the degree of decrease in edema [ 2 , 3 , 31 ] . however , the relation between the compression pressure and its effects on hemodynamics is not systematically demonstrated [ 3133 ] . a possible explanation for this lack of relation is that hemodynamics may also be affected by other factors such as pressure gradient [ 24 , 32 ] and elastic properties [ 34 , 35 ] . for example , it has been shown that with the same pressure inelastic material is more effective than elastic [ 34 , 35 ] . by extension the seamless knitting could be in favor of long lasting effects on sto2 : no fraying and stitch defects making the sleeves loose fit and negatively affecting compression and elastic properties . this study provides support for the hypothesis that wearing compression sleeves from various brands may differently affect recovery , by virtue of their varied effects on sto2 . these results contribute to understanding well the large differences reported in recent meta - analysis [ 6 , 7 ] about effects of compression garments during recovery . since higher compression pressure was associated with higher sto2 this work suggests focusing on compression garments with high pressure level for recovery . in conclusion , this study shows that wearing compression sleeves from various brands differently increases tissue oxygen saturation . differences were linked to the compression pressure : higher compression pressures were associated with higher sto2 .

aim . the purpose was to examine the changes in tissue oxygen saturation ( sto2 ) in response to the application of different commercially available calf compression sleeves . methods . eight subjects came to the laboratory to complete a session in seated position including 10 min of quiet rest followed by 3 min measuring calf sto2 without compression sleeves and then alternating of 3 min of passive rest and 3 min measuring sto2 with calf compression sleeves . a total of 15 different commercially available compression sleeves were studied in a randomized order . calf sto2 was recorded using near - infrared spectroscopy . results . sto2 was significantly increased with all compression sleeves ( p < 0.05 ) compared with no compression ( from + 6.9% for the least effective to + 22.6% for the most effective ) . large differences were observed between compression sleeves ( p < 0.05 ) . sto2 was positively correlated with compression pressure ( p < 0.05 ; r = 0.84 ) . conclusion . this study shows that wearing compression sleeves from various brands differently affects tissue oxygen saturation . differences were linked to the compression pressure : higher compression pressures were associated with higher sto2 .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC3395253

thimerosal is a preservative that is widely used in medical products , including as a preservative in vaccines , immunoglobulin preparations , skin test antigens , antivenins , ophthalmic and nasal products , and tattoo inks , and is composed of 49.6 percent ethylmercury by weight . the widespread use of thimerosal exposes many to its potential toxic effects , especially in utero and in neonates . we report the results of a series of experiments using cultured normal human astrocytes ( nha ) exposed to thimerosal to study the compound 's effect on astrocyte mitochondria . the brain utilizes 20% of the oxygen consumed by the body but constitutes only 2% of the body 's mass . the majority of superoxide generated in cells comes from the reaction of molecular oxygen with flavin or quinone radicals , which are partly generated during respiration within complexes of the mitochondrial respiratory chain . the rate of reactive oxygen species ( ros ) production increases steeply with increased mitochondrial membrane potential . superoxide has a very short half - life in cells as it is rapidly dismutased by either the cytosolic cu - zn superoxide dismutase ( sod ) or the mn - sod in the mitochondrial matrix , producing molecular oxygen and hydrogen peroxide . thus , generation of superoxide is always accompanied by hydrogen peroxide production , and so opens up the possibility of hydroxyl radical ( ho ) generation via fenton / haber - weiss chemistry . fenton metals , including iron and copper , catalyze the production of ho from superoxide / hydrogen peroxide and so the free , unchelated levels of transition metals inside cells are very low and normally all stored in an oxidized state . normally , these metals are tightly bound to various metallochaperones , such as the ferric iron chelator ferritin . astrocytes are the major supporting cells of the brain and one of their key features is their ability to become reactive towards infectious agents and use chemical warfare , upregulating inos to generate high levels of nitric oxide and nadph oxidase to generate superoxide , hydrogen peroxide , peroxynitrite , and other oxidative per - species ( see and references within ) . the types and levels of antioxidant enzymes of nha are rather different from most other cell types and the levels of different enzymatic antioxidant enzyme change when nha transition from unreactive to reactive states . in many cell types the main defense against peroxide stress are selenol containing enzymes including the glutathione peroxidases ( gpx ) and thioredoxin reductase ( trxr ) . gpx is not present in detectable levels in human unreactive astrocytes in normal brain and it appears that gpx is only present in high levels in reactive astrocytes [ 8 , 9 ] . trxr levels in normal human brain are also low , but is significantly elevated in the brains of alzheimer 's patients , especially at the site of amyloid plaques where reactive astrocytes are present . it has been shown that in cultured nha that trxr expression is under tight regulation , with increases from very low basal levels , under the control of cytokines and growth factors . peroxiredoxins , including the mitochondrial peroxiredoxin v , are an important class of peroxide / peroxynitrite detoxification enzymes that are sensitive to organomercury . like the selenol - based antioxidant enzymes , these thiol - based antioxidant proteins are only found in very low levels in human astrocytes . there is much evidence to suggest that catalase , rather than cysteine or selenocysteine - based peroxidases , is the main enzymatic peroxidase in unreactive nha . nha also have high levels of reduced glutathione ( gsh ) , capable of detoxifying peroxides via direct chemistry , and high levels of all three superoxide dismutases [ 15 , 16 ] . catalase and the manganese superoxide dismutase are both upregulated when astrocytes are subjected to oxidative stress [ 14 , 16 ] . in cell types where selenol / thiol containing peroxidases are the major enzymes that detoxify peroxides , organomercury toxicity tends to result from loss of antioxidant enzyme function coupled with an increase in the rate of oxidant production [ 17 , 18 ] . there is a large literature examining the role of organomercury toxicity and the involvement of selenoenzymes trxr and glutathione peroxidase , gpx , see and references therein ; however , these data may not apply to nha , especially unreactive nha which appear not to make extensive use of these organomercury sensitive detoxification enzymes . ethylmercury is a lipophilic cation which can cross the blood - brain barrier [ 1922 ] . the octanol / water partition coefficients of methyl and ethylmercury are 1.4 to 1.8 [ 21 , 23 ] , at intracellular ph and [ cl ] , thus both organomercury compounds will predominately exist as lipophilic cations inside cells . mitchell demonstrated that lipophilic cations accumulate inside mitochondria , in a nernstian fashion , driven by the steady state membrane potential . given that the typical mitochondrial membrane potential of astrocytes and neurons is between 140170 mv , one would , a prior , expect the concentration of these organomercury compounds within mitochondria to be approximately 1000 times greater than the cytosolic concentration . we postulate that this compound is preferentially taken up into the mitochondria of nha causing damage to the respiratory chain and subsequent ros production . the damage of a cell 's mitochondria leads to the activation of the apoptotic cascade and subsequent cell death [ 3 , 4 , 24 , 2631 ] . this may be clinically relevant in the setting of a patient who harbors a known or unknown mitochondrial disorder . in the setting of a mitochondrial disorder we designed this series of experiments to examine the effects of thimerosal - derived ethylmercury on human astrocyte apoptosis by choosing a time course of cell examination after treatment that would showcase the early stages of apoptosis . we proposed that by examining the cells in an early phase , sixty minutes after ethylmercury dosing , we could visualize the compound 's effect on the mitochondria and mitochondrial dna ( mtdna ) . normal human astrocytes ( nha ) were obtained from lonza ( walkersville , md , usa ) and grown subject to their recommendations . nha were grown to confluency in astrocyte cell basal medium supplemented with 3% fbs , glutamine , insulin , fhegf , ga-1000 and ascorbic acid in 16-well lab - tek slide chambers ( nalge nunc , rochester , ny , usa ) , in a total volume of 240 l . firstly , a 50 l aliquot of ice - cold 8% pfa was added to each well , then gently aspirated and the wells were twice washed with ice - cold 2% pfa and then allowed to completely fix at 4c . after fixation cells were washed twice in x1 pbs ( thermo fisher scientific , rockford , il , usa ) . the tanks were then removed from the slides , the well area covered with fluoromount - g ( southernbiotech , il , usa ) , cover - slipped and sealed with nail varnish . h1398 ) , mitochondrial membrane potential with 500 nm mitotracker red ( cat no . m22425 ) , hydrogen peroxide using 5 m h2dcfam [ 31 , 32 ] ( cat no . d399 ) , mitochondrial superoxide generation with 5 m mitosox red ( cat no . m36008 ) ; ho was assayed using 5 m hydroxyphenyl fluorescein ( hpf ) ( cat no . h36004 ) , with reagents obtained from molecular probes ( eugene , or , usa ) . hydrazine reactive aldehyde / ketones were labeled using 225 m biotin - xx hydrazide ( cat no . the activity of caspase-3 in fixed , 0.1% triton permeabilized cells was measured using the molecular probes r110-enzchek assay kit ( cat no . the measurements and quantification of dna 3oh ( ddtunel ) , oxidized dna bases ( fpg - ddtunel ) and blunt ended breaks by use of the ddtunel and blunt - ended ligation were performed as described in our recent publications [ 35 , 36 ] . biotinylated ddutp and biotinylated blunt ended oligonucleotide probe a tdt reaction buffer was prepared daily diluting a stock solution 1 : 5 of tunel buffer ( 125 mm tris - hcl , 1 m sodium cacodylate , 1.25 mg / ml bsa , ph 6.6 ) and a 25 mm cobalt chloride stock solution , 1 : 25 . each well was twice washed in this reaction buffer and then incubated with 50 l of reaction buffer containing 20 units / ml of tdt and 250 nm of biotin-16-ddutp ( roche , in , usa ) . each sample , having previously undergone ddtunel , was washed and incubated with ne buffer 3 for 30 minutes and then with 50 l of the same buffer containing 100 units / ml of calf intestinal alkaline phosphatases ( sigma ) for 2 hours and the newly generated , 3po43oh , ends . following ddtunel / ciap - ddtunel , capping all 3oh/3po4 ends with authentic , unlabeled avidin , samples were washed twice in 10 mm hepes , 10 mm nacl , 2 mm edta and 0.1% bsa and then 50 l of the same buffer containing 100 units / ml of formamidopyrimidine dna glycosylase ( fpg ) ( usb , cleveland , oh , usa ) was applied to each of the wells , then incubated in a humidified box 2 hours . each sample was washed twice in x1 pbs ( thermo fisher scientific , rockford , il , usa ) twice in nebuffer 3 and 50 l of the same buffer containing 100 units / ml of ciap was applied to each section and incubated for 2 hours ; samples then underwent a third round of ddtunel and labeling with fitc - avidin . the wells were preincubated in the ligation buffer without the probe ( 66 mm - tris hcl , ph 7.5 , 5 mm mgcl2 , 0.1 mm dithioerythritol , 1 mm atp , and 15% polyethylene glycol-8000 ) to ensure even saturation . the buffer was aspirated , and the full ligation mix containing the ligation buffer with probe , 35 g/l , and 0.5 u/l t4 dna ligase ( new england biolabs , ipswich , ma , usa ) was applied to the sections , which were then incubated in a humidified box overnight . thimerosal 97% ( hplc ) and all unspecified regeants were obtained from sigma - aldrich ( st . louis , mo , usa ) , unless otherwise specified . thimerosal solutions were prepared in x1 pbs ( thermo fisher scientific , rockford , il , usa ) to a maximum concentration of 360 m and 10 l were added to the 240 l astrocytic volume . 3% fcs was present in the nha media throughout the time course . to generate the time course shown in figure 1 , nha were exposed to mitotracker , h2dcfam and hoechst at t = 0 . additions of 10 l aliquots were added at 10 minute intervals , to different wells in sequence , so that all the cells had the same length of exposure to the reporters , but different temporal exposure to thimerosal . the signal was acquired using a nikon eclipse te2000-e fluorescent microscope equipped with a coolsnap es digital camera system ( roperscientific ) containing a ccd-1300-y / hs 1392 1040 imaging array cooled by a peltier device . images were recorded and analyzed using nikon nis - elements software and images were stored as both .jpeg200 and .jpg files . the effect of ethylmercury on the fluorescence levels of the three reporters was investigated in two ways . the concentration dependence of ethylmercury towards nha was studied by adding to 014.4 m thimerosal to the cell media at t = 0 . in addition , we investigated the temporal changes caused by the addition of 14.4 m thimerosal at t = 0 , 10 , 20 , 30 , 40 , and 50 minutes before fixation at 60 minutes . we imaged the center field of three independent wells , at each time point or concentration , collecting the fluorescence levels of the three reporters of an average of 44 18 individual astrocytes per center field . in figure 1 we show the changes in the levels of mt and ros ( via dcf formation ) as a function of thimerosal concentration ( figure 1(a ) ) and of changes induced by incubation with 14.4 m thimerosal over time . it can be seen that low concentrations of ethylmercury cause an increase in both signals . the finding that ethylmercury increases ros generation is not surprising , given the well - known effects this agent has in disrupting cellular thiol / glutathione - based antioxidant defenses [ 20 , 22 , 30 ] . the hyperpolarization of mitochondrial membrane potential was unexpected , given that depolarization of mitochondria has been observed in most cell types prior to apoptosis . at higher concentrations ( > 7.2 m thimerosal ) a loss of mitochondrial signal and of dcf is observed . this loss of signal , when comparing > 7.2 m with < 7.2 m thimerosal , correlates well with changes in cell morphology , cell shrinkage and the formation of a ruffled plasma membrane and blebs . in the time course of ethylmercury - induced changes shown in figure 1(b ) , we see that the generation of ros species is an early event and that there is an increase in ros generation prior to changes in mitochondrial membrane potential . the levels of cellular dcf begin to fall at > 40 minutes , and this drop in the levels of the cellular ros reporter also correspond the observation of cell shrinkage and the formation of cytoplasmic blebs . in figure 2 we show the colocalization of mitochondrial and ros signals in high resolution images of control nha treated for 60 minutes with 14.4 m thimerosal . in figure 2(a ) , upper panels , we present the mitotracker ( red ) , ros - induced dcf ( green ) , and nuclear hoechst staining ( blue ) of nha taken at magnifications of 60 in the absence ( left ) and presence ( right ) of 14.4 m thimerosal . the fluorescence levels of all three panels are matched in the two images , so that the color levels absolutely reflect signal levels and show that thimerosal causes an approximately 50% drop in mitochondrial membrane potential and a two - fold increase in ros . it is clear that the majority of mitochondria in the cells are in a vermiform network and that there appears to be a strong colocalization of the mitochondrial and ros signals . in figure 2(b ) the images of control and treated cells obtained at 150 magnification are shown . here , the red mitochondrial signals are multiplied by a factor of four in the 14.4 m thimerosal - treated astrocytes , so as to allow visual identification of the distribution of the mitochondria within these cells . the three treated cells shown are reasonably representative of the population with the central cell being shrunken and with a highly distorted nucleus . the square outlines are areas of the cells where we present individual mitotracker and ros images , and the overlaid images of these fluorophores of these chosen areas , figure 2(c ) . these images clearly show that an orange colored horseshoe shaped signal in the control cell , shown in figure 2(b ) , consists of a network of mitochondria and that this mitochondrial network is mirrored in the dcf , ros , image . the correlation of mitochondrial signaling in the treated cells is also indicated , and in one of the treated cells we have identified a lightening - bolt shaped mitochondrial network . one can observe that this lightening bolt feature consists of mitotracker positive network of mitochondria and dcf signals . both images in figure 2(b ) have a diagonal line running from top - left to bottom - right . the bottom panel , figure 2(d ) , shows the intensity profile of mt , dcf , and hoechst along these two lines ( with the mt signal 4 in the thimerosal treated image ) . the red lines correspond to the fluorescence signal of mt , the blue lines to hoechst and the green lines to ros generated dcf . in both plots there is an additional black line , which matches the line shape and amplitude to the dcf signal . this black line is our fit to the ros signal , based on the amplitudinal changes of mt and hoechst . in the control panel the ros signal is best simulated by 0.44 multiplied by the mt signal and 0.39 multiplied by the hoechst signal . in the thimerosal - treated cells the relationship between hoechst staining and dcf levels is within 3% of that in the control cells modeled at 0.38 multiplied by hoechst fluorescence . however , the fit with mt labeling is strikingly different , with the best simulation generating a value of 0.117 for the ratio of actual mt signal to ros . we compared the cross - correlation of the simulated fit with the actual dcf signal and found the slopes were 1 0.01 in both cases and that the r values were greater than 0.99 in both controls and treated cells . thus , thimerosal treated astrocytic mitochondria are generating four times the amount of ros as the control mitochondria , but the steady state generation of ros in areas with no mitochondria , especially the nucleus , is unchanged . in figure 3 we show that damage from ros , in the form of aldehyde / ketones ( carbonyls ) , is also colocalized with mitochondrial membrane potential , and that more carbonyls are present in thimerosol treated nha . figure 3(a ) shows control and 14.4 m thimerosal prepared using mt and hoechst , then treated with biotin - xx hydrazide carbonyl labeling , which was visualized using fitc - avidin . figure 3(a ) shows control / thimerosal - treated cells where all three fluorophores have the same scale . we chose to present the images of a large ethylmercury - treated cell , somewhat unrepresentative of the population size distribution , as larger cells allow easier discrimination of the mitochondrial network . what is noticeable is that there is an increase in green ros damaged cell contents as a function of distance from the nucleus , in both images . the two boxes in figure 3(a ) show areas we have chosen to highlight the correlation between mt and carbonyl signals . these areas are shown as single images of mt and carbonyls , and as a merged image in figure 3(b ) . in the control cells it is clear that the network of mitochondria is colocalized with some networks of carbonyls , but there are some well - defined structural networks which show evidence of oxidative stress that do not correlate with mitochondria . we observe a similar pattern in thimerosal - treated astrocytes ; there are quite clearly networks of mitochondria and carbonyls and structures that contain evidence of ros damage , but without polarized mitochondria . the two vertical lines in figure 3(a ) indicate the position the fluorophores were interrogated to generate the fluorophore profiles shown in figure 3(c ) . again the three colors represent different fluorophores , mt ( red ) , carbonyls ( green ) and hoechst ( blue ) , and the black line is a simulation of the levels of ros damage generated by combining fractions of the mt and hoechst signals . in both samples the simulation is a poor match for the actual ros - induced signal , but control cells give a much poorer fit than do thimerosal - treated astrocytes . cross - correlations of ros versus our simulation of carbonyl levels generate slopes of 0.75 and 1.1 for controls and ethylmercury - treated cells , and prove r values of only 0.68 and of 0.86 , respectively . therefore , although we observe that generation of ros is highly localized to mitochondria ( figure 2 ) , the cellular distribution of markers of ros damage is poorly localized with mitochondria . it therefore appears that proteins suffering ros damage , and so having carbonyls , are transported from the regions where they have been damaged . vesicles containing high levels of carbonyls are present in both the controls and treated cells ; however , in the cells that have been incubated with ethylmercury we observe a large number of small , < 500 nm , clumps of oxidized material . a possible origin of this material is that it represents flocculated damaged mitochondria that are unable to maintain a membrane potential , such as that which occurs following the mitochondrial permeability transition . this clumping of mitochondria has previously been described during the early stages of apoptosis and has shown to be a result of the activation of the bh3 domain of bax . we initially postulated that cationic , lipophilic ethylmercury should partition into the mitochondrial matrix and that accumulation should be driven by the mitochondrial membrane potential . as mtdna is restricted to the mitochondrial matrix , an increase in the steady state of ros in this compartment should act as a reporter of this oxidative stress . we examined the presence of 3oh dna breaks or of fpg - labile modified dna bases using ddtunel and fpg - ddtunel , and additional aldehyde / ketones ( carbonyls ) using biotin - xx hydrazide . cells grown in the absence or presence of 14.4 m thimerosal were labeled for the presence of 3oh dna nicks ( ddtunel ) or for oxidized / acylated dna bases ( fpg - ddtunel ) using biotinylated ddutp . these dna ends were visualized using fitc - avidin and carbonyls with texas red - avidin , and nuclei were again labeled with hoechst , figure 4 . the signals in figure 4 show ( blue ) nuclei , ( red ) carbonyls , and ( green ) 3oh dna ends ( figure 4(a ) ) or ( green ) fpg - labile dna bases / apurinic or apyrimidinic sites ( figure 4(b ) ) , reflecting the levels of fluorophore in all four images . the insert , figure 4(c ) , is of a cell that has entered the final stages of apoptosis and so shows large numbers of 3oh dna ends . in figure 4(c ) the ros signal has been divided by a factor of four and the ddtunel signal by a factor of five , compared to figure 4(a ) and ( b ) . comparison of figure 4(c ) with 4(a ) and 4(b ) reveals that at one hour of incubation we are not observing full blown apoptosis , which is characterized by nuclear dna fragmentation , and are indeed observing the early phases of cell death . what figure 4 demonstrates is that there is a clear colocalization of dna damage and the presence of carbonyls . the damaged dna is cytosolic , not nuclear , suggesting mitochondrial dna damage . by demonstrating a colocalization of mitochondrial dna damage and ros in the cytosol of the nhas , we show that the mitochondria may be responsible for the generation of ros in the presence of ethylmercury and are the primary inducers of the apoptotic cascade . we measured the production of ros , using the mitochondrial superoxide probe mitosox , and additionally measured ho via hydroxyphenyl fluorescein ( hpf ) , 3oh dna ends with ddtunel and blunt - ended dna breaks in nha incubated for 1 hour with 14.4 m thimerosal , figure 5 . figure 5(a ) shows that reporters for both superoxide and ho are highly colocalized , giving r values of > 0.98 , and thus superoxide generation leads to fenton / haber - weiss chemistry inside mitochondria . treatment of nha with ethylmercury leads to a 90% increase in superoxide generation per cell , even though under the same conditions we observe a 50% drop in mitochondrial membrane potential . deconvolution of superoxide and ho signals show that the presence of ethylmercury results in 60% more ho generation per superoxide . figures 5(b ) and 5(c ) show that superoxide generation correlates with nonnuclear , thus mtdna damage in the form of ddtunel 3oh dna ends and also of highly damaging blunt - ended dna breaks . the scaling of the two green channels in figures 5(b ) and 5(c ) differ by a factor of 4 , and this indicates that there are on average 9 times as many 3oh ends as there are dna breaks in the control mitochondria . in figure 6 we present a bar plot showing the summarized changes we observe in nha following a one - hour exposure to 14.4 m thimerosal . five images were taken from three parallel experiments with an average of 44 18 individual astrocytes per visual field , and the error bars represent the sd of the population . ethylmercury causes a 50% collapse in membrane potential in astrocytes at 1 hour . accompanying this collapse in membrane potential we observe a significant increase in the levels of various ros . the internal mitochondrial steady state level of superoxide increases by 70% in treated cells and is matched by an increase in cellular hydrazine reactive carbonyls . using h2dcf - am we observe a 200% increase in steady state production of reactive oxidants , which from deconvolution we know to be mitochondrially generated ( figure 2 ) . mitochondrial dna , and not nuclear dna , is far more vulnerable to ethylmercury - induced damage . we observe a 240% increase in the levels of mitochondrial dna breaks , a 300% increase in 3oh dna nicks and 460% increase in the levels of oxidized bases / apurinic or apyrimidinic sites . as mtdna is localized within the mitochondrial matrix , it follows that this is the main site of ros generation . as fenton / haber - weiss chemistry is the primary generator of ho in biological systems , this finding suggests that ethylmercury is also increasing the levels of fenton metals , such as iron and copper , inside the astrocytes ' mitochondria . in the final pair of bars were shown the change in the levels of caspase-3 activity , measured by examining the cleavage of a z - devd - r110 substrate . we also find a five - fold increase in caspase-3 activity , indicating that this pathway has been activated in thimerosal - treated cells . we find that treatment of nha with ethylmercury causes an increase in mitochondrial superoxide generation as shown in figure 5 . however , the increase in superoxide generation is identical to the increase in the levels of protein carbonyls as shown in figure 6 . h2o2-induced formation of dichlorofluoresein from h2dcf - am is only approximately 20% greater than superoxide / carbonyl formation , which suggests that the loss of peroxidase function is not a feature of nha ethylmercury toxicity . this is consistent with the effect of methylmercury on hela cells , where mitochondrial matrix generation of superoxide was implicated as the most damaging ros . hela cells can be protected from methylmercury toxicity by upregulating mitochondrial mn - sod but not cytosolic cu / zn - sod , gpx or catalase . the majority of protein carbonyls in controls and in ethylmercury - treated nha are also colocalized with mitochondria , as shown in figure 2 . the peroxides measured via h2dcf - am and protein carbonyls are derived from mitochondrial ros generation , as shown by colocalization of signals with the specific mitochondrial superoxide probe , mitosox , as shown in figure 5 . these findings are in broad agreement with the known generation of ros on either side of the inner mitochondrial membrane in normal mitochondria and effects of methylmercury on rodent astrocytes observed by shanker and coworkers , as they too identified that mitochondria are the main production sites of increased superoxide generation . in addition to measuring peroxide / superoxide generation we also examined the formation of ho using the specific probe hpr and using the fpg - ddtunel assay which measures oxidized dna bases . the conversion of guanine to 8-hydroxyguanine and 8-hydroxyguanine to more oxidized dna hydantoin lesions , spiroiminodihydantoin and guanidinohydantoin , is generally believed to be due to ho or to fenton 's reagent ( oxy - ferry ; fe = o ) and oxy - cupryl cu = o ) . 8-hydroxyguanine , spiroiminodihydantoin , and guanidinohydantoin are substrates from the fpg - ddtunel assay [ 35 , 42 ] . in figure 4 we demonstrated that while the levels of damaged nuclear dna and mtdna are very low in untreated cells , ethlymercury induces a large increase in oxidized mtdna lesions . the highest levels of damaged mtdna and protein carbonyls occur in structures that appear to be flocculated mitochondria . these grainy , oxidized , structures are not present as bright grains when viewed using mitotracker , when carbonyl rich grains can be identified , shown in figure 2 . these same vermiform structures are also identified in treated cells labeled with specific probes for both superoxide and ho seen in figure 5 . however , although we observe an increase in the levels of cytosolic ( hence mitochondrial ) blunt - ended breaks and nicks in figure 5 , very high levels of dna breaks are not present in granular form . thus , these flocculated mitochondria represent a dead - end mitochondrial state and given the close correlation between fpg - ddtunel and caspase-3 upregulation shown in figure 6 , it is reasonable to conclude these are mitochondria that have undergone the permeability transition , resulting in the release of proapoptotic proteins like cytochrome c and diablo from the intermembrane space , mitoposis , and the initiation of the caspase-3 apoptotic cascade . it has long been known that organomercury reacts with iron sulfur centers ; indeed methylmercury has been used as an aid to identify mercury adducts in iron - sulfur protein crystal structures for decades . the reaction of organomercury with iron sulfur centers in proteins such as aconitase results in loss of enzymatic function , the formation organomercury thioether adducts , and exposure to the bulks aqueous phase to redox active iron or release of free iron . it has been shown that , in mouse brain , the mitochondrial iron - sulfur complex rich enzyme nadh / quinone oxidoreductase ( complex i ) is highly sensitive to methylmercury . in a study by lebel et al . the potent iron - chelator , deferoxamine , protected rat cerebellum from ros following an injection of methylmercury . iron chelation also protected neurons from ros following in vitro exposure to methylmercury , but there was no evidence of deferoxamine - mercurial complex formation . methylmercury treatment of isolated mitochondria , from the cerebrum , the cerebellum and from liver , causes an inhibition of respiration and increased superoxide / hydrogen peroxide formation , mostly via damage to succinate dehydrogenase . the three iron - sulfur centers of succinate dehydrogenase on the matrix side of the inner mitochondrial membrane are the likely site of inhibition and possible iron release given that these clusters are sulphide / iron labile towards the thiophilic reagent , p - chloromercuribenzoate . based on the work reported here and by others , we suggest a mechanism for the toxicity of organomercury , which is shown in diagrammatic form in figure 7 . as a lipophilic cation , ethylmercury will become concentrated inside astrocytes , with respect to the bulk extracellular phase , following the plasma membrane potential of 45 mv by a factor of 5.6 fold , and cytosolic ethlymercury will partition into the mitochondria by a factor of 1,000 fold , its accumulation driven by the approximate 180 mv mitochondrial membrane potential , figure 7(a ) . inside the mitochondria the ethylmercury will react with iron - sulfur centers , causing the release of iron into the mitochondrial matrix , figure 7(b ) . the role of ethlymercury in ros species formation and detoxification is shown in figure 7(c ) . the iron - sulfur centers of oxidoreductases ( e.g. , succinate dehydrogenase ) when damaged by organomercury not only generate free iron , ( figure 7(b ) i ) , but also form intraenzyatic carbon radical species ( figure 7(b ) ii ) that will react with molecular oxygen to give rise to superoxide , ( figure 7(b ) iii ) . superoxide can react with either free iron generation , the ferrous ion , or be dismutated into hydrogen peroxide by the mitochondrial mn - sod . ferrous ion , and hydrogen peroxide react to generate the highly oxidizing radical , hydroxyl radical , ( figure 7(b ) iv ) , an agent implicated in pathology and ageing [ 47 , 48 ] . the levels of hydrogen peroxide would be generally lowered by the mitochondrial antioxidants , including glutathione - dependent selenol / thio - based peroxidases , like gpx and trxr . however , these enzymes are inhibited by organomercury indirectly by depletion of glutathione , ( figure 7(b ) v ) , and directly by the capping of the active site selenol / thiol by organomercury , ( figure 7(b ) vi ) . thus , the release of iron catalyzes fenton / haber - weiss chemistry leading to the formation of the highly oxidizing ho . ho has multiple targets , including sensors of the permeability transition complex and also mtdna . high levels of ho cause mitoposis , leading to cytochrome c release from the mitochondria and the initiation of apoptosis . we find that a consequence of ethylmercury exposure to nha is damage to the mitochondrial genome . we find an increase in dna nicks , breaks and most importantly , in the level of oxidized bases . mitochondria typically have 150 copies of mtdna and during aging or with exposure to environmental stressors , the number of error free copies of mtdna undergoes a decline . according to harman 's free radical / mitochondrial theory of aging [ 47 , 48 ] , the production of ros by mitochondria leads to mtdna damage and mutations . these in turn lead to progressive respiratory chain deficits , which result in yet more ros production , producing a positive feedback loop . the results of this study suggest that ethylmercury is a mitochondrial toxin in human astrocytes . we believe that this finding is important , particularly since the number of diseases in which mitochondrial dysfunction has been implicated are rapidly increasing .

thimerosal generates ethylmercury in aqueous solution and is widely used as preservative . we have investigated the toxicology of thimerosal in normal human astrocytes , paying particular attention to mitochondrial function and the generation of specific oxidants . we find that ethylmercury not only inhibits mitochondrial respiration leading to a drop in the steady state membrane potential , but also concurrent with these phenomena increases the formation of superoxide , hydrogen peroxide , and fenton / haber - weiss generated hydroxyl radical . these oxidants increase the levels of cellular aldehyde / ketones . additionally , we find a five - fold increase in the levels of oxidant damaged mitochondrial dna bases and increases in the levels of mtdna nicks and blunt - ended breaks . highly damaged mitochondria are characterized by having very low membrane potentials , increased superoxide / hydrogen peroxide production , and extensively damaged mtdna and proteins . these mitochondria appear to have undergone a permeability transition , an observation supported by the five - fold increase in caspase-3 activity observed after thimerosal treatment .

1. Introduction 2. Methods 3. Results 4. Discussion

PMC3308306

participants performed aerobic and resistance exercise at least three times per week and had been doing so for at least 6 months ( table 1 ) . participant characteristics the research took place in the human and environmental physiology research unit at the university of ottawa . after being informed of the purpose , protocol , and possible risks of the study , participants gave written consent and completed physical activity readiness questionnaires ( american heart association / american college of sports medicine health / fitness facility preparticipation screening questionnaire ) . hand - held glucose meters ( onetouch ultra , lifescan , johnson & johnson , milpitas , ca ) and test strips ( with identical code ) were provided for capillary glucose tests . on a separate visit , participants underwent an incremental workload running test on a treadmill with a monitored electrocardiogram ( quinton q4500 , quinton , bothell , wa ) to determine peak oxygen consumption ( vo2peak ) . vo2peak was determined by measuring the volume and concentration of expired oxygen and carbon dioxide ( ametek model s-3a/1 and cd 3a , applied electrochemistry , pittsburgh , pa ) . muscular strength ( eight repetition maximum [ 8-rm ] ) was recorded as the maximum weight that participants could lift eight times with good form for chest press ( pectoralis major ) , leg press ( quadriceps , biceps femoris , gluteus maximus ) , seated row ( latissimus dorsi , rhomboids , trapezius ) , leg curl ( biceps femoris ) , shoulder press ( deltoids ) , and lat pull - down ( latissimus dorsi ) . a venous blood sample was drawn for determination of hba1c , which was measured by automated heterogeneous immunoassay with latex - enhanced turbidimetric detection on a roche cobas integra 800 analyzer ( roche diagnostics corp . , indianapolis , in ) . the cgms system gold ( medtronic , northridge , ca ) was used in this study . participants were blinded to their glucose values and could not change their regular behavior patterns based on real - time glucose monitoring . twenty - four hours before each experimental session , the cgm sensor was inserted subcutaneously in the abdomen or in the upper gluteal area . participants performed capillary glucose tests and calibrated the cgm unit four times daily using the hand - held glucose meter provided . on the third day , 24 h postexercise , participants removed the sensors and the researchers retrieved the monitors . data were downloaded using a medtronic com - station and minimed solutions software version 3.0 ( medtronic ) . they ate the same breakfast , same lunch , and same supper each day of sensor wear and kept their insulin doses the same each of these days to the greatest extent possible . participants avoided exercise ( apart from that performed in our laboratory ) for 24 h before inserting the sensor ( 48 h before each study exercise session ) as well as during the 3 days of sensor wear . participants arrived at the laboratory at 1600 h. intravenous catheters were inserted soon after arrival . each participant performed two experimental sessions in random order separated by at least 5 days : aerobic exercise before resistance exercise session ( ar ) : a 45-min bout of moderate - intensity aerobic exercise ( treadmill running at 60% of their predetermined vo2peak ) , followed by a 45-min bout of resistance training ( three sets of eight repetitions with 90 s of rest between sets ) . resistance exercise before aerobic exercise session ( ra ) : the same exercises as above were performed , with the resistance exercise completed before the aerobic exercise . women were using monophasic oral contraceptives and were tested during the active pill consumption phase . on the days that exercise was scheduled ( day 2 of each 3-day monitoring period ) , participants were asked to decrease their insulin doses ( a 10% decrease in long- or intermediate - acting for patients receiving multiple daily injections and a 50% decrease in basal rate 1 h pre - exercise for patients receiving a continuous subcutaneous insulin infusion ) . a further 25% basal rate decrease was made for continuous subcutaneous insulin infusion patients if their capillary glucose was 5 standardized snacks ( glucerna chocolate graham snack bars , 150 calories , 25 g of carbohydrate ; abbott laboratories , abbott park , il ) were provided and consumed at 1600 h each day of monitoring . before starting exercise capillary glucose tests were performed upon arrival at the laboratory , 30 min before and immediately before exercise . if capillary glucose levels were < 4.5 mmol / l , participants were provided with 32 g of glucose ( dex4 , amg medical , montreal , qc , canada ) before levels were checked again 15 min later . if initial readings were between 4.5 and 5.4 glucose concentrations during exercise were monitored by applying a drop of venous blood to a test strip inserted in the study hand - held glucose meter when venous blood samples were collected . when levels were < 4.5 mmol / l , exercise was interrupted and participants were provided with 16 g of glucose . capillary glucose tests were then performed every 10 min , and an additional 16 g of glucose was provided when necessary until a level of 5.5 mmol / l was achieved and exercise resumed . oxygen consumption was measured using a portable gas analysis system ( oxycon mobile , jaeger , hoechberg , germany ) . venous blood samples were collected at baseline , 5 , 10 , 15 , 30 , 45 , 50 , 55 , 60 , 75 , and 90 min during exercise and at 5 , 10 , 15 , 20 , 30 , 40 , 50 , and 60 min after exercise . blood was drawn using 5 ml sterile plastic syringes and transferred immediately into 5.4 ml plasma ( k2edta ) bd vacutainer tubes ( bd , franklin lakes , nj ) , mixed by inversion and centrifuged immediately . plasma aliquots were transferred into 1.5 ml microcentrifuge tubes and stored at 80c until analyzed . plasma glucose concentrations were determined using the hexokinase timed end point method on the beckman coulter unicel dxc600 synchron clinical analyzer ( beckman coulter inc . , plasma glucose concentration was compared between treatments using two - way repeated - measures anova with the factors of time ( exercise : 5 , 10 , 15 , 30 , 45 , 50 , 55 , 60 , 75 , and 90 min ; recovery : 5 , 10 , 15 , 20 , 30 , 40 , 50 , and 60 min ) and treatment ( ra or ar ) . paired sample t tests were used to perform pairwise post hoc comparisons between treatments for each time point to examine within - treatment changes from baseline and changes throughout recovery . energy expenditure during the exercise sessions ( including the recovery ) was compared using a paired sample t test . cgm data were grouped and summarized as follows : 24 h and overnight ( 2400 to 0600 h ) pre - exercise , as well as 24 h and overnight postexercise . hypoglycemia was defined as any value < 3.5 mmol / l detected by cgm , and values > 10.9 mmol / l were categorized as hyperglycemic . total time spent in hypoglycemia , euglycemia , and hyperglycemia for the predetermined periods and the area under the curve ( auc , defined as the absolute distance from the described limits , multiplied by the time spent outside those limits ) for time spent hypo- and hyperglycemic was determined along with the maximum , minimum , and mean interstitial glucose for each time period . variables were compared between exercise treatments , and pre- and postexercise values were compared within treatments using related - samples wilcoxon signed rank tests . these tests were also used to examine differences in insulin and carbohydrate intake ( calculated from the participants food and insulin diaries ) between days within exercise treatments ( day 1 vs. 2 ) , and between exercise treatments ( days 1 through 3 ) . pearson correlation analyses were performed comparing capillary glucose values recorded by the participants during nonexercise periods to cgm data to assess the accuracy of the sensors throughout each 3-day measuring period . the research took place in the human and environmental physiology research unit at the university of ottawa . after being informed of the purpose , protocol , and possible risks of the study , participants gave written consent and completed physical activity readiness questionnaires ( american heart association / american college of sports medicine health / fitness facility preparticipation screening questionnaire ) . hand - held glucose meters ( onetouch ultra , lifescan , johnson & johnson , milpitas , ca ) and test strips ( with identical code ) were provided for capillary glucose tests . on a separate visit , participants underwent an incremental workload running test on a treadmill with a monitored electrocardiogram ( quinton q4500 , quinton , bothell , wa ) to determine peak oxygen consumption ( vo2peak ) . vo2peak was determined by measuring the volume and concentration of expired oxygen and carbon dioxide ( ametek model s-3a/1 and cd 3a , applied electrochemistry , pittsburgh , pa ) . muscular strength ( eight repetition maximum [ 8-rm ] ) was recorded as the maximum weight that participants could lift eight times with good form for chest press ( pectoralis major ) , leg press ( quadriceps , biceps femoris , gluteus maximus ) , seated row ( latissimus dorsi , rhomboids , trapezius ) , leg curl ( biceps femoris ) , shoulder press ( deltoids ) , and lat pull - down ( latissimus dorsi ) . a venous blood sample was drawn for determination of hba1c , which was measured by automated heterogeneous immunoassay with latex - enhanced turbidimetric detection on a roche cobas integra 800 analyzer ( roche diagnostics corp . , indianapolis , in ) . the cgms system gold ( medtronic , northridge , ca ) was used in this study . participants were blinded to their glucose values and could not change their regular behavior patterns based on real - time glucose monitoring . twenty - four hours before each experimental session , the cgm sensor was inserted subcutaneously in the abdomen or in the upper gluteal area . participants performed capillary glucose tests and calibrated the cgm unit four times daily using the hand - held glucose meter provided . on the third day , 24 h postexercise , participants removed the sensors and the researchers retrieved the monitors . data were downloaded using a medtronic com - station and minimed solutions software version 3.0 ( medtronic ) . they ate the same breakfast , same lunch , and same supper each day of sensor wear and kept their insulin doses the same each of these days to the greatest extent possible . participants avoided exercise ( apart from that performed in our laboratory ) for 24 h before inserting the sensor ( 48 h before each study exercise session ) as well as during the 3 days of sensor wear . participants arrived at the laboratory at 1600 h. intravenous catheters were inserted soon after arrival . each participant performed two experimental sessions in random order separated by at least 5 days : aerobic exercise before resistance exercise session ( ar ) : a 45-min bout of moderate - intensity aerobic exercise ( treadmill running at 60% of their predetermined vo2peak ) , followed by a 45-min bout of resistance training ( three sets of eight repetitions with 90 s of rest between sets ) . resistance exercise before aerobic exercise session ( ra ) : the same exercises as above were performed , with the resistance exercise completed before the aerobic exercise . women were using monophasic oral contraceptives and were tested during the active pill consumption phase . on the days that exercise was scheduled ( day 2 of each 3-day monitoring period ) , participants were asked to decrease their insulin doses ( a 10% decrease in long- or intermediate - acting for patients receiving multiple daily injections and a 50% decrease in basal rate 1 h pre - exercise for patients receiving a continuous subcutaneous insulin infusion ) . a further 25% basal rate decrease was made for continuous subcutaneous insulin infusion patients if their capillary glucose was 5 standardized snacks ( glucerna chocolate graham snack bars , 150 calories , 25 g of carbohydrate ; abbott laboratories , abbott park , il ) were provided and consumed at 1600 h each day of monitoring . before starting exercise , participants were required to have blood glucose levels between 5.5 and 13.9 mmol / l . capillary glucose tests were performed upon arrival at the laboratory , 30 min before and immediately before exercise . if capillary glucose levels were < 4.5 mmol / l , participants were provided with 32 g of glucose ( dex4 , amg medical , montreal , qc , canada ) before levels were checked again 15 min later . if initial readings were between 4.5 and 5.4 mmol / l , participants were given 16 g of glucose . glucose concentrations during exercise were monitored by applying a drop of venous blood to a test strip inserted in the study hand - held glucose meter when venous blood samples were collected . when levels were < 4.5 mmol / l , exercise was interrupted and participants were provided with 16 g of glucose . capillary glucose tests were then performed every 10 min , and an additional 16 g of glucose was provided when necessary until a level of 5.5 mmol / l was achieved and exercise resumed . oxygen consumption was measured using a portable gas analysis system ( oxycon mobile , jaeger , hoechberg , germany ) . venous blood samples were collected at baseline , 5 , 10 , 15 , 30 , 45 , 50 , 55 , 60 , 75 , and 90 min during exercise and at 5 , 10 , 15 , 20 , 30 , 40 , 50 , and 60 min after exercise . blood was drawn using 5 ml sterile plastic syringes and transferred immediately into 5.4 ml plasma ( k2edta ) bd vacutainer tubes ( bd , franklin lakes , nj ) , mixed by inversion and centrifuged immediately . plasma aliquots were transferred into 1.5 ml microcentrifuge tubes and stored at 80c until analyzed . plasma glucose concentrations were determined using the hexokinase timed end point method on the beckman coulter unicel dxc600 synchron clinical analyzer ( beckman coulter inc . , plasma glucose concentration was compared between treatments using two - way repeated - measures anova with the factors of time ( exercise : 5 , 10 , 15 , 30 , 45 , 50 , 55 , 60 , 75 , and 90 min ; recovery : 5 , 10 , 15 , 20 , 30 , 40 , 50 , and 60 min ) and treatment ( ra or ar ) . paired sample t tests were used to perform pairwise post hoc comparisons between treatments for each time point to examine within - treatment changes from baseline and changes throughout recovery . energy expenditure during the exercise sessions ( including the recovery ) was compared using a paired sample t test . cgm data were grouped and summarized as follows : 24 h and overnight ( 2400 to 0600 h ) pre - exercise , as well as 24 h and overnight postexercise . hypoglycemia was defined as any value < 3.5 mmol / l detected by cgm , and values > 10.9 mmol / l were categorized as hyperglycemic . total time spent in hypoglycemia , euglycemia , and hyperglycemia for the predetermined periods and the area under the curve ( auc , defined as the absolute distance from the described limits , multiplied by the time spent outside those limits ) for time spent hypo- and hyperglycemic was determined along with the maximum , minimum , and mean interstitial glucose for each time period . variables were compared between exercise treatments , and pre- and postexercise values were compared within treatments using related - samples wilcoxon signed rank tests . these tests were also used to examine differences in insulin and carbohydrate intake ( calculated from the participants food and insulin diaries ) between days within exercise treatments ( day 1 vs. 2 ) , and between exercise treatments ( days 1 through 3 ) . pearson correlation analyses were performed comparing capillary glucose values recorded by the participants during nonexercise periods to cgm data to assess the accuracy of the sensors throughout each 3-day measuring period . energy expenditure was measured during exercise and recovery together for both sessions in 9 of the 12 participants . there were no differences in energy expenditure between ar ( 4,277 729 kj ) and ra ( 4,247 589 kj ) . plasma glucose levels for exercise and recovery ( supplementary table 1 ) are plotted in fig . 1 . a significant effect of time ( p = 0.001 ) and an interaction of treatment and time ( p = 0.004 ) the aerobic exercise performed in the ar treatment caused a substantial decline in blood glucose concentration , resulting in plasma glucose levels that were lower than baseline within the first 10 min of exercise , persisting until the end of aerobic exercise ( 9.1 2.4 at baseline ; 5.5 2.4 mmol / l at 45 min ; p < 0.01 ) and continuing into resistance exercise . glucose then increased during resistance exercise , producing levels that were similar to baseline by the end of exercise . conversely , the ra treatment did not produce significant changes from baseline during resistance exercise . after the change in exercise modality in ra , glucose levels were only significantly different from baseline after 75 ( p = 0.044 ) and 90 ( p = 0.018 ) min of exercise . glucose was lower in the ar treatment than in the ra treatment until the end of exercise , with differences achieving statistical significance between 30 and 60 min ( p < 0.05 ) . mean se plasma glucose during exercise and recovery for aerobic exercise performed before resistance exercise ( ar , dashed line with ) and resistance exercise performed before aerobic exercise ( ra , solid line with ) ( n = 11 ) . change throughout recovery from end - exercise level where p < 0.05 . during the postexercise recovery period , there was a significant effect of time ( p < 0.01 ) for changes in plasma glucose , but no effect of treatment or interaction of treatment and time . significant increases in plasma glucose from the end of exercise were seen throughout recovery after ar where none were observed after exercise in ra ( fig . total daily insulin doses did not differ significantly between treatments on the first 2 days of cgm wear before the experimental exercise session , or between the first and second day within each treatment . insulin adjustments for exercise were similar between treatments ( supplementary table 2 ) . on the day after the exercise testing session , insulin intake was lower after the ar session compared with ra ( 36.1 16.3 vs. 38.8 18.5 units , p = 0.028 ) . ten of 12 participants required carbohydrate supplementation during the ar session compared with only 6 of 12 during ra ; however , there were no statistically significant differences between groups in total carbohydrate intake during exercise and recovery in the laboratory ( supplementary table 3 ) and in the 6 h after exercise ( supplementary table 4 ) . pearson correlations between capillary glucose readings from nonexercise periods and sensor readings over the monitoring period were 0.95 for ar and 0.91 for ra ( p < 0.001 ) . there were no significant differences between treatments with respect to hypoglycemia and hyperglycemia ( number of excursions , time , auc ) as well as mean , maximum , and minimum glucose on the night before or 24 h before exercise . average maximum nocturnal glucose levels were significantly lower after exercise than the previous ( nonexercise ) night ( pre - exercise = 9.5 3.0 mmol / l , postexercise maximum = 8.8 4.0 mmol / l ; p = 0.04 ) . within the ar treatment , there was a trend toward greater auc postexercise for nocturnal hypoglycemia ( p = 0.06 ) compared with ra . although the frequency of nocturnal hypoglycemic events did not differ between the two exercise sessions , the duration and depth of hypoglycemia tended to be longer and more severe after ar than after ra ( table 2 ) . mean glucose ( n = 12 ) as measured by continuous glucose monitoring from 1 to 12 h after exercise following aerobic exercise performed before resistance exercise ( ar , dashed line ) and resistance exercise performed before aerobic exercise ( ra , solid line ) . ( a high - quality color representation of this figure is available in the online issue . ) summary of overnight ( 2400 to 0600 h ) continuous glucose monitoring data for the night before and the night after exercise * plasma glucose levels for exercise and recovery ( supplementary table 1 ) are plotted in fig . 1 . a significant effect of time ( p = 0.001 ) and an interaction of treatment and time ( p = 0.004 ) the aerobic exercise performed in the ar treatment caused a substantial decline in blood glucose concentration , resulting in plasma glucose levels that were lower than baseline within the first 10 min of exercise , persisting until the end of aerobic exercise ( 9.1 2.4 at baseline ; 5.5 2.4 mmol / l at 45 min ; p < 0.01 ) and continuing into resistance exercise . glucose then increased during resistance exercise , producing levels that were similar to baseline by the end of exercise . conversely , the ra treatment did not produce significant changes from baseline during resistance exercise . after the change in exercise modality in ra , glucose levels were only significantly different from baseline after 75 ( p = 0.044 ) and 90 ( p = 0.018 ) min of exercise . glucose was lower in the ar treatment than in the ra treatment until the end of exercise , with differences achieving statistical significance between 30 and 60 min ( p < 0.05 ) . mean se plasma glucose during exercise and recovery for aerobic exercise performed before resistance exercise ( ar , dashed line with ) and resistance exercise performed before aerobic exercise ( ra , solid line with ) ( n = 11 ) . change throughout recovery from end - exercise level where p < 0.05 . during the postexercise recovery period , there was a significant effect of time ( p < 0.01 ) for changes in plasma glucose , but no effect of treatment or interaction of treatment and time . significant increases in plasma glucose from the end of exercise were seen throughout recovery after ar where none were observed after exercise in ra ( fig . total daily insulin doses did not differ significantly between treatments on the first 2 days of cgm wear before the experimental exercise session , or between the first and second day within each treatment . insulin adjustments for exercise were similar between treatments ( supplementary table 2 ) . on the day after the exercise testing session , insulin intake was lower after the ar session compared with ra ( 36.1 16.3 vs. 38.8 18.5 units , p = 0.028 ) . ten of 12 participants required carbohydrate supplementation during the ar session compared with only 6 of 12 during ra ; however , there were no statistically significant differences between groups in total carbohydrate intake during exercise and recovery in the laboratory ( supplementary table 3 ) and in the 6 h after exercise ( supplementary table 4 ) . pearson correlations between capillary glucose readings from nonexercise periods and sensor readings over the monitoring period were 0.95 for ar and 0.91 for ra ( p < 0.001 ) . there were no significant differences between treatments with respect to hypoglycemia and hyperglycemia ( number of excursions , time , auc ) as well as mean , maximum , and minimum glucose on the night before or 24 h before exercise . average maximum nocturnal glucose levels were significantly lower after exercise than the previous ( nonexercise ) night ( pre - exercise = 9.5 3.0 mmol / l , postexercise maximum = 8.8 4.0 mmol / l ; p = 0.04 ) . within the ar treatment , there was a trend toward greater auc postexercise for nocturnal hypoglycemia ( p = 0.06 ) compared with ra . although the frequency of nocturnal hypoglycemic events did not differ between the two exercise sessions , the duration and depth of hypoglycemia tended to be longer and more severe after ar than after ra ( table 2 ) . mean glucose ( n = 12 ) as measured by continuous glucose monitoring from 1 to 12 h after exercise following aerobic exercise performed before resistance exercise ( ar , dashed line ) and resistance exercise performed before aerobic exercise ( ra , solid line ) . ( a high - quality color representation of this figure is available in the online issue . ) summary of overnight ( 2400 to 0600 h ) continuous glucose monitoring data for the night before and the night after exercise * this study evaluated , in the context of a combined resistance and aerobic exercise session , the effects of exercise order on blood glucose levels in individuals with type 1 diabetes . as we had anticipated , performing resistance exercise before aerobic exercise rather than the reverse resulted in attenuated declines in glucose concentration during exercise , fewer exercise - induced hypoglycemic events , and less need for carbohydrate supplementation . furthermore , we observed beneficial effects from this sequence on subsequent 12-h glycemic trends where the duration and severity of hypoglycemia was reduced . the benefits of performing resistance exercise before aerobic exercise instead of the reverse were observed despite overall energy expenditure being equal between experimental sessions . other types of high - intensity exercise combining aerobic and anaerobic metabolism ( e.g. , high intensity cycling ) can increase the rate of glucose appearance to a greater extent than the rate of glucose utilization ( seven and four times , respectively ) during exercise in type 1 diabetes ( 18 ) . this may cause glucose levels to increase during exercise , producing postexercise hyperglycemia if intense exercise is sustained for 12 or more minutes ( 19 ) . shorter anaerobic exercise bouts ( intermittent 4-s sprints or 10-s sprints before or after low - intensity aerobic exercise ) attenuated declines in blood glucose both during and after exercise when combined with low - intensity ( 40% vo2peak ) cycling ( 68 ) . elevated glucose production from very high - intensity exercise is generally attributed to increased levels of circulating epinephrine [ known to triple with short sprints ( 6,8,9 ) and increase up to 14 times its resting value ( 18 ) after 12 min of exhaustive cycling ] and norepinephrine , which augment glycogenolysis throughout exercise and early recovery ( 18,19 ) . although we did not measure catecholamines during the sessions , responses to high - intensity exercise are known to be comparable ( 18,20 ) or slightly attenuated ( 19,21 ) in individuals with type 1 diabetes compared with nondiabetic counterparts . catecholamines can increase to three or four times resting values during moderate - intensity resistance exercise in individuals without diabetes ( 22 ) , with responses increasing in proportion to exercise intensity ( 23 ) . if our participants experienced similar responses to resistance exercise as individuals without diabetes , then increases in epinephrine may have contributed to the attenuated rate of decline in blood glucose during the first 15 min of aerobic exercise in ra , and to the increase in glucose during resistance exercise in ar ( fig . 1 ) . the latter should be interpreted with caution , because most participants needed glucose supplements to prevent hypoglycemia during aerobic exercise in this session . it is also possible that exercise - related growth hormone ( gh ) secretion differed between treatments , potentially affecting fuel selection during exercise . goto et al . ( 24 ) found that in nondiabetic individuals , endurance exercise performed before resistance exercise produced lower gh secretion than resistance exercise alone . they also found that resistance exercise performed 20 min or less before endurance exercise produced elevated levels of gh and greater rates of lipolysis during the subsequent aerobic activity compared with endurance exercise alone ( 16 ) . because higher gh levels are known to decrease muscle glucose uptake and increase lipolysis in nondiabetic individuals ( 25 ) , this may have been a factor in the attenuated declines in blood glucose during aerobic activity in ra . high - intensity cycling increases blood lactate levels during and up to 40 min after exercise in individuals with type 1 diabetes ( 6,7,9,18,19 ) . we are unaware of published data describing lactate responses to resistance exercise in this population . resistance exercise protocols similar to the one we used have produced lactate concentrations up to four times those measured at rest , with levels remaining significantly higher than baseline until 30 min postexercise in trained nondiabetic individuals ( 26 ) . because elevated lactate could serve to increase gluconeogenesis ( 7 ) , it could be a contributing factor in the attenuated decline in glucose during the first 15 min of aerobic exercise in ra as well as in the increases in postexercise glucose levels in ar . studies suggest that high - intensity exercise may be associated with a greater frequency of nocturnal hypoglycemia in type 1 diabetic individuals ( 10,11 ) . nocturnal hypoglycemia has been identified as a risk inherent with intensive insulin therapy ( 27 ) , and it is possible that overnight hypoglycemia in our study was more related to insulin therapy than to exercise . it is noteworthy that hypoglycemic events occurring after ar tended to be longer and more severe than those experienced in ra , as demonstrated by a greater auc . studies using glucose clamp found that counter - regulatory responses to subsequent hypoglycemia were blunted after exercise , even in the absence of significant changes in glucose levels during exercise ( 28 ) . in addition , even mild hypoglycemia ( 3.9 mmol / l ) in nondiabetic individuals is sufficient to elicit counter - regulatory reactions that can blunt neuroendocrine responses to subsequent hypoglycemia within 24 h ( 29 ) . because decreases in blood glucose were greater during ar ( reaching a mean of 5.5 2.4 vs. 6.9 3.1 mmol / l in ra ) , it is plausible that subsequent responses to declining blood glucose could have been subject to impairment after exercise . although there are advantages to admitting study subjects the night before testing to control participant activity and food intake , we chose a study design more reflective of real - life conditions . participants controlled their meals and insulin but were asked to eat the same breakfast , lunch , and dinner at the same time for every day of sensor wear and to match their insulin intake as closely as possible . exercise took place at 1700 h when many individuals who work during the day opt to exercise , unlike several other studies where midmorning exercise was performed ( 69,18,19 ) . glucose responses may be different if exercise is performed at another time of day because hormone and exogenous insulin concentrations are both likely to be different . our participants were fit , habitual exercisers , and the effects of exercise may be less pronounced in unfit individuals exercising at the same relative intensity because the activity would be at a lower absolute intensity . in nondiabetic subjects running at very high relative intensity , glucose production and catecholamine concentrations increase more in athletes than in physically untrained individuals , resulting in hyperglycemia after exercise in the former group because glucose production falls more slowly than glucose utilization when exercise ends ( 30 ) . further research on different subpopulations of type 1 diabetic individuals , including those with lower fitness levels and poorer glycemic control , is warranted . this study is limited by its small sample size ( n = 12 ) , which may have prevented us from finding all of the significant differences in plasma glucose levels during exercise . to examine our participants in a real - life scenario , we compromised a certain amount of experimental control such as having complete control over all food and insulin intake . the ability to interpret the data would have been improved by having catecholamine , lactate , and gh measurements . finally , having a relatively fit sample with moderate to good control of their diabetes makes the applicability of the outcomes to individuals who are inactive or have poor glycemic control uncertain . in summary , our findings suggest that trained individuals with type 1 diabetes who perform both resistance and moderate aerobic exercise should consider performing their resistance exercise first if they tend to develop exercise - associated hypoglycemia because doing so may attenuate declines in glucose levels during subsequent aerobic exercise . this order of exercise could lead to a lower reliance on glucose supplementation during exercise and might also decrease the severity of potential nocturnal hypoglycemia . conversely , individuals having exercise - associated hyperglycemia may wish to perform aerobic exercise before resistance training . both approaches should still be accompanied by careful monitoring of blood glucose levels , both during and after exercise .

objectiveto determine the effects of exercise order on acute glycemic responses in individuals with type 1 diabetes performing both aerobic and resistance exercise in the same session.research design and methodstwelve physically active individuals with type 1 diabetes ( hba1c 7.1 1.0% ) performed aerobic exercise ( 45 min of running at 60% vo2peak ) before 45 min of resistance training ( three sets of eight , seven different exercises ) ( ar ) or performed the resistance exercise before aerobic exercise ( ra ) . plasma glucose was measured during exercise and for 60 min after exercise . interstitial glucose was measured by continuous glucose monitoring 24 h before , during , and 24 h after exercise.resultssignificant declines in blood glucose levels were seen in ar but not in ra throughout the first exercise modality , resulting in higher glucose levels in ra ( ar = 5.5 0.7 , ra = 9.2 1.2 mmol / l , p = 0.006 after 45 min of exercise ) . glucose subsequently decreased in ra and increased in ar over the course of the second 45-min exercise bout , resulting in levels that were not significantly different by the end of exercise ( ar = 7.5 0.8 , ra = 6.9 1.0 mmol / l , p = 0.436 ) . although there were no differences in frequency of postexercise hypoglycemia , the duration ( 105 vs. 48 min ) and severity ( area under the curve 112 vs. 59 units min ) of hypoglycemia were nonsignificantly greater after ar compared with ra.conclusionsperforming resistance exercise before aerobic exercise improves glycemic stability throughout exercise and reduces the duration and severity of postexercise hypoglycemia for individuals with type 1 diabetes .

RESEARCH DESIGN AND METHODS Experimental design CGM Experimental sessions Measurements Blood analyses Statistical analyses RESULTS Plasma glucose Carbohydrate intake and insulin dosage Interstitial glucose levels CONCLUSIONS

PMC3125737

scientific workflows attempt to automate repetitive computation and analysis by chaining together related processes . automating repetitive time - consuming tasks allows scientists to keep pace with ever - growing volumes of data ( 1 ) . furthermore , workflows can aid in the reproducibility of scientific computations by providing a formal declaration of an analysis . reproducibility is central to the scientific method , and detailed workflow provenance information ensures an analysis can be reproduced and extended ( 1 ) . it has been suggested that in addition to references , scientific publications should include documentation of computational methods that allows readers to easily reproduce their results , increasing reader engagement with scientific publications ( 2 ) . researchers need to access exponentially growing amounts of dna and protein sequence data along with a large number of bioinformatic analytic tools ( 3 ) . the data is distributed in different formats and in heterogeneous data structures and information systems . analytic tools are made available to scientists in a variety of ways including as downloadable tools for local installation , web pages and web services . by enabling researchers to work effectively through multiple interconnected tools and handling multiple data formats and large data quantities , workflows with embedded data pipelines and analysis scripts are a powerful alternative to using standalone , off - grid applications ( 4 ) . the bioextract server ( 5 ) is a web - based system that offers researchers a flexible environment for analyzing genomic data . the bioextract server provides : ( i ) a flexible querying and retrieval interface to national center for biotechnology information ( ncbi ) nucleotide and protein databases , european molecular biology laboratory ( embl - bank ) non - redundant nucleotide databases , and the european bioinformatics institute 's ( ebi ) universal protein resource ( uniprot ) and uniprot reference clusters ( uniref ) databases ; ( ii ) the ability to filter search results and use them as input into analytic tools ; ( iii ) the facility to save search results as data extracts that are automatically integrated into the system as searchable data resources ; ( iv ) access to analytic tools including a large list of curated web services , as well as generic access to soap - based web services and command line tools residing on the user 's local workstation ; ( v ) the ability to save a series of bioextract server tasks ( e.g. query a data source , save a data extract and execute an analytic tool ) as a workflow ; and ( vi ) the opportunity for researchers to share their data extracts , analytic tools and workflows with collaborators . queries are constructed by first checking the box for one or more data sources and composing a query via a web form . figure 1 presents an example of querying ncbi nucleotide and protein databases for the r2r3-myb genes in pinus taeda ( loblolly pine ) . queries executed against data sources residing at the bioextract server respond quickly . for example , a query of viridiplantae returns in a matter of seconds . response times for queries against data sources residing at other sites ( e.g. ncbi ) are consistent with response times for queries made directly at those sites . figure 1.bioextract server queries are executed through the query tab of the system by checking the box for the desired data resource(s ) , constructing the query using the query form , and submitting the query . bioextract server queries are executed through the query tab of the system by checking the box for the desired data resource(s ) , constructing the query using the query form , and submitting the query . the execution of the query results in the creation of a data extract that is displayed on the extracts tab of the system . individual records may be viewed locally or through an external link to the original data source . in addition , data extracts may be filtered , exported and used as input into analytic tools . while most of the bioextract server 's functionality is available to all users , the ability to save a data extract is available only to users who have registered with the system . users who are signed into the system as registered users ( figure 1 , top right ) also have the option of saving a data extract simply by clicking the save extract button on the extracts tab and entering the name and description of the extract . once saved , the researcher 's privately owned data extracts are integrated into the bioextract server platform and are listed with other available data resources on the query tab for the registered user only . data extracts may also be created within the system by providing a list of sequence record identifiers ( ids ) such as accession numbers . on the query tab , researchers are able to fetch sequence records by entering or uploading a list of ids and specifying from what data source the records should be retrieved . all data extracts created within the system are privately owned by the researcher and are only made available to others by explicitly sharing them with a group . groups tab ; ( ii ) creating a group ( under additional actions ) ; ( iii ) clicking on the new group ; ( iv ) selecting the extracts tab for the new group ; and finally ( v ) clicking the add elements button to select the extract to share . a number of well - established and unique bioinformatic analytic tools are made available through the bioextract server , with the majority integrated as curated web services . researchers access analytic tools through the list of available tools on the tools tab . the source of the analytic tool 's input(s ) may be : ( i ) the current data extract ( i.e. the records listed on extracts tab ) ; ( ii ) the output from a previously executed tool ; or ( iii ) private data provided by the researcher ( uploaded or entered in a text box ) . analytic tool parameters may be selected or modified before execution and resulting output files may be viewed , downloaded and used as input into subsequently executed analytic tools . the bioextract server offers researchers the ability to add additional analytic tools to their workspace . users may select such tools from a large list of web services , including emboss soaplab , biomoby and kegg , with the integration of biomart ( www.biomart.org ) currently in development . the system also offers generic support for other soap - based web services and lets users integrate local command line tools residing on their own workstations through the use of a client - side java applet . customize your tools functionality , which allows users to provide detailed descriptions of the tools as well as a help link to additional information . as researchers perform tasks ( e.g. execute a query , save a data extract or execute an analytic tool ) within the bioextract server , their actions are recorded and saved in the background . the series of recorded tasks may be saved as a workflow through the workflow tab after the user provides a name and description of the workflow . workflows are represented within the bioextract server as directed acyclic graphs with the graph nodes representing specific tasks and the arcs representing the data dependencies . reuse of workflows is realized through the easily modified workflow inputs , queries and analytic tool parameter settings . copying a workflow for future editing is accomplished by executing the workflow and providing a name and description for the copy . once a task within a workflow has been executed , specific task information may be accessed by clicking on the executed node within the workflow graph . additionally , a provenance report related to an executed workflow provides detailed information such as a general description of the workflow , analytic tool descriptions , parameter settings , input / output data , query information and saved data extract descriptions . bioextract server workflows may be shared with the research community in a variety of ways . first , researchers can export a workflow as an xml file and share it with collaborators . when a workflow xml file is imported into the bioextract server by a registered user , a new instance of that workflow is created and privately owned by the user . myexperiment is a virtual research environment designed to provide a mechanism for collaboration and sharing workflows . this was achieved by adopting a social web approach that is tailored to the particular needs of the scientist ( 6 ) . bioextract server workflows imported into myexperiment may be additionally annotated through their system and executed directly through their platform . third , along with data extracts and analytic tools , workflows can be shared with collaborative groups through the bioextract server groups tab . to explain how a simple workflow is created within the bioextract server , consider the task of carrying out a multiple sequence alignment analysis using a known protein sequence record as input . to complete this analysis , a researcher would : sign into the bioextract server by clicking the sign in link in the upper right corner of the screen.execute the ncbi blastp tool ( 7 ) located under the similarity search tools node in the available tools tree on the tools tab . the execution of blastp results in the creation of a data extract that can be viewed on the extracts tab.execute the xmknr analytic tool , a simple shell script that employs vmatch ( 8) ( www.vmatch.de ) to remove duplicate sequences , found under the edit tools node . the input into this tool is specified as use records on extract page formatted as fasta. its execution results in the modification of the data extractexecute the clustalw ( 9 ) tool to create the multiple sequence alignment with the input specified as use records on extract page formatted as fasta.navigate to the workflow tab and click the create and import workflows node within the researchers are able to annotate the workflow with information that will assist others in understanding decisions made during its creation.the multiple sequence alignment could serve as input for subsequent steps ; for example , phylogenetic or motif analyses . sign into the bioextract server by clicking the sign in link in the upper right corner of the screen . execute the ncbi blastp tool ( 7 ) located under the similarity search tools node in the available tools tree on the tools tab . the execution of blastp results in the creation of a data extract that can be viewed on the extracts tab . execute the xmknr analytic tool , a simple shell script that employs vmatch ( 8) ( www.vmatch.de ) to remove duplicate sequences , found under the edit tools node . the input into this tool is specified as use records on extract page formatted as fasta. its execution results in the modification of the data extract execute the clustalw ( 9 ) tool to create the multiple sequence alignment with the input specified as use records on extract page formatted as fasta. navigate to the workflow tab and click the create and import workflows node within the researchers are able to annotate the workflow with information that will assist others in understanding decisions made during its creation . the multiple sequence alignment could serve as input for subsequent steps ; for example , phylogenetic or motif analyses . more information related to the creation of workflows ( including this video demonstration ) can be found through the follow link : http://bioservices.usd.edu/bioextract/videos/blastn-clustalw-example.html . the top node denotes a tool that has completed execution , the middle node represents an executing tool , and bottom node represents an idle tool waiting to execute . the top node denotes a tool that has completed execution , the middle node represents an executing tool , and bottom node represents an idle tool waiting to execute . to further illustrate the advantages of the bioextract server platform this workflow represents typical steps undertaken in the primary analysis and comprehensive annotation of a set of transcript sequences . such sets are currently abundantly generated from full - length cdna sequencing projects or assembly of est sequences , and with increasing read lengths of novel sequencing technologies , there will be similar assemblies of rna - seq data from many species and sampling conditions . using ncbi blast ( 7 ) , museqbox ( 10 ) and linux shell scripting , the workflow partitions the input transcript set successively according to matching targets . specifically , mrna markup identifies potential vector or bacterial contaminants ( sequencing artifacts ) , potential chimeras ( assembly artifacts ) , likely full - length protein - coding mrnas , as well as matches from a comprehensive protein database and a protein domain database . what remains after these identifications include presumably novel transcripts for further analysis with other programs . while conceptually simple , implementation and maintenance of the programs in individual labs would be hugely time- and cost - ineffective compared to a publicly available workflow system . for illustration , the workflow is implemented with a sample configuration consisting of a transcript set of arabidopsis est assemblies derived from plantgdb ( www.plantgdb.org ) and target databases consisting of univec ( www.ncbi.nlm.nih.gov/vecscreen/univec.html ) for vector contaminants , an escherichia coli genome for determining bacterial contamination , the arabidopsis reference protein set tair10 ( www.arabidopsis.org ) , a comprehensive protein subject database containing viridiplantae entries from uniref90 ( www.uniprot.org/help/uniref ) and the conserved domain database ( www.ncbi.nlm.nih.gov/structure/cdd/cdd.shtml ) . the final step of the workflow produces a summary report detailing how many sequences were matched during each step as well as how many potentially novel sequences remain . the workflow is presented graphically at bioextract.org / scripts/. when the user clicks the mrna markup label within the list of workflows , a workflow graph will be displayed . the user can click any node in the workflow graph to acquire more information regarding the analytic step it represents as well as the output it produces . there exist many biological data and analytic tool integration systems that offer powerful and comprehensive workflow creation functionality . taverna ( 11 ) , kepler ( 12 ) , triana ( 13 ) , ugene ( 14 ) and trident ( 15 ) are some examples of workflow management systems that are primarily stand - alone applications requiring installation of client software . through a drag - and - drop user interface , these systems allow researchers to develop complex workflows from distributed and local resources . the taverna workbench , part of the mygrid project ( 16 ) , is arguably the most well - known bioinformatic workflow development system . it is an application that gives users the ability to easily integrate the growing number of molecular biology tools and databases available on the web , especially web services . genepattern ( 17 ) , mobyle ( 18 ) and galaxy ( 19 ) are web - based workflow management systems . genepattern is a genomic analysis platform that provides access to tools for gene expression analysis , proteomics and common data - processing tasks . a web - based interface provides easy access to these tools and allows the creation of multi - stepanalysis pipelines . mobyle provides a flexible web environment for defining and running bioinformatics analysis by embedding management features allowing users to combine tools using a hierarchical typing system . galaxy is an open source workflow system with which researchers are able to create complex workflows that can be shared , cloned , annotated and edited . the primary objective of the galaxy system is to provide support for accessible , reproducible and transparent science ( 19 ) . the bioextract server is entirely web - accessible , and while allowing completely flexible customization including integration of locally accessed scripts and tools , the interface and functionality of the site are specifically designed for the novice and implementation detail - uninterested user whose primary need is ease of use and quality results . these design criteria seek to explicitly circumvent the gap between workflow developers and the user community , which from our experience is still substantial . future enhancements to the bioextract server will focus on improving workflow reproducibility , flexibility and provenance information . functionality is currently being developed that will provide better feedback regarding a tool 's compatibility with the user - selected input file format . in order for workflows to be reproducible , it is imperative that the required analytic tools be reliable . this underscores the need for curation of analytic tools to ensure they are both available and well defined ( 4 ) . biocatalogue is a comprehensive , curated registry of biological web services that aims to improve process reliability and validation through individual , automatic and community curation ( 3 ) . recognizing that the application of web services within a computational experiment can compromise its reproducibility ( 19 ) , version information will be incorporated into all of the bioextract server 's publicly accessible analytic tools . the intent of a workflow to the research community is an important aspect of a computational experiment . providing researchers with the capacity to annotate and search workflow provenance information , advances the ability to understand the decisions made during the experimental design and execution . the bioextract server provenance information will be expanded to allow for additional user annotations and ad hoc querying of provenance data . national science foundation ( dbi-0606909 to v.b and c.m.l . ) funding for open access charge : national science foundation .

the bioextract server ( bioextract.org ) is an open , web - based system designed to aid researchers in the analysis of genomic data by providing a platform for the creation of bioinformatic workflows . scientific workflows are created within the system by recording tasks performed by the user . these tasks may include querying multiple , distributed data sources , saving query results as searchable data extracts , and executing local and web - accessible analytic tools . the series of recorded tasks can then be saved as a reproducible , sharable workflow available for subsequent execution with the original or modified inputs and parameter settings . integrated data resources include interfaces to the national center for biotechnology information ( ncbi ) nucleotide and protein databases , the european molecular biology laboratory ( embl - bank ) non - redundant nucleotide database , the universal protein resource ( uniprot ) , and the uniprot reference clusters ( uniref ) database . the system offers access to numerous preinstalled , curated analytic tools and also provides researchers with the option of selecting computational tools from a large list of web services including the european molecular biology open software suite ( emboss ) , biomoby , and the kyoto encyclopedia of genes and genomes ( kegg ) . the system further allows users to integrate local command line tools residing on their own computers through a client - side java applet .

INTRODUCTION QUERYING DATA SOURCES AND MANAGING DATA EXTRACTS EXECUTING, ADDING AND MODIFYING ANALYTIC TOOLS BIOEXTRACT SERVER WORKFLOWS BIOEXTRACT SERVER WORKFLOW EXAMPLE CONCLUSION FUTURE DIRECTION FUNDING

PMC4901838

sincipital encephaloceles , also called frontal or anterior encephaloceles , are rare neural tube defects and are more common in south and southeast asian populations . in children with sincipital encephaloceles there are often significant associated intracranial anomalies , including intracranial cysts , complete / partial corpus callosal agenesis , interhemispheric lipomas , facial clefts , and migrational anomalies . occipital encephaloceles may be associated with chiari or dandy - walker malformations and callosal or migrational anomalies . it is reported that younger siblings of patients affected with encephaloceles have up to 6% risk of a congenital central nervous system abnormality . after approval from the institutional ethics review committee , a hospital - based cross - sectional study was conducted over 8 years from june 2007 to may 2015 on 28 patients in the departments of radiodiagnosis and pediatric surgery in a tertiary care hospital . the study comprised of outpatients and in patients , of both sexes presenting with sincipital encephaloceles . patients were only allowed plain water preceding the imaging so as to keep the patient ready for sedation / anesthesia if needed . the patients were evaluated by either computed tomography ( ct ) or magnetic resonance imaging ( mri ) whichever was feasible . the ct scanner used in this study was siemens somatom spirit dual slice ct scanner ( siemens healthcare , erlangen , germany ) . unenhanced ct scans of head and faciomaxillary region were obtained in all patients . scanning parameters used were spiral mode with slice thickness of 6 mm and collimation 6 mm 2.5 mm , pitch : 1.4 ; kvp : 130 ; mas : 80 . mri imaging was performed with a 1.5-t superconducting magnet system 1.5 tesla siemens magnetom avanto b15 system ( siemens healthcare , erlangen , germany ) using a transmit / receive body coil with a 15-mt / m gradient . the imaging parameters were t1 , t2 , and fluid - attenuated inversion recovery ( flair ) sequences obtained in all three planes : axial , coronal and sagittal . parameters for t1-wi ( t1-weighted image ) : 400600/15 ( repetition time ms / echo time ms ) , 220240-mm field of view , 256 192 matrix , and slice thickness of 5 mm with a gap of 1 mm . parameters for t2-wi : 3200/428 ( repetition time ms / echo time ms ) , 256-mm field of view , 256 256 matrix , and slice thickness of 5 mm with a gap of 1 mm . parameters for flair : 10,000/70130 ( repetition time ms / echo time ms ) , 240-mm field of view , 256 192 matrix , and slice thickness of 5 mm with a gap of 1 mm . the areas of interest in the study were type of encephalocele , its contents , extracranial extension and effect on adjacent structures and to look for associated brain malformations . after approval from the institutional ethics review committee , a hospital - based cross - sectional study was conducted over 8 years from june 2007 to may 2015 on 28 patients in the departments of radiodiagnosis and pediatric surgery in a tertiary care hospital . the study comprised of outpatients and in patients , of both sexes presenting with sincipital encephaloceles . patients were only allowed plain water preceding the imaging so as to keep the patient ready for sedation / anesthesia if needed . the patients were evaluated by either computed tomography ( ct ) or magnetic resonance imaging ( mri ) whichever was feasible . the ct scanner used in this study was siemens somatom spirit dual slice ct scanner ( siemens healthcare , erlangen , germany ) . scanning parameters used were spiral mode with slice thickness of 6 mm and collimation 6 mm 2.5 mm , pitch : 1.4 ; kvp : 130 ; mas : 80 . mri imaging was performed with a 1.5-t superconducting magnet system 1.5 tesla siemens magnetom avanto b15 system ( siemens healthcare , erlangen , germany ) using a transmit / receive body coil with a 15-mt / m gradient . the imaging parameters were t1 , t2 , and fluid - attenuated inversion recovery ( flair ) sequences obtained in all three planes : axial , coronal and sagittal . parameters for t1-wi ( t1-weighted image ) : 400600/15 ( repetition time ms / echo time ms ) , 220240-mm field of view , 256 192 matrix , and slice thickness of 5 mm with a gap of 1 mm . parameters for t2-wi : 3200/428 ( repetition time ms / echo time ms ) , 256-mm field of view , 256 256 matrix , and slice thickness of 5 mm with a gap of 1 mm . parameters for flair : 10,000/70130 ( repetition time ms / echo time ms ) , 240-mm field of view , 256 192 matrix , and slice thickness of 5 mm with a gap of 1 mm . the areas of interest in the study were type of encephalocele , its contents , extracranial extension and effect on adjacent structures and to look for associated brain malformations . fifty percent of the patients presented before the age of 3 years , with the youngest presenting on day 7 . rest of the patients ( 42% ) presented between 3 and 10 years of age . our study did not show any gender predominance , with both males and females being affected equally . nasofrontal encephalocele was the most common type , seen in 13 ( 46.4% ) patients . the naso - orbital and the combined type were the least common with 4 ( 14.2% ) patients each . two patients had evidence of herniation of lateral ventricle along with brain tissue through the cranium into the encephalocele sac [ table 1 ] . type of sincipital encephaloceles corpus callosal agenesis with colpocephaly was the most common associated malformation in our study [ table 2 ] . 6/10 archnoid cysts were hemispheric , 4/10 were inter hemispheric and 2/10 were located in posterior fossa . associated brain malformations seven patients had evidence of hydrocephalus , of which five cases were due to the obstruction by large arachnoid cysts and two cases were due to aqueduct stenosis . subdural collection was noted in 6 patients . in 4 patients it was subdural csf collection due to hyperfunctioning of shunts and in 2 patients it was blood due to rupture of bridging cortical veins . of these 6 patients , ventriculoperitoneal ( vp ) shunts were performed in 2 patients and cystoperitoneal shunt in 2 patients with large interhemispheric cysts causing mass effects . corpus callosal agenesis with colpocephaly was the most common associated malformation in our study [ table 2 ] . 6/10 archnoid cysts were hemispheric , 4/10 were inter hemispheric and 2/10 were located in posterior fossa . associated brain malformations seven patients had evidence of hydrocephalus , of which five cases were due to the obstruction by large arachnoid cysts and two cases were due to aqueduct stenosis . subdural collection was noted in 6 patients . in 4 patients it was subdural csf collection due to hyperfunctioning of shunts and in 2 patients it was blood due to rupture of bridging cortical veins . of these 6 patients , ventriculoperitoneal ( vp ) shunts were performed in 2 patients and cystoperitoneal shunt in 2 patients with large interhemispheric cysts causing mass effects . encephaloceles occur due to the failure of surface ectoderm to separate from the neuroectoderm , resulting in a bony defect in the cranial and/or facial bones , which allows herniation of meninges or brain tissue . encephaloceles occur in one of every 4000 live births and have no sex predilection . according to location , these are occipital ( 75% of cases ) , sincipital ( 15% ) , or basal ( 10% ) . sincipital encephaloceles involve the mid - face and occur about the dorsum of the nose , orbits , and forehead . these sincipital encephaloceles are more common in south and southeast asian populations whereas the occipital encephaloceles are more common in the western population . the sincipital encephaloceles are further subdivided into nasofrontal , nasoethmoidal , and naso - orbital types . the nomenclature used for encephaloceles is based on the origin of their roof and floor ; thus , for example , the roof and floor of frontonasal encephaloceles are the frontal and nasal bones , respectively . nasofrontal encephaloceles result from herniation through both the foramen cecum and the fonticulus frontalis and projecting along the nasal bridge between the nasofrontal sutures into the glabella ( nasofrontal region ) . nasoethmoidal encephaloceles occur when there is persistent herniation through the foramen cecum into the prenasal space and nasal cavity under the nasal bones and above the nasal septum ( nasoethmoidal region ) . moreover , naso - orbital types occur along the medial orbit at the level of the frontal process of the maxilla and the ethmoid - lacrimal bone junction ( naso - orbital region ) . affected children may present with nasal obstruction , mass in the frontal region , proptosis , or cerebrospinal fluid ( csf ) leaks from ruptured encephalocele sac with recurrent meningitis . the prognosis for the sincipital encephalocele patients is generally good and is usually associated with normal intelligence and motor development . surgery is the best treatment option for encephaloceles , and mri is the choice of imaging modality for defining the contents of an encephalocele prior to surgery . high - resolution ct may also be used to display the bone anatomy , but the intracranial extension is best defined with mri . the extent of cerebral tissue in an encephalocele is also better defined with mri , which aids in prognosis and surgical planning . the aim of the radiologist is to precisely define the type of the encephalocele ; delineate the size , contents , extension ; and identify associated anomalies . there are often significant associated intracranial anomalies , including intracranial cysts , complete / partial corpus callosal agenesis , interhemispheric lipomas , facial clefts , and migration anomalies . earlier studies postulated theories such as adhesive theory , which suggest failure of mesodermal migration into midline leading to herniation of the brain matter and resulting failure of closure of anterior neuropore . as corpus callosum forms near the site of final closure of anterior neuropore , many of these patients have associated corpus callosal agenesis which was also observed in our series . rapport et al . , postulated that this condition may have several etiological factors because mesodermal abnormalities alone can not account for the observed abnormalities . lemire et al . , thought that anterior encephaloceles result from abnormal dedifferentiation , whereas some other studies suggest neuroectodermal abnormality . from the array of associated malformations that we have observed in our series the most common associated malformation with sincipital encephaloceles is the agenesis or hypogenesis of the corpus callosum . corpus callosum is the largest cerebral commissure connecting neocortical areas and develops between 12 and 20 weeks of gestation . development occurs from front to back with the exception of the rostrum which develops after the splenium . imaging by ct and mr in complete callosal agenesis reveals widely separated and nonconverging lateral ventricles , disproportionately enlarged occipital horns ( colpocephaly ) , and an elevated third ventricle which is continuous superiorly with the interhemispheric fissure [ figure 1 ] . in sagittal mr studies , corpus callosum , cingulate gyrus , and sulcus are not seen . gyri on the medial hemispheric surface radiate outward from the high - riding third ventricle . in partial agenesis of corpus callosum , the splenium and rostrum are absent . 3-year - old male child with a swelling at nasal bridge , ( a ) axial computed tomography scan shows a nasofrontal encephalocele ( * ) , ( b and c ) axial computed tomography sections show corpus callosal agenesis as evidenced by parallel running lateral ventricles with colpocephaly ( arrow in c ) . they are congenital lesions that occur as a result of the splitting of the arachnoid membrane . the cysts are extra - axial fluid - filled sacs , which are sharply marginated somewhat scalloped appearing lesions that parallel the csf intensity on all sequences of mri . some suprasellar cysts become very large [ figures 2 and 3 ] and cause obstructive hydrocephalus . the arachnoid cysts , particularly the interhemispheric cysts , are usually associated with corpus callosal agenesis . 2-year - old male child with a forehead swelling , ( a ) axial computed tomography bone window shows nasoethmoidal and naso - orbital type of encephalocele ( * ) . ( b ) axial , ( c ) coronal and ( d ) sagittal computed tomography sections show associated large hemispheric arachnoid cyst ( arrow ) . ( b ) axial ct section also reveals the parallel orientation left lateral ventricle ( upward arrow ) suggestive of corpus callosal agenesis . 2-year - old female child with swelling at nasal bridge , ( a ) sagittal t1-weighted image shows a large interhemispheric arachnoid cyst ( # ) . ( b ) axial t1-weighted image in another patient with nasoethmoidal encephalocele ( * ) shows an arachnoid cyst in right temporal fossa ( arrow ) . in our series , subdural collection of the six patients four had subdural csf collection and it was thought to be due to hyperfunctioning of the shunts [ figure 4 ] , ( two had been operated with ventriculo - peritonial shunt for obstructive hydrocephalus and in the other two , ventricular catheter of the shunt was placed in the large interhemispheric cysts i.e. cysto - peritoneal shunt which caused mass effects ) . other two patients had small subdural hematomas probably due to rupture of bridging cortical veins around the herniation . 2-year - old female child with forehead swelling , ( a ) axial computed tomography section shows left subdural collection ( arrow ) in a patient with ventriculoperitoneal shunt . ( b ) axial computed tomography section in another patient with a large arachnoid cyst showing bilateral frontal subdural collection ( arrow ) with calcification of the arachnoid membrane . agyria - pachygyria complex is a defect in migration of cerebral neurons , resulting in failure of cortical gyri to develop [ figure 5 ] . the extent , distribution , and detailed structure of agyria - pachygyria vary widely , and the clinical features are accordingly very diverse . rather than one entity , pachygyric cortical abnormalities form a broad spectrum that raises different clinical and genetic problems . this complex can be of generalized / bilateral or localized / unilateral gyral malformations . generalized seizures with increased incidence of spastic quadriparesis and microcephaly are associated with the generalized / bilateral type , whereas partial seizures are more common in localized / unilateral gyral malformations and hemiparesis is the most frequent neurologic deficit . patients with bilateral or generalized gyral anomalies have poor prognosis for outcome of epilepsy and neurologic disability . the recognition of these lesions with high - resolution techniques of mri is important for planning proper treatment and genetic counseling . 4-year - old child presented with swelling over the nose and seizure , ( a ) axial computed tomography scan shows nasofrontal type of encephalocele ( * ) with mild orbital extension . ( b ) axial computed tomography section also shows agyria and pachygyria complex involving left parietooccipital lobe ( arrow ) with corpus callosal agenesis . ( c ) higher axial computed tomography section also shows ventriculoperitoneal shunt ( # ) in interhemispheric fissure with left subdural collection ( arrow ) . we could not perform mri in many cases , which is the investigation of choice due to cost constraints . we could not perform mri in many cases , which is the investigation of choice due to cost constraints . encephaloceles are often associated with significant intracranial anomalies , with agenesis of corpus callosum being the most common . hence , a precise analysis of each case is necessary to recognize the associated malformations and establish the prognosis and risk of recurrence .

objective : the aim of this study was to evaluate the associated intracranial malformations in patients with sincipital encephaloceles.materials and methods : a hospital - based cross - sectional study was conducted over 8 years from june 2007 to may 2015 on 28 patients . the patients were evaluated by either computed tomography or magnetic resonance imaging whichever was feasible . encephaloceles were described with respect to their types , contents , and extensions . a note was made on the associated malformations with sincipital encephaloceles.results:fifty percent of the patients presented before the age of 3 years and both the sexes were affected equally . nasofrontal encephalocele was the most common type seen in 13 patients ( 46.4% ) , and corpus callosal agenesis ( 12 patients ) was the most common associated malformation . other malformations noted were arachnoid cyst ( 10 patients ) , hydrocephalus ( 7 patients ) , and agyria - pachygyria complex ( 2 patients).conclusion : capital brain malformations are frequently encountered in children with sincipital encephaloceles . detail radiological evaluation is necessary to plan treatment and also to prognosticate such rare malformations .

INTRODUCTION MATERIALS AND METHODS Study group Patient preparation Technique of study Computed tomography Magnetic resonance imaging RESULTS Associated brain malformations DISCUSSION Limitation CONCLUSION Financial support and sponsorship Conflicts of interest